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Introduction Articular cartilage of higher animals does not regenerate, but under some circumstances mesenchymal cells can be inducted to behave phenotypically as chondrocytes. Tissue engineering has produced only partial cartilage repair and inconsistent outcomes. This study reports full thickness articular cartilage regeneration induced by freeze-dried particulate cartilage. Methods and Materials Full thickness cartilage defects in the femoral condyles of 22 baboons were filled with freeze-dried cartilage particles. Defects in two animals were filled with frozen cartilage particles. Four animals served as controls. Experimental animals were sacrificed at 2 weeks (2), 6 weeks (6), 9 weeks (1) 12 weeks (3), 14–16 weeks (4) and at 7 and 14 months. The knees were photographed and x-rayed. The condyles were frozen, sectioned, photographed again, fixed, decalcified and processed for histology. To determine if cartilage preparations were osteogenic samples were implanted intramusculary in 16 athymic rats. The specimens were examined at 1,2,4 & 8 weeks. Cartilage particles were also implanted intraosseously into 2 baboons. Regenerating cartilage was graded in accordance with a modified Mankin's method. Results Cartilage regeneration proceeded from the edges of the defect. The 12 week and older defects were covered with smooth and glistening new cartilage. Control defects remained open or contained uneven patches of cartilage. Animals with frozen cartilage implants appeared identical to controls. Cartilage particles implanted into athymic rats remained inert and elicited neither osteogenesis nor chondrogenesis, as did cartilage placed intramedullary in baboons. Conclusions The data demonstrates induction of articular cartilage regeneration by freeze-dried hyaline cartilage particles implanted orthotopically., Introduction Long-term maintenance of cartilage allograft tissue is challenging. This study examines if dexamethasone, a synthetic adrenal corticosteroid, can play a role in maintaining the mechanical properties of cartilage explants in serum-free tissue culture. Methods and Materials In Study 1, middle zone explant disks were harvested from juvenile (4∼6 month) bovine cartilage plugs. In Study 2, full-thickness osteochondral plugs were harvested from mature (1∼2 year-old) bovine femoral condyles. Explants were cultured with continuous dexamethasone (0.1 microM) supplementation; with dexamethasone removed after 2 weeks of culture; or dexamethasone-free throughout 28 days of culture. Results In Study 1, Youngs modulus (Ey) of explants grown with continuous dexamethasone supplementation increased ∼50% from initial values (reaching 2800 kPa) while dexamethasone-free explants decreased ∼80% (down to 400 kPa). Removal of dexamethasone after 14 days resulted in significant Ey reduction of juvenile explants. In Study 2, Ey remained similar (at day 0 levels) for all groups. Modulus changes, when observed, were accompanied by corresponding changes to GAG and collagen levels in some cases. Conclusions Our encouraging findings with the well-established bovine model support the use of serum-free tissue culture as a preservation technique for cartilage grafting. Dexamethasone proved to be necessary in maintaining the mechanical stiffness of juvenile cartilage explants during long-term culture, even improving properties over time, whereas mature bovine explants maintained their initial properties irrespective of steroid supplementation. This outcome suggests that age can be a significant factor in the selection of culture supplements. These findings need to be verified with human cartilage allografts., Introduction The objective of the present study is to investigate the repairing effects of the implanted scaffold-free chondrocyte plate on the osteochondral defects of the articular cartilage. Methods and Materials Thirty eight rabbits were used and 76 osteochondral defects were created. These rabbits were classified into two groups; a chondrocyte plate insertion group (CP group, n=26), and a noninsertion group (defect group, n=12). Histological evaluations were done at 4, 12and 24 weeks after surgery. The histological evaluations contain safranin O staining, immunohistochemical analyses for chondromodulin-I (ChM-I), vascular endothelial growth factor (VEGF) and superficial zone protein (SZP), and the histological scoring using modified O'Driscoll ICRS grading system. Results The repair sites appeared to be filled with cartilaginous tissues and much expressed ChM-I and little VEGF in the CP group 4 weeks after surgery. Conversely in the defect group, the defects were filled with fibrous tissue, and repair sites expressed VEGF and did not expressed ChM-I. The implantation sites kept the phenotype of the articular cartilage in the CP group 24 weeks after surgery. Moreover, SZP was expressed at the surface of repair sites. After 12 and 24 weeks, histological scores in the CP group were significantly higher than those in the defect group. Conclusions The chondrocyte plate contributed to repair the osteochondral defect. The repair site was altered to acquire the cartilage phenotype by implantation of the chondrocyte plate., Introduction Monolayer-grown chondrocytes can keep phenotype only with a limited number of passages. Mechanical stimulus may improve to keep the phenotype of the cultured chondrocytes. The purpose of this study is to investigate the effect of cyclic tesile strain (CTS) on the glycosaminolgycan (GAG) synthesis of primary cultured rabbit articular chondrocytes. Methods and Materials Chondrocytes were isolated from the knee joints of six one-month rabbits. Chondrocytes from each rabbit were divided into three parts and cultured in a Flexercell 4000 strain unit. The cultured cells were loaded with CTS (sinusoidal wave, 0.3Hz) in 0%, 5% and 15% strain for 6 hours per day. The GAG concentrations of supernatants were measured by precipitation with alcian blue at 24, 36, 48, 60h after the beginning of the first CTS loading. (Fig. 1). The data was analyzed with ANOVA of repeated measures. Results The GAG concentrations of the supernatants increased with time (P⇠0.001). It increased with the increase of strain rate (P⇠0.001). And post hoc tests showed the significant difference between different strain rate in any time point (P⇠0.05). (Fig. 2) Conclusions The CTS (sinusoidal wave, 0.3Hz, 6h/d) regimen enhance the GAG synthesis of cultured primary rabbit articular chondrocyte. Higher strain rate produce larger stimulus with more GAG synthesis. The CTS may be an effective way to prevent the dedifferentiation of chondrocytes in monolayer culture., Introduction The aim of this study was to quantify the incidence and microbial spectrum after contamination in the operating room, assess the most effective sterilizing agent, and to identify the effect on bone and cartilage. Fragments were dropped onto the OR floor and cultured. Next, bone fragments were inoculated with three organisms to assess the different sterilizing agents. The contamination rate was 70%, with coagulase negative staphylococcus as the most common organism. Bactericifal agents, and scrubbing were the most effective methods of decontamination. Methods and Materials Phase I, Osteoarticular bone fragments were dropped onto the operating room floor, and permitted to rest on the OR floor for a total of 30 seconds before being cultured. Each positive culture was then speciated by a hospital microbiologist. Phase II, bone fragments were inoculated with different species and exposed to different sterilizing agents based on a protocol. Results Coagulase Negative Staphylococcus: For both betadine and chlorhexidine groups, there was no growth in any group. In the isopropyl alcohol and saline groups, only those rinsed with saline had growth. Bacillus species: There was no growth for betadine, chlorhexidine, or isopropyl alcohol groups. Those rinsed with saline, and all controls had growth. Corynebacterious species: There was no growth in any of the groups, except in the control group. Conclusions We found 70% contamination rate for dropped bone grafts. The most common species in our study was coagulase negative staphylococcus. Bactericidal agents, and scrubbing were most effective. There is no difference in the duration of exposure to sterilizing agent., Introduction The purpose of this study was to evaluate the performance of a novel allograft cartilage scaffold (ACS) in critically sized goat osteochondral defects. ACS is a regionally specific acellular construct comprised of articular cartilage and both demineralized and non-demineralized cancellous bone. Methods and Materials Two (2) 6mm chondral or osteochondral defects, one in the lateral proximal trochlear sulcus (LPTS) and one in the medial femoral condyle (MFC), were created in the knees of skeletally mature goats. The chondral defects were microfractured. The osteochondral defects were filled with ACS, Tru-FitTM, or left empty. Implanted knees were immobilized in a modified Thomas splint for 7 days post-op. After 6 months, gross and histological analyses were done. In addition, stiffness of the chondral-subchondral bone complex was assessed by durometer testing and compare to the contralateral non-operated side. Results Defects treated with either the ACS or Tru-Fit BGS plug had better repair than the empty defect and the microfractured defects. Stiffness results of the joint surface surrounding the MFC lesion sites were generally within the normal range. Histologically, type II collagen repair tissue and positive Safranin-O staining were evident in ACS-filled defects, along with integration to the host. Subchondral bone repair was also observed in ACS-filled defects while cysts were seen with the Tru-FitTM and empty defects. Conclusions This clinically relevant model of osteochondral defect fill demonstrated that the ACS implant is capable of eliciting hyaline or hyaline-like cartilage repair at 6 months, along with endochondral repair of subchondral bone., Introduction Compare the fill of osteochondral defects using a novel allograft sponge with autograft transplants. Methods and Materials Two full-thickness defects (4.5 mm ø, 10 mm depth) were created in both the medial femoral condyle and the lateral patellar groove of adult goats [Lane, 2004]. In each location, one defect was filled with an autograft osteochondral plug while the other was filled with an acellular allograft sponge [Bacterin International]. The sponge is demineralized goat cancellous bone created similarly to commercially-available human allograft products. Animals were splinted for 2 weeks. After 3 and 6 months, the animals were sacrificed and the defects underwent MRI morphological analysis, macroscopic scoring [ICRS] and histological scoring [Sellers, 2000]. Data were analyzed using three-way ANOVA for time, condition and site. Results Sponge-treated defects were filled with white, glistening repair tissue and the quality of repair improved with time. Results achieved using the sponge in the groove were similar to those of the autograft controls in macroscopic appearance and most histological assessments at 6 months. Repairs in the groove were flush with surrounding cartilage (based on histology and MRI). The results in the condyle were less optimal. Remarkably, integration was significantly better in the sponge-treated defects when compared to the autograft controls, and that outcome was true for both sites and both timepoints. Conclusions This study demonstrates the use of a novel allograft sponge to successfully fill osteochondral defects and improve integration with the surrounding cartilage in goats. These results have not been validated in human joints., Introduction Bupivacaine has been shown to be cytotoxic to articular chondrocytes in a dose- and time-dependent fashion in vitro. The purpose of this study was to determine whether a single intra-articular injection of bupivacaine induces chondrolysis in vivo. Methods and Materials Thirty-six Sprague-Dawley rats received a 100μl single-intrarticular-injection of saline and 0.5% bupivacaine or 0.6% MIA (positive control) per IACUC approved protocols. After 1, 4, 12, and 24 wks, 6 rats were euthanized and femurs were harvested. Cartilage health was assessed by chondrocyte viability staining, gross and india ink imaging, gross histology, modified Mankin score, and quantitative histological analysis for cell density. Data were analyzed by one-way ANOVA with Bonferroni post-test. Results Chondrocyte viability was similar to saline following bupivacaine injection (p⇢.05). MIA injections acutely reduced viability 78% compared to saline (p⇠0.01). Gross histological analysis showed no difference with bupivacaine injection vs. saline. However, at 24 weeks MIA treated knees had elevated total Mankin scores and clear cartilage degeneration compared with saline (p⇠.001). Histology post-bupivacaine injection demonstrated a 43–50% reduction in cell density (p⇠0.05) at 24 weeks, but an intact articular surface. In contrast, MIA injected knees exhibited marked cartilage loss with replacement by fibrous scar. Conclusions No chondrolysis was seen with bupivacaine injection in an in vivo rat model. A subtle reduction in overall chondrocyte density was observed six months post-bupivacaine suggesting possible chondrocyte injury. In contrast, MIA injection had acute chondrocyte necrosis followed by degeneration/chondrolysis. These results demonstrate minimal toxic effects of a single intra-articular injection of 0.5% bupivacaine on articular chondrocytes in vivo., Introduction Osteoarthritic change is one of the major complications associated with joint reconstruction using frozen osteoarticular grafts after tumor resection. We employed a frozen autologous whole rabbit knee joint graft model to investigate the influence of freezing on joint components. We then modified a vitrification method utilized for embryo cryopreservation and tested its ability to protect cartilage from cryoinjury. Methods and Materials (Study 1) 30 rabbit knee joints immersed in liquid nitrogen (F) or saline (C) were replanted. Histological and histochemical findings were assessed after 4, 8 or 12 weeks. (Study 2) Full-thickness articular cartilage harvested from rabbit femoral condyles was immersed in liquid nitrogen with and without vitrification. Histologic findings, ultrastructure and chondrocyte viability were examined before and 24hrs after culture. Results (Study 1) Both groups exhibited bone healing. In group F, despite restoration of cellularity to the menisci and ligaments, no live chondrocytes were observed, and cartilage deterioration progressed over time. (Study 2) Vitrified cartilage cell viability was ⇢85% that of fresh cartilage. Transmission electron microscopy demonstrated preservation of original chondrocyte structure. Conclusions Cryoinjury to chondrocytes of articular cartilage causes osteoarthritic changes following joint reconstruction with frozen osteoarticular grafts. To prevent osteoarthritic changes, articular cartilage needs to be protected from cryoinjury during freezing. Our vitrification method is effective for protecting chondrocytes from cryoinjury leading to cartilage deterioration. Reconstructing joints with osteoarticular grafts containing living cartilage may avert osteoarthritic changes. Vitrification is useful for long-term storage of living cartilage for allografts or, in Asian countries, for reconstruction with frozen autografts containing tumor., Introduction The aim of this study was to establish an in vitro quantitative magnetic resonance (MR) imaging technique of 3D tissue engineered cartilage with a combination of delayed gadolinium-enhanced MR imaging of cartilage (dGEMRIC), transverse relaxation time (T2) mapping, and fat-suppressed three-dimensional spoiled gradient-recalled (3D-SPGR) MR imaging. Methods and Materials Chondrocytes were isolated from knee cartilage of rabbits. Tissue engineered cartilage with different thickness of collagen scaffold and chondrocyte concentration were prepared, and quantitative MR imaging techniques was performed with a custom made micro-imaging coil, which could kept the samples almost similar conditions to that used for cultivation while the MR imaging. To investigate whether the T2 of tissue engineered cartilage had dependence of T2 on the angles relative to the static magnetic field (B0), which normally observed in native articular cartilage, T2 measurements with the samples oriented at different angles relative to B0 were performed. Histological and biochemical assessments of samples were then performed. Results The GAG concentration and histological findings of the tissue engineered cartilage correlated with the findings assessed by dGEMRIC. The water concentration of the tissue engineered cartilage correlated with the findings assessed by T2 mapping. On the other hand, variation of T2 due to the orientation-dependent dipolar interaction was not observed. The thickness of the tissue engineered cartilage evaluated with histology well correlated with the findings assessed with 3D-SPGR MR imaging. Conclusions A combination of dGEMRIC, T2 mapping and 3D-SPGR MR imaging can be a useful in vitro evaluation method for the quality and morphology of the tissue engineered cartilage., Introduction The purpose of this study was to evaluate the effect of splinting time on the retention of allogenic cartilage particle preparations implanted into full thickness cartilage defects. Methods and Materials Three full thickness critical sized cartilage defects were created in the trochlear sulcus and one in the medial femoral condyle. Subchondral bone was subjected to microfracture procedure. Post operative splinting was either 3 days, 7 days, or 14 days. One of four cartilage particle preparations (allogenic cartilage particles in either phosphate buffered saline (PBS), hyaluronan, hyaluronan and proprietary growth factor, or hyaluronan and insulin) was implanted. At 6 weeks, gross and histological analyses were performed to evaluate degenerative changes. Results Slightly increased degenerative articular cartilage changes corresponded to longer casting interval, regardless of treatment. Test article retention was highest in the distal trochlear sulcus site, followed by the MFC site, with the least retention in the middle trochlear site. Microfracture brought bone marrow and blood oozing into the allograft particle preparation filled defects, though no benefit was detectable. Normalized gross scores and histological evaluation demonstrated the best healing occurred with 7 day splint time. Conclusions Four defects per animal were a severe test. At this early 6 week time point, all subjects exhibited some degree of circumferential healing which varied depending on splinting time and treatment. The optimal splinting time was 7 days. The best treatment preparations were the allogenic cartilage particles in PBS and allogenic cartilage particles hyaluronan plus proprietary growth factor., Introduction Medical and industrial attempts for the treatment of articular cartilage pathologies have been increasing. But, they are still a major locomotor problem. Our aim was to determine the effects of synovium on cartilage proliferation as “in-vivo” culture medium and to anticipate a new, biological and cheap treatment method Methods and Materials In this study, 12 New Zealand male rabbits were used. Cartilage samples were taken from both knees: right (osteochondral, 4×4×6mm), left (chondral, 4×4×4mm). Two groups were formed: In group I (synovium group), the cartilage samples were placed into the synovium in the supracondylar groove, and in group II (intraarticular group), behind the patellar tendon. After 4 months, we sized and analyzed samples histologically with the camera lucida method to count the chondrocyte numbers. Mann-Whitney U-Test and regression analysis were used. Results For both groups, the chondrocyte numbers in the osteochondral samples were found to be higher than chondral samples (p⇠0.05) and the chondrocyte number increase was only found in the osteochondral samples, compared with preoperative samples (p⇠0.05). For the osteochondral samples, the chondrocyte numbers were found to be significantly increased more in the group I than in the group II (p⇠0.05). A logarithmic correlation was found between size and chondrocyte numbers (p⇠0.05). Conclusions This is the first study showing histologically the chondrocyte number increase with the camera lucida method. It also showed firstly that the synovium is an excellent chondrogenetic “in-vivo” culture medium. Arthroscopy-assisted implantation of “in-vivo” produced cartilage would probably be a new, cheap and biologically efficient treatment alternative for this clinical problem., Introduction The purpose of the present study was the evaluation of the effect of autologous chondrocyte transplantation placed on non-absorbable, polysulphonic and absorbable collagen mebrane in the treatment of leasion in hyaline cartilage in rabbits. Methods and Materials 80 White New Zealand rabbits, 4 month old, 2- 3.5 kg of weight were used in this research. Articular cartilage defects penetrating into the subchondral bone and superficial were created on the patellar groove of the femur. The defects were filled with chondrocytes placed on polysulfonic or collagen membrane. The expression of gene encoding procollagen type II that is a molecular marker of chondrocytes was evaluated. To estimate a weight of tissue grown on the polysulphonic membrane the burning analysis was performed. The reparative tissue was analyzed macroscopically and histologically after 12, 25 and 52 weeks since the performance of the operation. Results The RT - PCR analysis of the mRNA isolated from the cells detached from the bottom of the culture flask as well as polysulphonic membrane revealed the expression of type II procollagen. The burning analysis of polysulphonic membrane slices after two weeks of culture with chondrocytes revealed the concentration 0.23 mg of tissue on one part of membrane. Conclusions As far as macroscopic and microscopic evaluation is concerned the quality of regenerated tissue was similar after transplantation of cells placed on polysulphonic and collagen membranes. The regenerated tissue in full thickness as well as superficial defects reached maturity after 12 weeks and revealed morphology of hyaline - like cartilage even after 52 weeks., Introduction Meniscus loss leads to degenerative arthritis attributed to load distribution changes. Thus, there is a substantial need to protect the articular cartilage by either repairing or replacing the menisci. A novel Polycarbonate-Urethane (PCU) meniscal-implant was developed to comply with these requirements. Our goal was to elucidate whether a PCU-meniscal-implant is able to provide adequate protection to delay degenerative changes post-meniscectomy. Methods and Materials Seven ewes underwent full medial meniscectomy and were implanted with the PCU-meniscal-implant. Animals were euthanized at 3, 6 and 9 months. Range-of-motion (ROM), macroscopic and histological evaluations were performed post-implantation. The contralateral knee served as control. Results In general, periodic examination showed full ROM, no weight loss or signs of distress. The PCU-implant was durable and remained well-secured throughout 9-month trial. Gross and microscopic examinations of the implant's surfaces did not reveal any structural or material property changes. The PCU did not appear to exert any negative effect on cartilage and the degree of inflammation was minimal and primarily associated with the presence of foreign matter. The main pathological changes seen at 6 and 9 months post-implantation were similar to 3-months post-implantation observations. Cartilage in direct contact with the implant was preserved well and did not show significant degeneration. Conclusions To conclude, PCU did not exert any adverse effects and our results show that PCU-meniscal transplantation may protect, but not completely prevent degenerative changes of the medial articular cartilage. We believe that a PCU-meniscal-implant could counter the occurrence of major degenerative cartilage changes following meniscectomy., Introduction To determine the profile of Coll2–1, Coll2–1NO2 and myeloperoxydase (MPO) serum concentrations in experimental knee OA induced in the dog by transection of the anterior cruciate ligament. Methods and Materials Surgical transection of the ACL of the right knee was performed on 16 adult crossbred dogs. The dogs were sacrificed 8 weeks after the surgical procedure. Coll2–1, Coll2–1NO2 and MPO were measured by specific immunoassays in 16 dogs at baseline and every 2 weeks during the 8 weeks. The results were expressed as median (range). Results Immunostainings with D3 and D37, the antiserum recognizing Coll2–1 and Coll2–1NO2, respectively, labelled extracellular matrix in the superficial layer of fibrillated cartilage. After the transection of the ACL, the concentration of 3 biomarkers increased significantly (Friedman test: p⇠0.001). The concentrations of Coll2–1 and MPO were significantly increased at week 2 compared to baseline [Coll2–1 baseline: 281.57 (131.02–384.67) nM vs Coll2–1 week 2: 345.52 (181.15- 589.25) nM (p⇠0.01) and MPO baseline: 5.16 (⇠0.4–14.7) ng/ml vs MPO week 2: 14.54 (3.28–31.50) ng/ml (p⇠0.001)] and remained stable until week 8 [Coll2–1 week 8:318.89 (117.95–492.28) nM and MPO week 8: 11.55 (2.87–42.94) ng/ml]. The Coll2–1NO2 concentration increased significantly at weeks 6 and 8 compared to baseline [Coll2–1NO2 baseline: 0.54 (0.29–1.48) nM vs Coll2–1NO2 week 6: 0.64 (0.40- 1.9) nM (p⇠0.001) vs week 8: 0.61 (0.37–1.79) nM]. Conclusions These findings suggest that Coll2–1 is a relevant marker for the detection of early structural changes in OA dogs. Interestingly, MPO and Coll2–1NO2 are increased in OA dogs indicating that an oxidative stress occurs in this OA model., Introduction This study aims to set up and validate a protocol for 3D high resolution imaging of rabbit knee joints based on the equilibrium partitioning of a contrast agent with μCT. Accurate segmentation and 3D quantification require consistent scan and threshold parameters. Methods and Materials Five rabbit knee joints were obtained from unrelated studies, dissected and immersed in a 40% /60% solution of Hexabrix/PBS at room temperature, rinced in normal saline solution before scanning. EPIC-μCT scans were performed with a GE Locus μ-CT before immersion and at day 2, 4 and 6. Histology was then performed. Results 20μm isotropic resolution was achieved. Scanning parameters were 50kV, 450mA, 2000ms. The best contrast between subchondral bone and cartilage was found after 4 days of immersion in the contrast agent, showing a clear bimodal distribution of both cartilage and sub-chondral bone Hounsfield Units. An appropriate threshold was selected to perform the cartilage segmentation and 3D morphology modelisation using the GE Micro-View software. Quantification of the 3D local cartilage thickness was obtained and displayed with a color scale related to this parameter, and finally confirmed by histology. Conclusions In this preliminary study, we present the effectiveness of the EPIC-μCT for the quantitative 3D morphometric analysis of the rabbit knee joint. It is a promising complementary non invasive technique to monitor surface morphology, and obtain local and global 3D quantitative information of rabbit articular cartilage since accuracy and resolution are improved by 4 in the plane and by 20 in slice thickness with respect to common 7T μ-MRI acquisitions., Introduction The Göttingen Minipig is a well established large animal model for preclinical evaluation of novel articular cartilage repair procedures. Bone marrow derived mesenchymal stem cells hold great promise for future tissue engineering based articular cartilage repair strategies. The purpose of this study was to identify optimal cell culture conditions during MSC expansion in order to maximize cell proliferation and osteo- and chondrogenic cell differentiation. Methods and Materials Mesenchymal stem cells were isolated, cryopreserved and expanded from 3 adult Göttingen Minipigs. Cells were expanded until passage 2 using three different standard culture media with or without additional FGF-2 supplementation (10ng/ml): DMEM-LG+10%FCS, alpha-MEM+10%FCS and Verfaillie's Media (DMEM-HG, 2 % FCS, MCDB, Transferrin, Selenite, Dexamethason, A2P, Insulin, 10ng/ml EGF and PDGF). MSCs were evaluated for their potential to differentiate into chondro-, osteo- and adipognic cell lineages. Results During cell expansion each culture media displayed a distinct type of cellular morphology. With additional FGF-2 supplementation cell proliferation rate was significantly higher using alpha-MEM and DMEM-LG when compared to Verfaillie's media. Furthermore, FGF-2 did significantly stimulate chondrogenic and osteogenic differentiation in all three media with significantly more GAG and calcium deposition found in alpha-MEM expanded MSCs. Conclusions Our data suggests that using FGF-2 supplemented alpha-MEM media for MSC-expansion in the Göttingen Minipig model does maximize cell yield and boosts chondro- and osteogenic tissue formation., Introduction Frozen osteoarticular grafts treated with liquid nitrogen are often utilized for joint reconstruction after tumor resection, but the joint may develop osteoarthritic changes. Cryoinjury is one of the major complications of frozen osteoarticular grafts. In this study, we modified a vitrification method utilized for embryo cryopreservation in order to preserve articular cartilage from cryoinjury, and investigated the prognosis of the vitrified cartilage in rabbit model. Methods and Materials Osteochondral plugs (2.7mm diameter) were obtained from the medial condyle and patellar groove of the distal femur. These grafts were divided into 3 groups (control group, frozen group, and vitrified group), and were implanted as autografts. Histological findings, ultrastructure, and chondrocyte viability were examined 6 and 12 weeks after implantation. Results Histological findings and chondrocyte viability of the vitrified group showed no significant difference from those of the control group. Viable cells were absent in frozen group. Transmission electron microscopy demonstrated preservation of the original chondrocyte structure in the vitrified group. Conclusions Cryoinjury of chondrocytes from articular cartilage causes osteoarthritic changes following joint reconstruction with frozen osteoarticular grafts. To prevent osteoarthritic changes, the articular cartilage must be protected from cryoinjury during freezing. Our vitrification method was effective in protecting chondrocytes from cryoinjury leading to cartilage deterioration. Reconstructing joints with osteoarticular grafts containing living cartilage may help to avert osteoarthritic changes. Our vitreous preservation method is useful for long-term storage of living cartilage for allografts or, as in Asian countries, for reconstruction with frozen autografts containing tumor. In addition, the preserved grafts can maintain cartilaginous tissue., Introduction PVA-PEG theta hydrogel is a candidate material for synthetic articular cartilage. The strength of PVA-PEG theta hydrogel can be increased by increasing its crystallinity by dehydration followed by annealing to meet the requirements of the targeted application. The crystallization kinetics of PVA-PEG thetagels during high temperature annealing is not well understood; especially the changes that take place during the steps of dehydration, annealing, and rehydration. In this study we used wide angle x-ray diffraction (WAXD) to investigate these changes. Methods and Materials PVA-polyethylene glycol (PEG) hydrogels were prepared by theta gel method. One group was used in their ‘as-gelled’ form (AG). Second group was studied in ‘dePEGed’ form (DP) after removing the PEG in DI water. The crystallinity of the gels were measured during (i) gelation (ii) DePEGing, (iii) dehydration under vacuum at 25°C (iv) high temperature annealing under Ar at 160°C (v) rehydration subsequent to equilibrium dehydration and annealing using a PANalytical X'Celerator powder diffractometer. Results DePEGing process decreased the crystallinity. However, crystallinity of DP gel was increased markedly after dehydration and annealing. After rehydration, crystalline peaks of the dried DP disappeared and DP annealed gel did not show any difference in the WAXD. AG gels exhibited strong diffraction peaks after dehydration and annealing. AG annealed gel showed higher crystallinity than D P. Crystallinity of AG annealed gel decreased after rehydration. Conclusions Annealing and dehydration increased crystallinity of the PVA-PEG theta gels. Annealing resulted in a more stable crystalline network and mechanically stronger gels., Introduction This study was undertaken to evaluate muscle and cartilage damage after different running distances by marathon and ultra-marathon runners. Methods and Materials Ten male marathon and ten male ultra-marathon runners participated in the study. Serum cartilage oligomeric matrix protein (COMP) was measured as a marker of cartilage metabolism and/or damage. Plasma CPK and hs-CRP activities were measured as markers of muscle damage and inflammation. Results Serum COMP was increased 1.6-fold at 10km during a marathon race. In contrast serum COMP was increase 1.9-fold after a 200km race and this level was maintained until day 3 of recovery, only returning to the pre-race level on day 6. Plasma CPK was increased at 10km of the marathon race and up to 3-fold at the end of the race. This was further increased after 1 day of recovery, only returning to pre-race level on day 6. Plasma CPK was increased 35-fold at the end of the 200km race and remained significantly increased until day 5 of recovery. There was no change in plasma hs-CRP during the marathon race, but this was increased 3.4-fold by day 1 of recovery, returning to the pre-race level on day 4. Plasma hs-CRP increased 40-fold by the end of the 200km race and was still increased on day 6 of recovery. Conclusions Long distance running may induce more impact-stress on muscle than on cartilage at a given running distance. Further, the required time for recovery may vary with running distance and the tissue type, e.g. cartilage or skeletal muscle as in this case., Introduction Polyvinyl alcohol) (PVA) has been advanced as a biomaterial to be used as synthetic articular cartilage. Key material requirements for such devices are high creep resistance and high water content to maintain a lubricious surface to minimize damage of the cartilage counterface during articulation. The creep resistance of PVA hydrogels can be increased by high temperature annealing; however this process collapses the pores, reducing the water content and lubricity of the hydrogel surface. In this study we added hydrophilic poly(acrylamide) (PAAm) to prevent the pore collapse by filling the pores during annealing resulting in a highly lubricous hydrogel while still maintaining a high creep resistance. Methods and Materials PVA-AAm interpenetrating networks hydrogels were prepared by the combination of freeze-thaw and thermal polymerization. PVA-polyethylene glycol (PEG) hydrogels were prepared by theta gel method. Coefficient of friction (COF) of the hydrogels was determined using a rheometer. The equilibrium water content (EWC) of the gels determined by using Thermogravimetric Analyzer. The total creep strain was determined on a custom made mechanical tester. Results The equilibrium water content of the PVA-AAm gels showed an increase with increasing AAm content both before and after annealing. The creep resistance of the PVA-AAm gels was similar to PVA-PEG gels. The COF of the PVA-AAm gels was lower than that of the PVA-PEG gels. Conclusions Incorporation of AAm component in to PVA networks substantially improved the lubricity of the resulting gels while maintaining high creep resistance. PVA-AAm hydrogels are promising candidates for cartilage resurfacing., Introduction Fabricating the biphasic scaffold to benefit both bone and cartilage regeneration is potentially an optimal technique for osteochondral repair. This study describes our continuing effort to optimize the osteochondral lesion repair from adipose derived stem cells toward engineering articular cartilage repair in vivo. Methods and Materials We developed the integrated biphasic scaffold, of which the upper part is an extracellular matrix-derived of cartilage by combining a decellularized procedure with ice crystal orientation and freeze-drying technique and the lower part is true bone ceramic. The upper layer the scaffold was loaded with chondrogenically induced adipose-derived stem cells (ADSCs). MTT assay showed no cytotoxicity of the biphasic scaffolds. The structure of biphasic scaffolds were characterized by scanning electron microscopy and micro-computed tomography. Osteochondral defects (4.2 mm diameter ×6 mm deep) were created in the patellar groove of rabbit femurs, and the defects were filled with constructs of a biphasic scaffold with chondrogenically induced ADSCs (Group A, 2×107/ml, n=12) or cell-free scaffolds (Group B, n=12) or nothing (Group C, n=12). Results All of the defects of Group A showed good osteochondral repair, and macroscopic and histologic scores were superior to those of Group B and Group C. The subchondral bone showed mature trabecular bone regularly formed in Group A and Groups B at both 6 and 12 weeks. But the subchondral bone formation was not good in Group C. Conclusions The novel biphasic scaffold loaded with chondrogenically induced ADSCs successfully filled the osteochondral defects of rabbits with hyaline cartilage that is similar to normal cartilage biomechanically and biochemically., Introduction Polyvinyl alcohol) (PVA) is a candidate for synthetic articular cartilage to treat osteochondral defects. However PVA gels are not strong enough to serve this purpose. Annealing has been proven to increase the strength of PVA hydrogels; however it reduces the water content and consequently reduces the lubricity of the hydrogel. Keeping the articulating surface of the hydrogel lubricious is a key requirement to prevent damage against opposing cartilage. In this study we have investigated the effect of acrylamide and 2-acrylamido-2-methyl-1-propanesulfonic acid (AAMPS) on the lubricity of the PVA hydrogels by measuring coefficient of friction (COF) of these gels. We also investigated effect of lubricant on COF while testing these hydrogels in deionized water, saline, and bovine serum based storage media. Methods and Materials PVA-AAM PS, PVA-AAm IPNs and PVA hydrogels were prepared by the combination of freeze-thaw and thermal polymerization. PVA-polyethylene glycol (PEG) hydrogels were prepared by theta gel method. COF of the hydrogels was determined at 40°C using a rheometer. Results AAm and AAMPS addition increased EWC of PVA gels. PVA only gels showed the lowest EWC after annealing. Annealing increased the COF values in all types of gels. PVA-AAm and PVA-AAM PS hydrogels showed lower COF values than PVA only and PVA-PEG hydrogels. COF values measured in saline were lower than other media. Conclusions The addition of AAm and AAMPS in to PVA decreased the friction of the PVA hydrogels significantly. The lubricant in which the gels were tested has a strong influence on the COF., Introduction The purpose of the present study was to explore the surgical possibilities for replacement of the medial tibial plateau by a metal implant and to examine the implications for the opposing cartilage. Methods and Materials In 6 goats, the medial tibial plateau of one knee was replaced by a cobalt-chromium (CoCr) implant, using PMMA bone cement for fixation. The anterior part of the medial meniscus was dissected, without damaging the knee ligaments, and the femoral cartilage. The un-operated knee served as a control. After 26 weeks the animals were killed. Joints were evaluated macroscopically. Cartilage quality was analysed macroscopically, and microscopically. GAG content, synthesis, and release were measured in tissue and medium. Results Knees were loaded without limitations by all animals. Macroscopic articular evaluation scores showed worsening 26 weeks after inserting the implant (p⇠0.05). Macroscopic and microscopic scores showed more cartilage degeneration of the opposing medial femoral condyle in the experimental knee compared to the control knee (p⇠0.05). Higher GAG synthesis was measured in the experimental knees (medial femoral condyle) (p⇠0.05). GAG content and release were not different (p⇢.05). Conclusions Replacing the medial tibia plateau by a custom designed metal implant is surgically feasible, however this induces considerable damage of the opposing medial femoral cartilage. Given the results obtained, the current model is a viable tool in the evaluation of bearing materials for implants. However, the introduction of CoCr tibial implants in a human clinical setting for the treatment of post-meniscectomy cartilage degeneration of the medial tibial plateau does not seem a realistic option currently., Introduction The purpose was to investigate the feasibility of the application of defect-size femoral implants for treating localized cartilage defects and compare this treatment in terms of joint degeneration with microfracturing in a model of established cartilage defects. Introduction The purpose was to investigate the feasibility of the application of defect-size femoral implants for treating localized cartilage defects and compare this treatment in terms of joint degeneration with microfracturing in a model of established cartilage defects. Methods and Materials In 9 goats, a medial femoral condyle defect was created in both knees. After 10 weeks, the knees were treated by microfracturing or by placing an oxidized zirconium implant, and 26 weeks after surgery animals were killed. Joints were evaluated macroscopically. Implant osseointegration was measured by automated histomorphometry. Cartilage repair (after microfracturing) was scored microscopically. Cartilage quality was analysed macroscopically, and microscopically. GAG content, synthesis and release were measured in tissue and medium. Methods and Materials In 9 goats, a medial femoral condyle defect was created in both knees. After 10 weeks, the knees were treated by microfracturing or by placing an oxidized zirconium implant, and 26 weeks after surgery animals were killed. Joints were evaluated macroscopically. Implant osseointegration was measured by automated histomorphometry. Cartilage repair (after microfracturing) was scored microscopically. Cartilage quality was analysed macroscopically, and microscopically. GAG content, synthesis and release were measured in tissue and medium. Results Bone-implant contact was 14.6±5.4%. Healing of the microfractured defects was 18.4±0.4 (of 24). Macroscopically no differences were seen. Microscopically, more degeneration (medial tibial plateau) after microfracturing was seen as compared to joints with implants (p⇠0.05). Similarly, a higher GAG content, higher synthetic activity and decreased GAG release of the medial tibial plateau cartilage of implant-treated knees was measured (p⇠0.05). Microscopic degeneration was also found in cartilage of the lateral tibia plateau and condyle, but not different between treatments (p⇢0.05). Results Bone-implant contact was 14.6±5.4%; the amount of bone surrounding the implant was 40.3±4.0%. Healing of the microfractured defects was 18.38±0.43 out of 24. Macroscopically no differences were seen. Microscopically, more degeneration (medial tibial plateau) after microfracturing was seen as compared to joints with implants (p Conclusions Both microfracturing and using implants as a treatment for established localized cartilage defects in the medial femoral condyle caused degeneration in directly articulating cartilage as well as in more remote sites in the knee. Although this study shows that small metal implants may be more suitable than microfracturing, the generalised degeneration found for both treatments should be addressed first. Conclusions Both microfracturing and the use of implants as a treatment for established localized cartilage defects in the medial femoral condyle caused degeneration in directly articulating cartilage as well as in more remote sites in the knee. However, metal implants caused less damage than microfracturing. Although this study shows that small metal implants may be more suitable than microfracturing, the generalised degeneration found for both treatments should be addressed first, Introduction Hemiarthroplasy has been used widely in various joints since the 1950's. Histological studies on dogs confirmed that implants made of rigid materials cause progressive cartilage wear, while the histological study of a Polycarbonate-Urethane (PCU) implant against cartilage in sheep showed very little cartilage degeneration. We believe that PCU performance could be improved by a lubricious-polystyrene-brush-coating. Our goal was to evaluate whether this coating could be beneficial in reducing wear in PCU hemiarthroplasty. Methods and Materials Acetabular components of porcine hip joints (N=3) were placed in a rotating cell. The femur was fixed to the load axis and the artificial PCU buffer was tested by replacing the natural acetabular component (N=6). Simulations of 70,000 load-cycles were conducted on natural joints and hemiarthroplasties of the various PCU buffers. Using India-ink staining, scratches were measured and the total injured area in each specimen was calculated. Last, the difference in injured area between groups was evaluated and the moment developed in the fulcrum axis was measured. Results We found that cartilage-on-polystyrene-coated-PCU and cartilage-on-cartilage groups were significantly indistinguishable (p⩾0.05). In addition, microscopical examination of an intact femoral cartilage after 70,000 cycles showed negligible scratching. Interestingly, the moment measured for the coated-PCU was found to be ∼1.6-times lower, and less affected by lubricant depletion versus the non-coated-PCU. Conclusions To conclude, we evaluated the amount of cartilage damage associated with articulation of cartilage against polystyrene-brush-coated-PCU and showed that PCU performance can be improved further by an additional coating. Coated-PCU demonstrates exceptional wear results, comparable to those attained for articulation of cartilage-on-cartilage., Introduction Joint surface incongruities in the ankle can have a great response on stress gradients and contact area (CA). However, the response of small lesions seems limited. This study aims to determine the changes in contact area characteristics after a limited cartilage biopsy at the postero-medial rim of the talar dome. Methods and Materials Ten specimens were tested before and after harvest of a biopsy of 5 × 11 mm; using pressure sensitive flm; loading in neutral (N), plantar flexion (PF) and dorsiflexion (DF) while applying (sub-) physiological loads. The stains were further analyzed in the ImageJ environment. The surface area of the talar dome, the normalized tibio-talar CA (% of talar dome area), and the centroïd position of the CA were determined. Significant changes were checked using 95% Confidence Intervals. Results The mean changes in normalized CA were non significant. However, when considering the single specimens a significant change was observed 4 times in N and 3 times in PF. For the centroïd position, again, none of the mean changes was significant, although some single specimens showed a significant change especially for the antero-posterior direction in PF. Conclusions In general, harvesting a limited cartilage biopsy at the postero-medial rim of the talar dome does not result in a significant change in contact area characteristics. However, due to a great individual variability some specimens show significant changes. Future studies will have to determine the clinical importance of these changes and the long-term safety of the proposed biopsy site., Introduction Cartilage defect repair (CDR) is an attractive treatment option for osteochondral defects. In the design of such therapies, it is important to understand the very complex tribological regime of the natural joint, as well as the tribology of the CDR. Methods and Materials The friction and wear of CDR were investigated using our previously described tribological simulation of the medial compartmental knee [McCann, L.(2008)Tribology International(41),p:1126]. CDR using a cartilage-bone plug was compared to a metal pin, a blank defect in the femoral condyle, and the natural medial compartment. Friction was determined over 3600 cycles. Peak contact stresses were measured using prescale Fuji flm. A Talysurf Proflometer measured surface roughness. High resolution micro-MRI scans were taken to quantify wear. Results The frictional coefficient (u) was significantly lower (P⇠0.05) for the control bearing (u=0.07), than for the three defect bearings (u≥0.11). Overall, we found elevated frictional coefficient, friction shear stress and surface damage, for the defect bearings in comparison to the intact medial compartment. In the presence of a defect the biphasic nature of the joint was disrupted. The rehydration, which takes place during unloading in a natural joint could not take place, increasing the direct solid-to-solid contact which is known to cause an increase in frictional coefficient. Conclusions This investigation is the first tribological simulation of the knee examining CDR. It supports the use of biphasic materials for CDR, to minimize the solid-solid contact and rise in friction and shear stress. The experimental model has the potential to examine any potential CDR device - synthetic or biological., Introduction The biopsy site best used for an ACI at the talar dome remains an issue of debate. This study aims to determine the changes occurring in the pressure profile across the postero-medial rim of the talar dome after a biopsy has been harvested locally. Methods and Materials Ten specimens were (sub-) physiologically loaded pre- and post-biopsy; in neutral position, 10° of plantar-flexion and 10° of dorsi-flexion. Quantitatively, the pressure profile plots of each pre- and post-biopsy situation were analyzed. Qualitatively, the coverage of the biopsy by the tibial plafond, and changes in the shape of the contact area were analyzed. Finally, all parameters per specimen were compared to cross-check for consistency of the results. Results A lower pressure across the biopsy site after harvest was noted twice in N and 5-times in PF. No peak pressures near to the biopsy site developed. The biopsy site resulted (partially) covered by the tibial plafond 3-times in N and 8-times in PF. A change in the shape of the contact area was seen 7-times in PF. After comparing all results, a limited effect of the biopsy on the pressure profile was seen 6-times in PF and 2-times in N. Conclusions Only a part of the specimens showed a change in the local pressure profile after harvesting the biopsy. Nearly all changes were in PF. In this position the joint is loaded only a short time and in a limited way during the stance phase. Therefore, the investigated biopsy site seems safe to be used in clinical practice., Introduction Treatment of cartilage defects follows recommendations depending on defect-size. Exact sizing of defects is hard to achieve in daily practice. The critical size of a defect leading to pain, functional deficits and osteoarthritis is still unknown and depends on thickness/ stability of the surrounding cartilage, joint containment, loading forces and other factors. Methods and Materials The purpose of this study is to identify additional factors influencing the outcome of cartilage repair after a certain treatment by finite-element simulation with the following standards were used: a circular defect (diameter 5 and 10 mm)cartilage-thickness: 5mmCartilage Elasticity-Modulus: 18.8 MPaAxial-loading F=5000N (gait/running) Deformation and contact pressure were simulated for intact cartilage, cartilage defects with a diameter of 0.5 cm/1.0 cm and a defect filled with repair tissue with a reduced elasticity modulus of 14.8 MPa. Results Max.deformation (mm) and max.contact pressure (kg/mm sec2) was calculated for intact cartilage with 3.074 × 10−1 and 1.592 × 103,cartilage defect ø 5 mm with 4.504 × 10−1 and 1.831 × 103,cartilage defect ø 10 mm with 5.523 × 10−1 and 2.259 × 103,filled cartilage defect with 3.894 × 10−1 and 1.057 × 103. Edge-loading increases with increasing defect-size and decreases even with a defect-fill of inferior quality. Conclusions The model of finite-elements enables the simulation of various factors possibly affecting the outcome of cartilage repair (defect-size, cartilage thickness, elasticity, shape of joint-partners, loading/shear forces and others). Simulation-based individual treatment options after MRI-analyis could enhance the quality of medical recommendations., Introduction The purpose was to examine whether A-mode ultrasound signal intensity (SI, a measure of superficial cartilage integrity) detect fragility of superficial layer of reparative articular cartilage-like tissue in a full-thickness cartilage defect of rabbits (D-model). Methods and Materials A defect of 5-mm in diameter was created in the left trochlea groove in the D-model (n=18). A full-thickness osteochondral plug of 6-mm in diameter was removed from the right trochlea groove and grafted into the defect in an autologous osteochondral grafting model (OCG-model, n=36). Reparative tissue of the D-model and articular cartilage of the plug of the OCG-model was assessed by histology and an ultrasound system (center frequency 10 MHz) at 2-, 4-, 8-, 12-, 24- and 52-week postoperatively and was compared to normal control cartilage. Results In the D-model, the defect was repaired with fibrocartilage. SIs of control, 2-, 4-, 8-, 12-, 24- and 52-week were 3.29 (arbitrary unit), 0.75, 0.35, 0.65, 0.97, 0.96 and 0.23, respectively. SIs of 2-through 52-week were significantly lower than SI of control (P ⇠ .001). Cartilage of the plugs of the OCG-model retained features of hyaline cartilage throughout 52 weeks. SIs of control, 2-, 4-, 8-, 12-, 24- and 52-week were 3.18, 2.17, 2.04, 1.23, 1.01, 1.95 and 3.94, respectively. SIs of 8- and 12-week were significantly lower than SI of control (P ⇠ .001). There was no significant difference in SI between control and 2-, 4-, 24- or 52-week. Conclusions Low SI during 24 weeks might predict fragility of superficial layer of reparative cartilage-like tissue., Introduction There is a resurgence of interest in bone grafting procedures for patients with shoulder instability. We sought to investigate the alterations in glenohumeral contact forces to determine changes in pressure with proud, flush, and recessed Latarjet or ICBG augmentation, and to determine the optimal orientation of the coracoid bone graft. Methods and Materials Twelve fresh-frozen cadaveric shoulders were tested in abduction and ER with a compressive load of 440N. Glenohumeral contact area, mean pressure, and peak pressure were determined with a Tekscan sensor for: 1) intact glenoid, 2)30% defect, 3) 30% glenoid defect treated with Latarjet bone block placed 2mm proud, flush, and 2mm recessed to the glenoid, 4) 30% glenoid defect with ICBG placed 2mm proud, flush, and 2mm recessed to the glenoid, and 5) Latarjet bone block placed flush but oriented with either the lateral (Latarjet-LAT) or inferior (Latarjet-INF) surface of the coracoid as the glenoid face. Results Bone grafts in the flush position restored mean contact pressure to 85% (ICBG, p⇠0.04), 80% (Latarjet-INF, p=0.03), and 65% (Latarjet - LAT, p=0.02) of normal. Latarjet-LAT demonstrated statistically higher peak pressures than the ICBG and Latarjet-INF at all positions (p⇠0.02). With bone grafts in a proud position, contact pressure increased an additional 40% (p⇠0.01) in the anteroinferior quadrant with a concomitant 100% (p⇠0.01) increase in the posteroinferior glenoid pressure indicating a shift posteriorly. Bone grafts placed in a recessed position had high edge-loading. Conclusions The optimal restoration of glenohumeral forces was with the ICBG and the Latarjet-INF when the inferior aspect of the coracoid becomes the glenoid surface. Bone grafts placed in a proud position not only increased the peak pressure anteroinferiorly, but also shifted the articular contact forces to the posteroinferior quadrant. The optimal restoration of glenoid joint surface favors an optimally placed ICBG and inferior surface of the coracoid for glenoid bone augmentation., Introduction The tibio-talar contact area has been widely investigated to monitor biomechanical changes due to articular incongruities or an altered loading. This study aims to investigate for the first time in a systematic way the extent of the inter-specimen variability of the tibio-talar contact area, and its repercussions when analyzing data concerning this parameter. Methods and Materials Ten specimens were loaded to record the tibio-talar contact characteristics by use of pressure sensitive film. The size of the talar dome area, the size of the (normalized) tibio-talar contact area, the position of the tibio-talar contact area, and the shape of the latter were determined and analyzed. Inter-specimen variability was expressed as the Coefficient of Variation and was calculated for the datasets of previous studies as well. Results The size of the tibio-talar contact area showed a very high inter-specimen variability, in agreement with the range found in previous studies. This high variability persisted when a normalized tibio-talar contact area was calculated. The shape of the tibio-talar contact area showed some basic characteristics, but a high variation in details could be observed. Conclusions Every specimen can be considered to have its own “ankle print”. By this variability, articular incongruities are expected to have a different effect on local biomechanical characteristics in every single individual. Therefore, every single case has to be evaluated and reported for significant changes. In case of modeling, this also underscores the need to use subject specific models fed by sets of parameters derived from a series of single specimens., Introduction Microfracture is a marrow stimulation technique thought to produce less bone necrosis than drilling. Our study tested this hypothesis and investigated acute osteochondral characteristics, repair responses and effect of hole depth in adult rabbits. Introduction Microfracture is a marrow stimulation technique that is speculated to produce less bone necrosis than drilling. Our study tested this hypothesis and investigated acute osteochondral characteristics, repair responses and the effect of hole depth in a an adult rabbit model. Methods and Materials Trochlear cartilage defects were prepared and 0.9 mm diameter cylindrical drill holes were made either 2 or 6 mm deep (under cooled irrigation), or conically shaped microfracture holes were made 1mm diameter and 2mm deep. Sacrifices were at 1, 14 and 21 days, with assessments by micro-CT and histology. Methods and Materials Cartilage defects were prepared in rabbit trochleas (N=14) and further treated by marrow stimulation with 0.9 mm diameter cylindrical drill holes (2 or 6 mm deep, under cooled irrigation), or with conically-shaped microfracture holes (1 mm diameter and 2 mm deep). Rabbits were sacrificed at 1, 14 and 21 days, and defects assessed by micro-CT and histology. Results Microfracture induced compaction of surrounding bone, essentially sealing holes off from bone marrow; whereas drilling removed bone and debris from holes to provide access to marrow stroma. Microfracture induced substantial bone necrosis indicated by more empty osteocyte lacunae in adjacent bone around microfracture than drill holes, which were done under cooled irrigation. In our rabbit model, drilling deeper to 6 mm penetrated the epiphyseal scar and led to greater subchondral hematoma with increased access to marrow stroma. At 14 days post-operation, advanced new bone formation and remodelling were seen in drilled defects, in contrast to prolonged resorption of fractured bone around microfracture holes which impeded repair. New bone synthesis and chondrogenesis were observed in both deep and shallow drill holes on Day 21 post-operatively. Results Compaction of bone around microfracture holes essentially blocked connection with bone marrow. Drilling, however, removed bone and debris from holes to provide channels to marrow. Significantly more empty osteocyte lacunae (bone necrosis) were detected in bone lining the microfracture holes, compared to the drill holes generated under cooled irrigation. Deep drilling to 6 mm penetrated the epiphyseal scar in rabbits and led to greater subchondral hematoma and increased access to marrow stroma. At 14 days post-surgery, advanced new bone formation and remodelling were seen in drilled defects, in contrast to prolonged resorption of fractured bone around the microfracture holes, which impeded repair. New bone synthesis and chondrogenesis were observed in both deep and shallow drill holes at 21 days post-surgery. Conclusions Acute subchondral fractures and repair responses were distinct comparing microfractured and drilled defects. Surgical technique and hole depth may affect the patterns and connectivity of subchondral bone marrow channels, and influence long term cartilage repair properties. Conclusions Acute subchondral fractures and repair responses were distinct between microfractured and drilled defects. Surgical technique and hole depth may affect the patterns and connectivity of subchondral bone marrow channels, thus influencing cartilage repair processes and long-term outcomes., Introduction The understanding of cartilage development and its relation to chondrocyte function had been attained principally by biochemical and electronmicroscopic methods. Cartilage has been often viewed as a uniform structure, with ECM divided into pericellular, territorial and interterritorial zones, described by Benninghof in 1925, who accorded a central role to pericellular ECM investing one or a cluster of chondrocytes and forming units termed chondrons. In this scheme chondrocytes were viewed as static entities. In this investigation chondrocytes in cell culture and chondrocytes in hyaline articular and tracheal cartilage were studied to detect cell to cell communications and demonstrating their uniqueness. Methods and Materials Cell cultures were initiated from adult human cartilage using explant techniques. Cell cultures were viewed continuously in an incubated inverted microscope with time lapse photographs obtained from 5 to 15 minutes, utilizing Hoffman modulation contrast. Thin sections of adult human articular cartilage and those of cartilage from rat tracheas were studied utilizing structural coloration as previously described (Histochem Cell Biol 109:1, 1999) and high resolution transmitted light microscopy. Results Time lapse photography demonstrated intermittent contacts between chondrocytes by means of intercellular bridges of various configurations. Transmitted light microscopic studies of cartilage sections showed the presence of intrachondral tubules and intercellular bridges. Conclusions Chondrocytes in vivo and in-vitro demonstrated cell to cell communications by well recognized anatomic structures, suggesting chondrocytes are active dynamic cells, and not static structures., Introduction Honokiol is a small molecular weight ligand originally isolated from the Chinese medicinal herb Magnolia officinalis, a plant used in traditional Chinese and Japanese medicine. A previous study, the effect of honokiol was known as anti-angiogenic, anti-invasive and anti-proliferative activities in a variety of cancer. Methods and Materials MTT assay Western blot analysis Alcian Blue staining Imunohistochemistry Results we investigated whether honokiol inhibited NO-induced apoptosis in articular chondrocyes using by phase-contrast microscope, MTT assay and Western blot analysis. A previous study, SNP induced activation of p53 via activation of p38 kinase in articular chondrocytes. Treatment of honokiol inhibited SNP-induced activation of p38 kinase in articular chondrocytes. Inhibition of p38 kinase with SB203580 rescued SNP-induced apoptosis. Honokiol reduced p53 expression in dose-dependent manner. Also, treatment of honokiol was suppressed NO- induced dedifferentiation in articular chondrocytes as determined by the accumulation glycosaminoglycan by Alcian Blue staining, immunohistochemistry and Western blot analysis. Conclusions Our results collectively suggest that honokiol inhibited NO-induced apoptosis and dedifferentiation of rabbit articular chondrocytes via different mechamixms., Introduction Microtubule-interfering agents (MIAs), paclitaxel have been attributed in part to interference with microtubule assembly, impairment of mitosis, and changes in cytoskeleton. But the signaling mechanisms that link microtubule disarray to destructive or protective cellular responses are poorly understood. In this study, we investigated the effects of paclitaxel determined that is the mechanism of paclitaxel-induced differentiation and COX-2 expression in rabbit articular chondrocytes. Methods and Materials Chondrocytes were treated with various concentration of paclitaxel. And cells were determination of chondrocyte pheotype by western blotting, Immunofluorescence Microscopy, and detected cell cycle by flow cytometry of propidium iodide stained cells. Results Microtubule damages inhibit dedifferentiation, and increase COX-2 expression and Prostaglandin E2 production in chondrocytes by paclitaxel. The involvement of three major MAPK family members (ERK, p38 kinase and JNK), PKC and PI3/AKT kinase in the paclitaxel signaling to promote differentiation and COX-2 expression in chondrocytes. Activation of ERK-1/2 is suppressed the differentiation, whereas activation of p38 kinase signaling upregulated the differentiation. Interestingly, activation of ERK-1/2 and p38 kinase increased COX-2 expression by paclitaxel treatment in chondrocyte. In addition, paclitaxel suppressed is dedifferentiaion is necessary for PI3/Akt kinase activation, and COX-2 expression required for blocked the JNK signaling. Conclusions Therefore, these results collectively indicated that ERK-1/2, p38 kinase oppositely regulate paclitaxel-induce differentiaion but not COX-2 expression in chondrocytes., Introduction Leflunomide is an oral immuno-modulatory agent which is considered effective disease modifying anti-rheumatic drug(DMARD) in RA. Leflunomide known as a regulator of iNOS synthesis which largely increases NO production in diverse cell type. However, the effect of leflunomide on chondrocyte is still poorly understood. In our previous studies, we have shown that direct production of NO by treating chondrocytes with NO donor, sodium nitroprusside (SNP), causes apoptosis, dedifferentiation and COX-2 expression via p38 mitogen-activated protein kinase in association with elevation of p53 protein level, caspase-3 activation and extracellular signal-regulated kinases pathway. In this study, we characterized the molecular mechanism by which A77 1726 supress apoptosis, dedifferentiation and COX-2 expression through activation of Phosphoinositide 3-kinase signaling pathway. Methods and Materials We used that detection of NO-induced apoptosis by MTT assay and DNA fragmentation and FACS. Also, detection of inhibited dedifferentiation and COX-2 expression by A77 1726 were determined by Western blot and Alcian blue stain, Reverse transcription - polymerase chain reaction. Results We found that A77 1726 inhibited NO-induced apoptosis. Also, A77 1726 inhibited dedifferentiation and COX-2 expression. Inhibition of PI3K signaling pathway by LY294002, PI3K specific inhibitor, these effects of A77 1726 were completely inhibited. Thus, these data indicate that A77 1726 supress NO-induced apoptosis, dedifferentiation and COX-2 expression via PI3K signaling pathway. Conclusions Finally, our results suggest that A77 1726, active metabolite Leflunomide is benefit to prevent degradation of cartilage caused by OA or RA., Introduction 2-deoxy-D-glucose is a non-metabolizable glucose analogue in which the hydroxyl group at the second position carbon is replaced by a hydrogen. It is known as inhibitor the synthesis of glycoproteins by interference with the initial glycosylation steps at the ribosomal level. Glucose is generally believed to be an various crucial role of the numerous cell including the essential precursor in glycosaminoglycan biosynthesis. The purpose of this study was to characterize the effects of 2DG on dedifferentiation and expression of cyclooxygenase-2 (COX-2) in rabbit articular chondrocytes, focusing on the roles of ER stress. Methods and Materials 2DG reduced type â…¡ collagen and COX-2 in time- and dose- dependent manner as indicated by western blotting, prostaglandin E2 production, alcian blue staining and immunofluorescence. Results 2DG induced growth arrest and DNA damage induced-153 (GADD153) expression. in rabbit articular chondrocytes. Also, 2DG treatment inhibited activation of glucogen synthase kinase 3β (GSK3β) in dose and time dependent manner, accompanied by GADD153 expression. Inhibition of GSK3βwith Na3VO4 recovered 2DG induced N-glycosylation inhibited COX-2 but not effect on dedifferentiation. inactivation of GSK3β induced accumulation of β-catenin. Accumulation of β-catenin by the inhibition of GSK3beta with LICl recovered 2DG induced N-glycosylation inhibited COX-2. whereas addition of glucose recovered type â…¡ collagen and reduced GADD153 expression, but not N-glycosylation inhibited COX-2. Conclusions Our results indicate that 2DG-induced dedifferentiation and inhibition of N-glycosylation COX-2 by modulating ER stress pathway in rabbit articular chondrocytes., Introduction Time-lapse cinematography allows for the exploration of the dynamic features of cell populations in vitro. In the present study this technique was utilized to observe chondrocytes in the primary and low passage cultures. The aim of the study was to document unique features of articular cartilage chondrocytes propagated in vitro. Methods and Materials Cell cultures were initiated by explants of cartilage from cadaver donors. Cells were propagated in tissue cultures medium CMRL 1066 with 10% FCS. Only primary or low passage cultures were used. Chondrocyte cultures were also stained with fluorescent and non fluorescent vital stains. Fluorescent stains were acridine orange, and carboxy fluorescein ester. Non-fluorescent-neutral red, Janus green B and Nile blue sulfate. Cell culture, were viewed in a Nikon inverted microscope with Hoffman modulation contrast optics. Photographs were obtained from every 5 to 15 minutes for 24 to 96 hr period. Images were recorded using Windows version 4.1 and Insight Spot Advances programs. Results Chondrocyte cultures contained large cells with numerous projections. Single nucleated cells with bitemporal filopodia were migrating through the observation fields. When filopodial projections came into contact with chondrocytes the projections and their mother cells become united with the cells with which they established contacts. Cytonemeta were also formed after completion of cytokinesis by dividing cells. Vital stains showed normal patterns of fluorescent and vital stain uptake seen with viable cells. Conclusions Chondrocytes in culture establish cell to cell contacts by filopodia, cytonemeta and other types of intercellular bridges, and have unique morphologic characteristics., Introduction Evaluate the feasibility and efficacy of using autologous synovium tissues in chondral defect repair. Methods and Materials Synovium tissue fragments were harvested from the caprine knee joint using a custom made device. Cell viability in the fragments was assessed using the Live/Dead assay. Chondrogenesis of the synovium was assessed in an explant culture and in a caprine chondral defect repair model. In the explant culture, harvested synovium tissue was embedded in agarose, cultured in TGF-beta 3 supplemented medium for 28 days, and stained with Safranin O. In the repair model, a 4×5×0.45 mm chondral defect was created on the trochlear groove. Microfracture was performed in seven animals. In the other seven animals, synovium fragments were implanted into the defect following microfracture. Defects were covered by a devitalized fascia flap. Repair of the defect was evaluated histologically at 6 weeks post surgery. Results More than 90% of the cells in the harvested synovium appeared viable. Cells in synovium explant culture differentiated into chondrocytes with positive Safranin O staining. In the repair model, flap delamination was observed in 28% of control and 43% of synovium implant group, resulting in minimal tissue filling in the defect. Although defects with intact flaps were filled predominately with fibrous tissue, clusters of chondrocytes were observed in the defect with synovium implant. Conclusions We demonstrated the chondrogenic potential of synovial cells in vitro and the feasibility of harvest and implantation of autologous synovium tissue in a large animal model. Further investigation is required to optimize the repair efficacy mediated by synovium implant., Introduction Stem cell therapies are evaluated as a promising alternative for cartilage regeneration. Since adipose tissue provides a rich source of mesenchymal stem cells (ASCs) which can be harvested in clinically relevant quantities within a short time frame, we investigated whether a one-step surgical procedure for osteoarthritic treatment can be devised with ASCs from the infrapatellar (“Hoffa”) fat pad. Methods and Materials Infrapatellar fat pads were harvested with informed consent from patients undergoing knee arthroplasty. Colony-Forming Unit assays were performed, a.o. to calculate the percentage of stem cells and their multidifferentiation potential. Growth kinetics and the surface marker expression profile of ASCs were determined. Finally, according to the one-step surgical procedure, ACSs were induced into the chondrogenic lineage in a 3D poly-lactic acid-co-caprolactone (PLA-CPL) scaffold, and analyzed using RT-PCR, immunohistochemistry and glycosaminoglycan formation. Results The frequency of Colony-Forming Units in the stromal vascular fraction (SVF) of these fat pads was 2.6 ± 0.6%. These CFUs showed multilineage differentiation towards the osteogenic and adipogenic lineage. The population doubling time of approximately two days and the surface marker expression profile matched that of ASCs. Seeded in a 3D PLA-CPL scaffold material, freshly isolated stromal cells showed chondrogenic differentiation potential. Conclusions Due to the high ASC quantity in the stroma of the infrapatellar fat pad, the favorable proliferation rate and the high chondrogenic differentiation potential, this stroma is a suitable candidate for a one-step surgical procedure to regenerate cartilage tissue., Introduction Mesenchymal stem cells (MSCs) have been preferred to as immunological tolerant cells. Previously we demonstrated that both juvenile and adult articular chondrocytes were non-alloreactive and that they inhibited the mitogenic activity of activated CD4+ T cells by cell-to-cell contact (ORS 2004, 2006). Furthermore adult chondrocytes preserved their immunological characteristics independent of their osteoarthritic condition (ICRS 2007). Cultured articular chondrocytes (ACs) had immune characteristics that could be used for tissue repair; it is likely that allogeneic MSCs will be used as well. We hypothesize that mesenchymal-lineage cells preserve common immunological characteristics. The purpose of this study is to compare immunological properties of ACs with other mesenchymal-lineage cells derived from various origins in the same persons. Methods and Materials ACs and bone marrow stromal cells (BMSCs), meniscus cells (MCs) and synovial cells (SCs) were isolated from 5 knee joints when arthroplasty was performed. Each of the cells were cultured and expanded. To explore surface antigens on each cell, flow cytometric analysis was performed. To determine the allogeneic reaction, MLR assay and a proliferation assay in mitogenic activating CD4+T cells were performed. Results Each of the cells expressed MHC class I(+), MHC class II(−), CD34(−), CD45(−), CD73(+), CD90(+), CD105(+) and CD166(+). These expression patterns were the same as on MSCs. ACs, BMSCs, MCs and SCs failed to stimulate MHC mismatch response in MLR assay and they did not inhibit the proliferation of activated CD4+ T cells without cell-to-cell contact. Conclusions We demonstrated that mesenchymal-lineage cells isolated from various types of tissues preserved common immunological characteristics., Introduction We hypothesized that human cartilaginous constructs can be engineered from chondrocytes derived from osteoarthritic cartilage. Methods and Materials Chondrocytes were isolated from the cartilage removed from patients (n=5, 57±12 years) during total knee arthroplasty for osteoarthritis. Using culture conditions from previous studies, cells were expanded ∼20X over 2 weeks from a plating density of 5,000 cells/cm2 (medium with 2% human serum (HS), TGF-β1, FGF-2, and PDGF-bb), and then redifferentiated over 2 weeks in alginate beads (medium with 20% HS). Cells with associated matrix were released from alginate and seeded into 3.2 mm and 6.5 mm diameter cell culture inserts for 2 weeks to form constructs. Medium was serum-free or with 10% HS, and ±TGF-β1, ±IGF-I, ±BMP-14, individually and in combination. Constructs were evaluated by appearance, wet weight (WW), cell number, sGAG and collagen content. Large constructs were also measured for thickness and compressive modulus. Data were analyzed by 2-way ANOVA. Results During construct formation, inclusion of 10% HS in culture medium led to increased collagen/WW (+120%, p⇠0.05), decreased WW (−56%, p⇠0.001), decreased sGAG/construct (−47%, p⇠0.001), and decreased sGAG/cell (−39%, p⇠0.05). For large constructs, inclusion of 10% HS led to decreased construct thickness (−58%, p⇠0.05), and increased compressive modulus (+176%). Addition of growth factors, in particular the combination of TGF-β1, IGF-I and BMP-14, resulted in an increase in both sGAG/construct (+29%, p⇠0.05) and collagen/construct (+27%, p⇠0.05). Conclusions Using culture medium including 10% HS and additional growth factors including TGF-β1, cartilaginous constructs can be fabricated from culture-expanded chondrocytes that originate from osteoarthritic cartilage., Introduction Purpose: We hypothesized that co-culture of chondroprogenitor cells with chondrocytes could enhance their chondrogenic differentiation. The aim of this study was to investigate the efficacy of simply co-culturing synovial cells with articular chondrocytes and to assess the cells biochemically. Methods and Materials Materials and Methods: Rabbit articular chondrocytes, synovial cells and tenocytes(as control) were used in this study. Co-culture of synovial cells and articular chondrocytes were conducted either with or without cell-to-cell contact. PG content and the expression of mRNA of Sox-9, Aggrecan and type-II collagen were assessed. Results Synovial cells were shown to express chondrogenic phenotypes when grown in chondrogenic medium that contained essential growth factors for cartilage differentiation. When synovial cells were co-cultured with chondrocytes without cell-to-cell contact, their expression of chondrogenic proteins, such as Sox-9, aggrecan and type II collagen, was increased even in the absence of growth factors. furthermore, in the situation of cell-to-cell contact, expression of Sox-9 and type-II collagen in the synovial cells-chondrocytes mixture was significantly higher than that of the control. Conclusions These results suggest that co-culture of synovial cells with chondrocytes has a promotive effect on synovial cell chondrogenesis. It also implies that combined implantation of articular chondrocytes with isolated synovial cells could be an efficient method for repair of large chondral lesions, which need abundant implanted cells., Introduction The aim of this study is to characterize the temporal gene expression levels of different markers in order to determine its potential use as monitors of in vitro differentiation of 3D scaffolds. Methods and Materials Human articular chondrocytes were isolated, culture expanded in monolayer and seeded in Hyaff 11 scaffolds. During expansion DMEM-F12 supplemented with 10% human serum was used. During differentiaton in Hyaff 11 scaffolds DMEM high glucose, ITS, Linoleic acid, human serum albumin, 10 ngTGFβ1, dexamethasone and ascorbic acid was used. The constructs were cultured for 1, 7, 14 and 21 days. Alcian blue-Van Gieson staining was performed to evaluate GAG production. Real time PCR was used to evaluate the gene expression of the following markers: Cathepsin B, EGR1, SerpinA1, SerpinA3, MMP 1 and 13, SOX 9, Collagens I, IIa and IIb, Versican and COMP. Results Histologically it was possible to see increased GAG production within time. Cathepsin B and EGR1 were upregulated during dedifferentiation of chondrocytes. SERPINA1, SERPINA3, SOX 9, Collagen IIa and IIb, and COMP were upregulated during redifferentiation. SOX 9 had the highest expression level before the activation of the Collagen IIa gene. Versican was downregulated during redifferentiation. Conclusions Cathepsin B, EGR1 and SerpinA3 are potential markers to monitor the differentiation status of 3D tissue engineered constructs. Detection of these markers in the media would help to monitor the differentiation stage of the constructs and determine release criteria for clinical tissue engineering., Introduction Articular cartilage (AC) heals poorly and effective host-tissue integration after reconstruction is a concern. The current study investigated the ability of implanted chondrocytes to attach at the site of injury and incorporate into the devitalized host matrix adjacent to a defect in an in vitro human explant model. Methods and Materials Human osteochondral dowels received a standardized injury and were seeded with PKH 26 labeled passage 3 chondrocytes and compared to two control groups. All dowels were cultured in vitro, harvested at 0, 7, 14 and 28 days and assessed for chondrocyte adherence and migration into the region of decellularized tissue adjacent to the defects. Additional evaluation included cell viability, general morphology and collagen II production. Results Seeded chondrocytes adhered to the standardized defect and areas of lamina splendins disruption but did not migrate into the acellular region adjacent to the defects. There was a difference in viable cell density between the experimental group and one control group. A thin lattice-like network of matrix surrounded the seeded chondrocytes and collagen II was present, demonstrating cellular activity. Conclusions This work demonstrated that cultured human chondrocytes do adhere to regions of AC matrix injury but appear incapable of migration into the host tissue despite the presence of viable cells capable of generating a paracrine stimulus. Additionally, the explant model was viable during long-term culture and could be used to study the interaction of implanted cells and host tissue, providing insight to better optimize defect preparation and graft integration., Introduction The results after Autologous Chondrocyte Implantation (ACI) are evaluated using several outcome measures. The histological analysis of the repair tissue after ACI is an important and objective outcome measure to assess the quality of the characterization and the distribution of cells. Methods and Materials The characteristics of the repair tissue after ACI of 97 patients were evaluated histologically. All patients had a second look arthroscopy where a biopsy was taken. Biopsy specimens were obtained from the center of the defect in 97 patients, fixed in 4% formaldehyde for 24 hours, embedded in paraffin, cut into 8-μm sections and placed onto microscope slides. After deparaffinization the sections were stained with Alcian blue van Gieson, hematoxylin and eosin, Alcian blue, or safranine O. To perform a semiquantification of the repair tissue the ICRS visual score was used. Briefy the aspect of the surface, extracellular matrix, subchondral bone, cell distribution and presence of calcified matrix was evaluated by three experienced phisicians. Clinical correlation of the biopsies and the clinical outcome were performed when available. Results After evaluation the surface showed a mean score of 2.60, the matrix 2.02, the subchondral bone 2.57, the cell distribution 0.90, the viability 2.97 and the calcified cartilage 2.95. The maximum score of all the items is 3. Conclusions The quality of the repair tissue after ACI shows a hyaline like or a mixture of fibrohyaline and hyaline like tissue. The normal distribution of the cells was not restored; however in some cases the normal columnar distribution was restored over time., Introduction Autologous chondrocyte implantation has been a widely used clinical strategy in the repair of damaged cartilage. Follow-up showed good clinical results together with the formation of a new tissue with many hyaline features. To explore a new approach to treat also early degenerative lesions to hyaline cartilage in osteoarthritis patients (OA), we proposed to transplant in an experimental animal model of OA, a scaffold seeded with autologous mesenchymal stem cells, obtained from bone marrow and expanded in culture. Methods and Materials Rabbit knee joints were bilaterally subjected to Anterior Cruciate Ligament Transection (ACLT) to surgically induce OA. After 8 weeks, necessary to the development of cartilage surface damage, animals were treated with mesenchymal stem cells seeded onto Hyaff®-11 scaffold in the left condyle and unseeded Hyaff®-11 in the controlateral knee. Untreated rabbits were used as control. All the animals were sacrificed at 3 and 6 months after surgery. Histological, histomorphometric and immunohistological evaluations were performed. Results OA changes developed in all animals subjected to ACLT. The predominant macroscopically observed OA changes were mild (lateral femoral condyle) or moderate (medial femoral condyle) ulcerations. Statistically significant differences in the quality of the regenerated tissue were found between the implants with scaffolds carrying mesenchymal stem cells compared to the scaffold alone or controls in particular at 6 months. Conclusions From the observations, it is possible to demonstrate that Hyaffò-11, a hyaluronan-based scaffold, provides promise for mesenchymal stem cell implantation and that may have application for the treatment of early OA in humans., Introduction There is a need for diagnostic and predictive biomarkers in musculoskeletal disorders generally; in autologous chondrocyte implantation (ACI) it would be useful if a serological marker tested early post-treatment could predict long term success. We have measured levels of 2 molecules commonly investigated in arthritis studies, cartilage oligomeric matrix protein (COMP) and hyaluronan (HA), in synovial fluid and blood of ACI-treated patients. Methods and Materials Plasma (P) and synovial fluid (SF) were obtained from 4 patients (aged 26–41 years) at 2 timepoints: three immediately pre-op and at 8–14 months post-ACI and one at 19 and 31 months post-treatment. COMP, HA and urea were measured via ELISA; COMP and HA in SF were normalised to urea to allow for its dilution factor due to sampling or pathology. Results Levels of HA in plasma were 39±34ng/ml and in SF 10±4mg/ ml, with little difference in SF pre- and post-op (23±15, 55±44ng/ml respectively (plasma); 10±5, 9±4mg/ml (SF)). COMP levels, however, increased post-operatively in both plasma (613±206, 727±290ng/ml respectively) and SF (298±185, 569±438μg/ml). Levels in the fourth patient showed similar trends between sampling times. Conclusions Although patient numbers are small, this study suggests that ACI results in altered composition of the synovial fluid for some time post-treatment, with increased COMP at the second timepoint. Whilst serum levels have been shown to increase similarly for 3–6 months post-arthroplasty, the increase is sustained longer post-ACI. How this relates to COMP's function of organising collagen fibrils and binding other molecules within the repair tissue remains to be determined., Introduction Bone tendon healing in shoulder surgery provides 50% failure rate after repair. The first aim of this study was to validate a small animal model, and the second was to evaluate the effect of chondrocytes injections on healing. Methods and Materials Under general anesthesia, Achilles' tendon of thirty 3 month-old wistar rats were detached from the calcaneum and enthesis was destroyed mechanically. The tendon was then reattached to the bone by transosseous 4/0 non absorbable suture, skin was closed (G1). Cell therapy group consist of the same surgical procedure with local delivery of 4 Millions rat chondrocytes suspended in fibrin glue (G2, n=30). Animals were sacrificed at 15, 30 and 45 days to evaluate global healing rate, biomechanical and histological data. Results The global healing rate increased from 47% (G1) to 70% (G2) (p=0,11). At 45 days, failure to load was 82 N in G2 versus 62 N in G1, with a statistically significant difference (p⇠0,05). Typical aspect of chondrocytes in chain was found, from 15 days in G2 but never in G1. Immunostaining demonstrates local production of type II collagen at the bone tendon junction only in G2. Conclusions The current study validate a small animal model of tendon to bone repair with 47% healing rate, close to the value reported in human shoulder cuff surgery. Injection of chondrocytes induces type II collagen production, and is an efficient therapy to stimulate bone tendon healing by increasing global healing rate and providing a statistically significant difference in failure to load at 45 days compare to surgery without cell transplantation., Introduction To develop an accurate, reproducible lot release assay to evaluate cell viability of advanced cell therapy and tissue engineering products like MACI, which contain cells seeded in 3-D matrices. Methods and Materials A novel enzymatic reaction-based method was invented by correlating viability to relative levels of enzyme activity in cell-containing and non cell-containing fractions (conditioned media) of the culture. Results The new method shows excellent accuracy (15%), precision (15%), linearity (R2⇢.95), and specificity (Signal/Noise⇢3) over a large range of cell viabilities (e.g. 0%-100%) and cell densities (e.g. 8.75 e4 to 2.8 e6 per cm2 membrane for MACI). The working cell density range and applicable medium type of the new method can be easily adjusted using phenol red to meet the needs of different products. The new method does not require cell recovery or control cells, and is not limited by donor variability or matrix type. It can be applied to both 2-D and 3-D cultures of human or animal cells in all media types. Conclusions The new method solved a long-standing technical challenge for tissue engineering products such as MACI. Viability can be accurately determined without interference from the matrix material. The method has been developed and validated for use with MACI, and further enhances our rigorous quality control and assurance program for advance cell therapies., Introduction We developed an injectable implantation system, using a novel in-situ forming material based on alginate, which can be arthroscopically performed. We have demonstrated that this system enhances cartilage repair in a rabbit model. The aims of this study were to evaluate the reparative tissue in a canine model and to determine whether this material can be arthroscopically implanted in cadaveric knees. Methods and Materials ⇠Canine model⇢Two osteochondral defects (95×5mm) were created in the patella groove of beagle dogs. The defects were divided into three groups: no treatment group, material (material without cells) group, and material with BMSCs group. Dogs were euthanized at 16 weeks postoperatively. The sections were evaluated using macroscopic and histological scoring. The mechanical properties were measured by an indentation test. ⇠Cadaver model⇢A cartilage defect (10×20 mm) was made at the medial femoral condyle. A dyed material was arthroscopically implanted and evaluated after 24 hours of manual mobilization. Results ⇠Canine model⇢ The material groups were repaired mainly with fibrocartilage. The material with BMSCs group exhibited normal cartilaginous tissue with rich GAG matrix. The macroscopical and histological scores of the treatment groups were significantly higher than the defect group (p ⇠0.05). The compressive modulus in the material with BMSCs group was significantly higher, compared to that in other groups (p ⇠0.05). ⇠Cadaver Model⇢The implanted material maintained its initial shape. Conclusions The current study showed that the implantation of BMSCs using our novel system induced matured hyaline-like cartilage repair in a canine model. We successfully established an arthroscopic implantation technique using human cadaveric knees., Introduction The aim of this study was to determine the appropriate in vitro culture conditions that increase the in vivo chondrocyte differentiation. Methods and Materials Human articular chondrocytes were isolated, culture expanded in monolayer and seeded in Hyaff 11 scaffolds. Three different media conditions were used. The different media were Media A: DMEM-F12 supplemented with 10% human serum, Media B: DMEM high glucose, ITS, Linoleic acid, human serum albumin, 10 ng TGFβ1, dexamethasone and ascorbic acid, Media C was a composed of Media B and 10% human serum. After 14 days the constructs were transplanted into human osteochondral plugs which were then implanted into the subcutaneous tissue of nude mice for 6 weeks. An empty scaffold was used as negative control. In order to determine if the preculture had an effect on the in vivo differentiation two scaffolds were implanted after 1 or 28 days of in vitro culture. The results were semiquantified using the Bern score. Results The preculture affected the differentiation in vivo. Samples implanted 1 day after seeding showed no differentiation after 8 weeks in vivo whether precultured samples showed significantly higher differentiaiton grades. The media composition affects the in vivo differentiation capacity. In this study the formulations B and C containing TGFβ1 affected the differentiation grade and the integration to the surrounding cartilage. Empty scaffolds did not show differentiation in vivo in this model. Conclusions Tissue engineered constructs should be cultured in vitro with media formulations containing TGFβ1 in order to increase the in vivo differentiation and integration capacity., Introduction Monolayer cultures of chondrocytes as well as in vitro tissues using cells and scaffolds have been widely used to understand the mechanisms involved in cartilage matrix turnover and cell differentiation. However, detailed studies have been hampered by a number of factors such as the scarce availability of human tissue, the limited proliferative capacity of cultured chondrocytes and the enormous donor dependent variances in the differentiation status of cells. It is frequently reported that primary chondrocytes in monolayer culture undergo a phenotypic drift including down regulation of cartilage markers like type II collagen and aggrecan. Methods and Materials In order to establish a nontransformed human articular chondrocyte cell line possessing a remarkable extended life span, we transfected human articular chondrocytes with selected proliferation genes enhancing the replicative capacity. The successful transfection as well as the expression of markers characteristic for hyaline cartilage was verified on the mRNA level by RT-PCR and on the protein level by immunofluorescence. Results This chondrocyte cell-line which has the capacity to escape the Hayfick limit of cell proliferation and also displays a cartilage typical protein expression profile (type II collagen and aggrecan) may provide an ideal cell source for numerous donor independent studies. Conclusions Especially the possibility to bypass the dilemma of donor variances nominate this human chondrocyte cell line as suitable basis for pharmacological test systems relevant to screen the physiological behaviour of cartilage cells in healthy tissue and in arthritic diseases, particularly in response to putative therapeutical agents., Introduction Matrix metalloproteinases (MMPs) are catabolic enzymes involved in the remodeling of connective tissues. In osteoarthritis (OA), dysregulation of MMPs in joints results in the change from physiologic remodeling to pathologic destruction of cartilage. The mechanisms involved in regulating MMP activity are therefore attractive targets for therapeutic intervention. We determined whether MMP expression can be modulated in cytokine activated chondrocytes by the combination of avocado soybean unsaponifiables (ASU), glucosamine hydrochloride (GLU) and chondrotin sulfate (CS). Previous studies have shown the benefit of using ASU alone, or GLU and CS in the management of OA. Methods and Materials Chondrocytes (5×105) in serum free media were activated with IL-1β (10 ng/ml) and TNF-α (1 ng/ml) on days 1 and 4. Cells were next incubated on day 7 with: control media or the combination of ASU (NMX1000® 8.3 μg/ml), GLU (FCHG49®, 11 μg/ ml), and CS (TRH122®, 20 μg/ml). Secreted MMPs were analyzed by zymography and Western blots on day 8. Results Non activated chondrocytes enzymatically express latent and active forms of MMP 9. Cytokine activation induced a shift in the ratio of the latent form of MMP 9 (∼92 kD) to the active form (∼82 kD). The combination of ASU, GLU and CS inhibited the cytokine induced conversion from latent to active forms of MMP 9. Conclusions Our finding suggests that the combination of ASU, GLU and CS inhibits cytokine induced shift from latent to active forms of MMPs. Suppression of MMP activation may minimize cartilage breakdown that is beneficial in the management of OA., Introduction Plasma-mediated bi-polar radiofrequency is a low temperature radio-frequency based technique used to remove soft tissue. In arthroscopic surgery, it is used when treating partial-thickness defects in cartilage, where the method aims to supply a smooth and stable joint surface. Previous studies have shown that plasma-mediated bi-polar radiofrequency effects interleukin production from porcine disc cells in vitro and in vivo in a way that is suggested to promote the healing response of the tissue. The aim of the present study was to evaluate the effect of plasma-mediated bi-polar radiofrequency on human chondrocyte proliferation. Methods and Materials Surplus human chondrocytes from four patients (mean age 23yrs) undergoing autologous chondrocyte transplantation were cultured in 3D-alginate gel in chondrogenic media. IL-1α-supplemented media was also used to stimulate an inflammatory response. After four weeks the cells were exposed to a plasma-mediated bi-polar radiofrequency wand (ArthroCare Corp.) for five seconds, or a temperature sham, with a similar temperature as the wand, approx 50°C. The amount of DNA in the gels was determined three and six days post treatment. Results A significant (p=0.05) increase in cell proliferation was observed in the plasma-mediated bi-polar radiofrequency treated group with unstimulated cells, whereas no such effect was seen in the IL-1α stimulated cultures. Conclusions Our results suggest that the plasma-mediated bipolar radiofrequency treatment induces a proliferative response of chondrocytes in alginate gel culture. The increase in proliferation observed might have positive clinical effects on a tissue healing response. Continued studies are ongoing to further investigate the response on a cellular as well as gene regulatory level., Introduction Oxidative stress plays an important role in the pathogenesis of osteoarthritis. Epigallocatechin gallate has potent antioxidant activity. Oral curcumin efficacy has been shown for many conditions characterized by oxidative damage and inflammation. Although the protective effects of these two agents have been reported in various models, there are few studies about their protection against cartilage damage in vitro. Methods and Materials Articular cartilage was isolated from carpal joints of bovine. Cartilage fragments were cultured in 1ml of DMEM/F12 supplemented with 10% foetal bovine serum, 100units/ ml penicillin/streptomycin. 24h later medium was changed and different cultures were produced: first one with IL-1b, second added with IL-1b and EG, third made of IL-1b and CR, fourth with IL-1b, EG and CR; two concentrations of EG and CR (6+5 mg/ml and 3+2,5 mg/ ml respectively) were used for a new set of cultures. GAG and NO release from cultured tissues were assayed. Results The studied compounds showed a significant inhibition of the release of GAG when the cartilage was challenged with IL-1b; the association of the two components (EG-CR) induced a more powerful and significant effect at both concentrations. Our results also indicate that NO is inhibited with less efficacy, but the effect of the association is more evident when compared with the effect of single product. Only CR and EG+CR are statistically significant. Conclusions Combined use of EG and CR seemed to be effective in cultured bovine cartilage tissues in reducing NO and GAG production when tissues were challenged with IL-1b., Introduction Autologous Chondrocytes Implantation relies on chondrogenic capacities of implanted cells. Markers allowing for selection of human articular chondrocytes (HAC) with better chondrogenic potential would represent a major advantage. In this study we investigated HAC intrinsic chondrogenic capacities with surface marker expression and cell population doubling (PD). Methods and Materials Expression of surface marker CD90, CD166, CD14, CD54, CD105 and CD49c was monitored in HAC during early monolayer expansion via flow cytometry. For cell sorting HAC (n=4) were expanded for 5 days and labelled with anti-CD90 monoclonal antibody. Unsorted, CD90-positive and CD90-negative sorted cells were further expanded for up to 33 days. At weekly passages cumulative PD was determined, flow cytometry analysis performed and micromass pellet cultures prepared and incubated in serum-free medium with and without the addition of chondrogenic factors. Results Changes in surface marker expression correlated with early dedifferentation process, with a marked upregulation of CD105 and CD49c after 3 days, and CD166 and CD90 after 10 days of expansion. CD90 expression distinguished two chondrocyte subpopulations at days 3–5 with a complete shift from CD90-negative to CD90-positive cells at day 14. Sorted CD90-negative cells proliferated slower, reaching a 3.2 PD threshold later, and produced pellets with more mature neocartilagenous tissue compared to CD90-positive or unsorted cells. However, sorted CD90-negative cells became CD90-positive within 26 days of further expansion, and beyond PD 3.2 also produced immature fibrocartilagenous tissue. Conclusions HAC lacking CD90 expression have more intrinsic chondrogenic potential to produce cartilage-like matrix. Furthermore, higher chondrogenic potential correlates with a slower proliferation rate., Introduction Chondrocyte viability is essential for the maintenance of articular cartilage as they constitute the only cell type in articular cartilage. Apoptosis is processed by specific intracellular signaling cascades and thus has been an attractive therapeutic target for diseases in which apoptosis is an integral part of its pathogenesis. Transglutaminase2(TGase2) has been shown to be induced and activated during apoptosis. We have previously shown that TGase2 expression is increased in human chondrocytes undergoing apoptosis. We also have explored the role of TGase2 in chondrocyte apoptosis by using monodansylcadaverine(MDC). RNA interference(RNAi) is a process of sequence-specific gene silencing mediating specific mRNA degradation and thus allows the understanding of the function of specific gene of interest. The purpose of this study was to explore the role of TGase2 in human chondrocyte apoptosis by using RNAi. Methods and Materials Chondrocyte apoptosis was induced by treating with H2O2 for 24 hours. Apoptosis was assessed by DNA fragmentation ELISA and Annexin-V FACS analysis. The expression and enzyme activity of TGase2 was examined with Western blot and immunocytochemistry TGase2 was inhibited by MDC. siRNA targeting TGase2 was constructed with sequences of 5A′-GGGCGAACCACCUGAACAATT-3A′ and 3A′-TCCCGCUUGGUGGACUUGUU-5A′. Results MDC increased the amount of apoptosis, measured by DNA fragmentation ELISA, in H2O2-treated cells in a dose-dependent manner. Transfection of siRNA into human chondrocytes resulted in downregulation of TGase2 protein as shown by the Western blot and immunocytochmistry. The enzyme activity of TGase2 was also down-regulated in siRNA-transfected cells. Apoptosis in siRNA-transfected chondrocytes was increased, measured by Annexin-V FACS analysis, when compared to control-siRNA transfected chondrocytes. Conclusions These results implicate a protective role of TGase2 against apoptosis in human chondrocytes. These results provide new insights into the possibility of TGase2 as a potential modulator of osteoarthritis by protecting against chondrocyte apoptosis., Introduction The purpose of this work is to evaluate the maturation in vitro and in vivo of an engineered cartilaginous tissue obtained by isolated swine articular chondrocytes embedded in fibrin glue at different experimental times. Methods and Materials Isolated swine articular chondrocytes were embedded in fibrin glue. Sample groups were divided as follow: Some samples were left in standard culture conditions for one, five and nine weeks. Some others were implanted in nude mice for the same time schedule. The remaining samples were cultured in vitro for one and five weeks and then implanted in subcutaneous pouches of nude mice for four additional weeks. Gross evaluation, biochemical analysis (DNA and GAGs content) and gene expression (collagen type I and type II, aggrecan and Sox 9) were performed. Results Generally, samples retrieved from nude mice experienced shrinkage and mass reduction; they also resulted in an higher content of DNA and GAG. Collagen type II was higher in the 5- and 9-week samples with respect to those cultured in vitro. Opposite finding was recorded for aggrecan expression, while Sox9 was not significantly different from in vitro and in vivo. Conclusions The results of this study demonstrate that in vivo implantation of engineered cartilage composite results in increasing the cell proliferation and matrix formation. Pre-culturing the samples before implantation does not seem to interfere with the capacity of cell proliferation and synthesis, but, on the other hand, does not appear to ameliorate the quality of the engineered samples in this model., Introduction To repair cartilage, the source of cells used in tissue engineering includes human native chondrocytes, mesenchymal stem cells (hMSCs) and hMSCs-derived chondrogenic cells. The mechanical properties of these cells are important predictors of chondrocyte function. To distinguish the mechanical properties of these different cell sources from the same patient, an atomic force microscope (AFM) was used to probe the surface ultrastructure and to measure the adhesion force and stiffness. Methods and Materials The hMSCs-derived chondrogenic cells were induced by TGF-β1 from hMSCs. The chondrogenic specific marker, type II collagen, was evaluted by RT-PCR and immunohistochemical staining. We directly employed the AFM to image a single cell and to quantitatively measure the dimensions of the cells. The cellular surface proteins were monitored by flow cytometric analysis. Results The profiles of shapes, sizes and structures were different across these 3 groups. The mean adhesion forces of native, hMSC and induced groups were 6.86±2.91, 13.31±5.16 and 4.54±1.14 nN, respectively. The stiffness values were also different (0.134±0.029, 0.164±0.017 and 0.109±0.017 N/m, respectively). The expression of type II collagen and integrin β1 were higher in native chondrocytes than those in hMSCs-derived chondrogenic cells. Conclusions Human native chondrocytes had stronger mechanical properties than those of hMSCs-derived chondrogenic cells. The differential properties might be the results of different expression amounts of cell matrix proteins. This study is the first one to directly characterize the mechanical properties between native chondrocytes and hMSC-derived chondrogenic cells., Introduction In patients with Osteoarthritis, the intra-articular temperature elevates to higher degree due to local inflammation. It was reported that heat stress on chondrocytes were induced apoptosis, but it is still clearly unknown how chondrocytes apoptosis were induced by heat stress. In the present study, we investigated the responses of chondrocytes to heat stress, and how chondrocytes apoptosis were induced by heat stress. Methods and Materials To apply heat stress to NHAC-kn cells(human normal chondrocytes), they were cultured for 1 days in incubator at 37℃. And then, they placed in water bath set at 43℃ or at 37℃ as a control temperature for 30min, 1hour, and 2hours. After the heat stress, they were removed from the water bath and moved to incubator at 37℃. Chondrocytes apoptosis were detected by TUNEL staining and western blotting. To explore the function of p53, NHAC-kn cells were pre-treated with pifithrin-alpha which is inhibitor of p53. Results Apoptotic cells were increased by heat stress in a time depend manner. The expression levels of p53, p53AIP1 and cleaved caspase-9 were increased after induction of heat stress. Apoptotic cells were decreased when chondrocytes were incubated with pifithrin-alfa. The expression levels of p53, p53AIP1 and cleaved caspase-9 were decreased when chondrocytes were incubated with pifithrin-alpha. Conclusions Our results showed that expressions of p53 and p53AI P1 were increased by heat stress, and apoptosis were inhibited when chondrocytes were pre-incubated with pifithrin-alpha which is inhibitor of p53. These indicated that apoptosis by heat stress in chondrocytes were dependent on p53 pathway., Introduction Tissue-engineering may offer promising future alternative to obtain extra autologous cartilage for reconstructions in otorhinolaryngology. Expanded chondrocytes or mesenchymal stem cells (MSCs) could be used for this purpose. The aim of this study was to study the performance of auricular and nasal-septum chondrocytes, and compare tissue-engineered constructs of these cells with those of the more frequently studied MSCs and articular chondrocytes, concentrating on subtype and stability of the tissue-engineered constructs. Methods and Materials Expanded chondrocytes and MSCs were chondrogenically differentiated in vitro. Subtype of the tissue-engineered construct was evaluated using immunohistochemistry, and their stability was tested both in vitro and in vivo. Results Auricular and nasal-septum as well as articular chondrocytes and MSCs produce a collagen II containing matrix. Although auricular and nasal-septum chondrocytes produce collagen X, both form cartilage matrix that is stable and does not mineralize when challenged in-vitro or after subcutaneous implantation in vivo. This in contrast to cartilage produced by MSCs that does mineralize, making it unsuitable for cartilage reconstructions. Although articular chondrocytes produce a true hyaline-like cartilage, without collagen type X, this source is not preferred in otorhinolaryngology. Comparing the performance of auricular and nasal-septum chondrocytes, it was obvious that auricular chondrocytes produce more cartilage-like matrix. Although direct comparison is difficult because nasal septum chondrocytes proliferate faster, auricular chondrocytes have our preference because of the robustness of results and the ease to harvest. Conclusions Our results indicate that auricular cartilage is a suitable candidate to generate stable cartilage for future reconstructions with tissue-engineered cartilage., Introduction Chondrosarcoma classification relies upon a combination of tissue morphology evaluation and clinical features. Analysis of cell membrane proteins may help to better define disease subtypes or uncover clinically and/or therapeutically relevant biomarkers. In this study we have combined flow cytometry with cluster analysis to compare the immunophenotypic profile of chondrosarcoma cells from primary tumors (CS), chondrosarcoma cell lines (CCL), bone-marrow derived mesenchymal stem cells (MSC), normal articular chondrocytes (HAC), and primary fibroblasts (FIB). Methods and Materials Human CS, CCL, MSC, HAC and FIB cultured in monolayer were analyzed by flow cytometry for the expression of 20 cell surface markers. Data expressed as mean fluorescence intensity ratio over that of the control were clustered with wCLUTO (http://cluto.ccgb.umn.edu/cgi-bin/wCluto/wCluto.cgi). Results Selection of a panel of 9 cell surface markers allowed separation of HAC, MSC, and FIB in three well differentiated clusters. CS clustered mainly with either MSC or HAC. CD14 was selectively expressed on HAC while CH2879 was the only other analyzed cell type showing significant expression of CD14. The three CCL analyzed showed decreased or lost expression of the mesechymal marker CD90, and aberrant expression of β-4 integrin, normally associated with cells of epithelial origin. Conclusions Flow cytometry combined with cluster analysis provides a tool to identify optimal combinations of antibodies allowing differentiation between closely related cell lineages. Preliminary data with chondrosarcoma cells suggest the possibility of using the presented approach to discriminate between different chondrosarcomas based on their degree of proximity to MSC or HAC clusters. This knowledge is relevant for further understanding of these neoplasms., Introduction Passaging chondrocytes approaching confluence has been standard practice. The effect of passaging cell cultures at various levels of confluence on chondrocyte gene expression has not been fully investigated. The objective of this study was to determine the effect of cell density during passaging on chondrocyte gene expression. We hypothesized that passaging cells at lower cell densities would alter gene expression of chondrocytes compared to higher densities. Methods and Materials Outerbridge grade 0 and 1 articular cartilage was collected from three patients undergoing total knee arthroplasty. Chondrocytes were isolated and plated in chondrocyte growth medium until confluence was reached (P0). These cells were trypsinized and plated (P1) in five dishes at 20% confluence and allowed to grow until densities of 30%, 50%, 70%, 90% and 100% confluence were obtained. The cells were released from the dish and total mRNA was isolated. RT-PCR for collagen types I and II, aggrecan and GAPDH were performed. Results There was no difference in collagen I and II gene expression at any cell density although more variation occurred at 90% and 100% confluence. The gene expression of aggrecan was also not significantly different between samples. There was a statistically significant difference between the results of each patient resulting in the variability at each cell density. Conclusions The results of this study suggest that the level of confluence up to 70% does not alter chondrocyte gene expression of collagen types I and II. At densities higher than 70% confluence, more variability occurs. There is much variability between patients., Introduction The nature of hyaline cartilage has imposed constraints on its study by transmission light microscopy. Most histochemical techniques are based on the staining characteristics of cartilage matrix. Techniques allowing for direct observation by light microscopy of subcellular structure of chondrocytes would enhance the understanding of chondrocyte biology. Such a technique, based on interference of light transmission by laminated specimens (Progr Histochem Cytochem 34:163; 1999) has now been applied to articular cartilage. Methods and Materials Pieces of normal human articular cartilage was fixed in 4% paraformaldehyde and 2% gluteraldehyde in 0.05M cocodylate buffer. For comparative studies Carnoy's fixative and 10% formalin were used. Dehydrated tissue embedded in LR white resin was cut at 1 to 2 microns and processed as previously described. Parafin embedded tissue was cut at 5 microns. Results The method was applicable to articular cartilage. Many structural characteristics of articular and tracheal chondrocytes were similar, but there were also marked differences. In the former chondronal arrangement of chondrocytes was more pronounced then in latter. There were also differences in the nuclei with typically large birefringent inclusions absent in the articular chondrocytes. The nuclei of the latter contained densely packed birefringent granules. These also stained densely for DNA and were surrounded by tannophilic protein membranes. Cell to cell contracts by intrachondral tubules were not observed. Conclusions Technique of structural coloration is applicable to articular cartilage. Cytological details of articular chondrocytes are different from their tracheal counterparts., Introduction There is extensive data on chondrogenesis utilizing animal tissue. However, there are many unknown aspects using human chondrocytes for neocartilage formation and lesion repair. We hypothesize that human cartilage can be engineered with morphological, biochemical and biomechanical properties compared to native cartilage. Methods and Materials A cartilage-cell/gel-cartilage construct (n=36) and cell/gel nodules (n=30) were done for this study. Articular tissue from healthy (non-arthritic) donors was enzymatically digested for 16–18 hours with dilute collagenase. Cells were collected, washed in phosphate buffered saline, and placed in culture. After expansion, the cells were mixed with a fibrin saline solution. The cell/fibrin mixture was polymerized with an equal volume of bovine thrombin resulting in a final cell concentration of 60×10e6 cells/ml. Two 6mm discs of native devitalized human articular cartilage were placed on the top and bottom of the fibrin gel, creating a cartilage-cell/gel-cartilage construct. The same cell concentration was used to create human cell/gel nodules. Constructs were implanted into a subcutaneous pocket on the dorsum of nude mice for 6, 12 and 18 weeks. Results Constructs harvested at six weeks were examined histologically for the presence of neocartilage formation. New cartilage matrix formation was noted in all of the specimens with a positive stain for glycosaminoglycans and collagen type II. A tight interface between neocartilage and native cartilage was present. Conclusions Chondrogenesis, heavily documented in animal models, has been demonstrated using human cells in a predictable and reliable manner. Engineered cartilage integrates to devitalized native cartilage, creating a tight interface., Introduction SIRT1, a mammalian homologue of longevity factor sir2, is known to inhibit apoptosis and promotes cell survival in cardiac myocytes and neuron. Yet there have never been reported the expression of SIRT1 in human primary chondrocytes (HCs). The purpose of this study is to investigate the localization of SIRT1 in HCs and to elucidate the relations of SI RT1 with apoptosis of HCs. Methods and Materials Expression of SIRT1 in HCs was examined by RT-PCR, immunoblotting, and immunohistology. HCs were transfected with SIRT1-GFP plasmid constructs by electroporation method and cellular localization of SIRT1 were examined in confocal microscopy. The expressions of SIRT1 under stresses, such as oxidative, mechanical and SIRT1 inhibitors, were examined by immunoblotting. siRNA for SIRT1 was also performed in HCs. TUNEL staining and immunoblotting of cleaved PARP were performed to examine whether SIRT1 is related with apoptosis in HCs. Results Expression of SIRT1 was detected by RT-PCR, western blotting, and immunohistology in HCs. Human GFP tagged SIRT1 was detected in the nuclei in HCs. Oxidative and mechanical stresses inhibited SIRT1 expressions in HCs. SIRT1 inhibitors suppressed SIRT1 expression in dose-dependent manner. SIRT1 inhibitors and siRNA for SIRT1 significantly induced apoptosis in HCs in TUNEL staining and immunoblotting of cleaved PARP. Conclusions SIRT1 was expressed in the nucleus of HCs. The results of this study indicated that SIRT1 regulates the apoptosis in HCs. Further research of SIRT1 might contribute to resolve the pathogenesis of osteoarthritis. f, Introduction Current techniques for autologous chondrocyte transplantation (ACT) require large numbers of cells to provide effective defect coverage. Common in vitro cellular dedifferentiation may deteriorate the chondrocyte in vivo performance. The aim of this study was to compare the gene expression profiles of chondrocytes from different topographical regions of the knee joint to characterize specific population behaviour during monolayer expansion. Methods and Materials Bovine knee chondrocytes from eight different topographical locations, weight-bearing medial(MF)/lateral(LF) femoral condyle and tibia(MT, LT), patella(P)/trochlea(T) and non-weight-bearing femoral notch(B) and proximo-medial femoral condyle(X), were cultured until passage (P) 7 under monolayer conditions. The mRNA expression levels for Collagen-1,-2,-10, COMP, Aggrecan, Sox9, PRG-4, PTHrp and MMP-1,-3,-13 were analysed for native cartilage, P0, P1, P2, P3, P5 and P7 and normalized to P0, respectively. Results From the first passage, significant differences in gene expression levels were observed for Col1, Col10, COMP and all MMPs over all passages cultured. While for Col2, Sox9, Aggrecan, PRG4 and PTHrp differences appeared to be exclusively significant for passages 1 and 2. Collagen 1 expression levels, for example, were representative of a striking difference in their upregulation, being consistently strongly increased for the regions MF, LF, P, T compared to an apparent lesser increase for both tibia locations and more pronounced for both non-weight-bearing areas. Conclusions Cartilage specific gene expression patterns among the eight locations revealed differences in regulation levels depending on the gene and/or passage number. These results, demonstrating significantly different behavior during monolayer expansion, could be embraced to identify favorable locations for cartilage biopsies when using ACT., Introduction Stromal cell derived factor (SDF-1), a chemoattractant member of the intercrine-α family, induced by pro-inflammatory stimuli, is synthesized by mesenchymal precursors and synovial fibroblasts. Chondrocytes express its receptor, CXCR4, suggesting oriented signaling upon the onset of inflammatory events in the joint; indeed attempted repair of damaged cartilage by resident chondrocytes, via cell-cycling re-induction, is insufficient to restore the tissue ad integrum. However, a better chondrogenic differentiation was demonstrated in vitro by culturing chondrocytes in serum-free conditions. Our aim was the evaluation of the SDF-1/ CXCR4 ligand/receptor system in this process. Methods and Materials To this purpose human primary chondrocytes were expanded in control (CN; 10% serum), in serum-free (SF), and in TGFp-1-supplemented SF medium (TGF). The SDF-1 mRNA levels were assayed by real-time quantitative PCR. Immunohistochemistry and FACS evidenced cytoplasmic distribution of CXCR4. Immunocytostainings were performed on micromass cultures prepared using CN-, SF- or TGF-expanded cells. Results SF chondrocytes displayed a 6.4±0.08 (mean±SD) fold-increase in SDF-1 with respect to controls, calling for restoration of a “mesenchymal precursor-like” phenotype. 3D cultures were homogenously positive for CXCR4 and matrix components (type-II collagen, aggrecan, proteoglycans). Conversely, TGF-administration brought back the chemokine expression to CN levels (0.96±0.1). However, in micromass cultures, loss of matrix components, positivity for IH H, type X collagen and apoptosis revealed a sustained presence of pre-hypertrophic cells. Conclusions These chondrocytes are CXCR4-negative, indicating that cells undergoing TGF-induced terminal differentiation turn off the expression of both ligand and receptor, acquiring a more “unresponsive” phenotype. Studies are in progress to ascertain the molecular cascades that trigger these events., Introduction Along with the development of tissue engineered products follows a need for storage and preservation. The aim of this work was to develop a cryopreservation technique for tissue engineered cartilage constructs. Methods and Materials A hyaluronan scaffold (Hyaff11, Fidia Advanced Biopolymers, Italy) was seeded with in vitro expanded human articular chondrocytes and cultured for 14 days in a modified differentiation medium. The cell-scaffold construct was frozen with a cooling rate of 1°C/min and then stored at −152°C. The freezing medium used was DMEM/F12 supplemented with 20% human serum and 10% DMSO. After quick thawing, half the construct was analyzed directly to evaluate morphology, viability (Live/Dead staining), histology (Ab/vG) and handling properties. The other half was further cultured for 28 days. Results The cell-scaffold construct showed a high viability directly after thawing. The extracellular matrix was intact after thawing and additional growth was observed during the in vitro culture. Furthermore the cells continued to metabolize the culture medium which was seen as a significant acidification of the medium during further culture. No difference in histological appearance, handling properties and overall appearance was seen between the cryopreserved and the non cryopreserved controls. Conclusions This method used for cryopreservation of cell-scaffold constructs seems promising given that the properties of the construct are kept unchanged during the freezing-thawing process. A great benefit with this technique is that it could be implemented in a near future since the freezing medium with DMSO is already commonly and successfully used for cell preservation., Introduction In pediatric traumatology fracture healing is known to exert a stimulus to the nearby epiphyseal cartilage possibly leading to severe limb-length-discrepancy during childhood. The exact underlying mechanism of increased growth post-trauma has not been clarified, yet. To investigate growth plate changes post-fracture, we determined physeal chondrocytes' proliferation- and apoptosis-rate in a certain time interval in a living rat-model. Methods and Materials Male Sprague-Dawley-rats (1 month, ∼110g) sustained a unilateral closed diaphyseal tibial fracture according to a standardized guillotine protocol. After euthanasia (days 3, 10, 14, and 29 post-fracture), physeal chondrocyte-proliferation was analysed by BrdU-Labelling. Chondrocyte-apaoptosis was determined by TUNEL-staining. Proliferation and apoptosis rates were statistically evaluated. A p-value⇠0.05 was seen as statistically significant. All animal tests were approved by the Austrian Federal Ministry of Science and Research. Results Chondrocyte-proliferation was significantly higher in the growth-plates of the fractured versus contra-lateral bones on days 3, 10, and 14, with maximum rates on day 3 (p-value 0.018). This strong physeal turnover of the fractured bone was accompanied by a significant higher apoptosis rate, valid on all evaluated days, with maximum levels on day 29 (p-value 0.018). Interestingly, no significant differences in proliferation and apoptotis rates were detected between the contra-lateral and control bones. Conclusions This study clearly demonstrates that a diaphyseal fracture locally stimulates the cell turn-over at the nearby growth plate, emphasizing that accelerated growth is at least partly caused by a higher proliferation and apoptosis rate of physeal chondrocytes. Nevertheless, the driving stimulus still needs to be identified., Introduction Linear bone growth is a highly complex process regulated by a multiplicity of endocrine, paracrine and autocrine factors within the growth plate. Knowledge of developmental and local regulation of growth plate function has been derived mostly from rodents that differ from humans in many growth-related aspects. Furthermore in vitro analyses were performed under variable conditions leading to inconsistent results. The aim of this project was to develop a culture model for the analysis of growth plate chondrocytes, which is similar to the human physiology and easy to handle. Methods and Materials Porcine growth plate and articular chondrocytes were isolated from 6–8 weeks old piglets. After one week monolayer culture the cells were cultivated in alginate beads for four weeks. The expression of differentiation factors and the availability of growth factor receptors were assessed in comparison between native samples, monolayer culture and alginate beads by real-time PCR and immunofluorescence. Results Whereas in monolayer culture the differentiation marker Col2, Col10 and aggrecan dramatically decreased, a similar expression to the native tissue was observed in alginate culture. The estrogen receptors Esr1 and Esr2 showed similar characteristics. However, GHR, IGF1R and IGF2R were expressed at higher levels in monolayer than in alginate culture or native tissue. In comparison between growth plate and articular chondrocytes in particular the different expression patterns of estrogen receptor subtypes were striking. Conclusions In conclusion, the porcine alginate model is promising as defined by tissue availability, expression of relevant hormonal receptors and comparability to human conditions, in particular in the area of basic growth research., Introduction Chondrocytes synthesize and degrade components of articular cartilage connective tissue. In osteoarthritis (OA), the balance between synthetic and degradative processes is disrupted by overproduction of pro-inflammatory mediators such as prostaglandin E2 (PGE2) and its cyclooxygenase 2 (COX-2) regulator. PGE2 induces cartilage degrading enzymes and joint pain in OA. We determined whether PGE2 production and COX-2 expression can be down-regulated in cytokine activated chondrocytes by natural products known to have anti-inflammatory activity: Avocado Soybean Unsaponifiables (ASU) and epigallocatechin gallate (EGCG). Methods and Materials Chondrocytes (5×105) were incubated for 24 hrs with: control media; ASU (NMX1000®); EGCG; or combined ASU and EGCG. Chondrocytes were activated with IL-1β (10 ng/ml) and TNF-α (1 ng/ ml) for 24 hrs to assay PGE2 or; 1 hr for COX-2 mRNA real time qPCR. Data analysis by ANOVA used p⇠0.05. Results Activation significantly increased PGE2 production and COX-2 expression. ASU (8.3 μg/ml) significantly reduced PGE2 production but not EGCG (400, 40, 4 ng/ml). The combination of ASU and EGCG synergistically reduced PGE2 production. ASU (4 μg/ml) also showed synergy with EGCG (40 ng/ml). COX-2 expression was similarly inhibited by the combination of ASU (8 μg/ml) and EGCG (40 ng/ml) but not with either agent alone. Conclusions The combination of ASU and EGCG synergistically inhibits PGE2 production and COX-2 expression. ASU is used for the management of OA. EGCG, a major component of green tea polyphenol catechins is being evaluated for the management of neurodegenerative and cardiovascular diseases associated with inflammation. The synergistic interaction between ASU and EGCG could minimize disruption of cartilage homeostasis., Introduction 2D cultivated chondrocytes dedifferentiate towards a fibroblastic phenotype in vitro. With the proceeding dedifferentiation, collagen type I takes over the chondrocyte specific collagen type II expression. To follow the state of dedifferentiation, we now introduce a combination of collagen I and collagen II reporter plasmids. Methods and Materials The collagen I plasmid contains the red fuorescent protein dsRed driven by a collagen I alpha 1 enhancer/ promoter combination. The collagen II reporter contains a CMV-enhancer element for signal amplification linked to a collagen II-promoter fragment containing chondrocyte specific cis-acting activator and repressor recognition sequences. In contrast to the collagen I reporter, the collagen II plasmid includes the fuorescent protein EYFP. Therefore both markers can be detected separately. Furthermore, the differentiation kinetics can be monitored for both directions, de- and redifferentiation. We achieved the design of a reporter-vector system simultaneously indicating collagen expression-levels of transfected cells by cloning of the two reporter-cassettes into one plasmid. Results Initial transfection of porcine BMSC with the separate reporter-vector systems confirmed the functionality of the designed chimeric transcription activating elements by fluorescence microscopy. The collagen-expression specific response of the designed reporter-vector systems is still under investigation in human adipose derived stem cells. Conclusions The described systems will be suitable for investigating collagen I and collagen II expression of human mesenchymal stem cells and articular chondrocytes in vitro. This work was supported by the European projects Hippocrates (NMP3-CT-2003-505758) and Expertissues (NMP-CT-2004-500283), Introduction Bupivacaine has been shown to be cytotoxic to articular chondrocytes in a dose-/time-dependent fashion and to disrupt mitochondrial membrane potential. The goals of this study are to determine whether alterations in mitochondrial membrane potential and function contribute to bupivacaine-induced toxicity and if these alterations are reversible. Methods and Materials Bovine articular chondrocytes were cultured in DMEM/F12. Chondrocytes were labeled with a fuorescent-probe for calcium fux and mitochondrial membrane potential, placed in saline, and exposed to bupivacaine over 14 min (37°C). Peak concentrations ranged from 0.25%-0.0625% occurring for one minute (t=7min), followed by saline inflow. Time-lapse confocal microscopy was performed once a minute for 60 min and images were analyzed. Mitochondrial function was also determined by XTT assay. Results Chondrocytes exposed to 0.25% bupivacaine exhibited reduced mitochondrial membrane potential, membrane blebbing and failure, and calcium influx at peak exposure (7min). In contrast, 0.125% bupivacaine decreased mitochondrial potential during bupivacaine inflow, which then stabilized during outflow. No increase in calcium fux was seen and cell membranes remained intact. XTT assay also demonstrated that bupivacaine reduced mitochondrial function in a dose-dependent fashion (p⇠0.001). Mitochondrial function, potential, and calcium fux following exposure to 0.0625% bupivacaine were similar to saline (p⇢.05). Conclusions These results show that bupivacaine reduces chondrocyte mitochondrial membrane function in a dose-dependent fashion and that progressive loss of mitochondrial membrane potential coincided with calcium influx and chondrocyte death. This provides both a potential mechanism for bupivacaine induced chondrotoxicity and a potential explanation for the importance of dilution in reducing the potential for chondrotoxicity in the clinical setting., Introduction Chondrocytes isolated from its original extracellular matrix and cultured in vitro, may change the phenotype and therefore the gene expression profile. Therefore, after several passages, dedifferenciated cells are similar to fibroblasts. The purpose was to study along the time the dynamic of type I and II collagens (COL1, COL2) and aggrecan expression by qRT-PCR in monolayer chondrocyte cultures. Methods and Materials Articular cartilage from 16 patients. Chondrocytes were isolated after collagenase digestion and cultured in DMEM (Passage 0: P0). Cells were trypsinized at 80% of confluence and subcultured (Passage 1: P1). The procedure was repeated twice, so the cells were subcultured until P3. Total RNA was isolated from P0, P1, P2 and P3 and cDNA synthesis was carried-out. Relative expression of COL1, COL2 and aggrecan expression was performed using the expression of the housekeeping glyceraldehyde-3-phosphate dehydrogenase (GADPH) gene as the reference. Results In the basal samples (P0), the median expression of aggrecan and COL2 were 0.39 (range: 0.04 − 8.00) and 0.08 (range: 1.92 × 10–5 − 4.68) respectively. The median basal expression of COL1 was 1.45 (range: 1.45 − 2.08). The median expression of aggrecan and COL2 is decreasing during the different passages, while that of type I increased during the passages. Aggrecan and COL2 were downregulated from P1 to P3 for aproximately 6-fold and 14-fold, respectively, while COL1 was upregulated 3-fold. Conclusions Although aggrecan and COL2 genes are downregulated in cultured human articular chondrocytes the relative expression of these genes is mantained until the end of culture., Introduction Articular chondrocytes acquire a fibroblastic-like phenotype when cultured in monolayer, characterized by different cell morphology and by expression of non-cartilage specific genes such as type I collagen. We focused on some of the events which could affect the chondrocytes phenotype in vitro: the cell density and the time of culture. Methods and Materials Swine articular chondrocytes were isolated and seeded at different cell densities (from 12,000 cell/ cm2 to 100,000 cells/cm2). Samples were cultured for 6 and 8 days. Phenotype was evaluated in terms of aggrecan and collagen type I and II expression, while proliferation was assessed by cell counting. Results Chondrocytes proliferated more at lower cell densities and did not show any proliferation at highest cell density; moreover, no further proliferation was observed when chondrocytes were kept in culture for 8 days. Chondrocytes seeded at 50,000 and 100,000 cells/cm2 were able to maintain better level of differentiation, characterized by higher expression of type II collagen and aggrecan and lower expression of type I collagen, but in all conditions a significant loss of chondrocyte phenotype from 6 to 8 days of culture was observed. Conclusions Cell density influences chondrocyte capability of maintaining a differentiated phenotype in vitro; moreover, the proliferation is allowed only at low cell densities. Some other important modulators of proliferation and differentiation, such as growth factors and growth substrates, will be valuated in future studies in order to better modulate proliferation and the loss of phenotype in vitro., Introduction Parathryoid hormone-related peptide (PTHrP) induced chondrogenic differentiation of hBMSCs while inhibiting hypertrophic differentiation (Kafenah 2007). PTH and PTHrP bind to the same PTH/PTHrP receptor. As blocking hypertrophic differentiation is desirable for autologous chondrocyte transplantation, and increased chondrogenic differentiation may lead to higher proteoglycan (PG) content, we hypothesized that addition of PTH to in vitro cultured chondrocytes positively influences proteoglycan metabolism. Methods and Materials Healthy femoral cartilage of 9 human individuals was digested in 0.1% collagenase. Following expansion, chondrocytes were cultured on collagen type II- coated Millipore culture inserts. During the 28-day culture, PTH (Brunschwig) was added at concentrations of 0.1 or 1.0 μM from days 0 (d0), 9 (d9) or 21 (d21) onwards, or no PTH was added (control). After 28 days, PG content and release were measured. Results Addition of PTH to in vitro cultured chondrocytes seemed to result in decreased PG content, although this effect was only significant for 0.1μM PTH added at d9 compared to PTH added from d21, (p=0.015). Moreover, PG release was higher when PTH was added later (1.0μM PTH from d21 vs 1.0μM PTH from d9, p=0.037), or when PTH was not added at all (control vs 1.0uM PTH from d0, p=0.01). Conclusions Addition of PTH to in vitro cultured chondrocytes did not result in increased PG content, but did decrease PG degradation. Although PTH and PTHrP are assumed to exert analogous effects, signalling in BMSCs by PTHrP may differ from PTH in chondrocytes. Analysis of the hypertrophic markers collagen type-X and Runx-2 may provide definite answers., Introduction Chondral injuries of the knee are commonly seen at arthroscopy, yet there is no consensus on the most appropriate treatment method. However, untreated cartilage injury predisposes to osteoarthritis contributing to pain and disability. For cell-based cartilage repair strategies, an ex vivo expansion phase is required to obtain sufficient cells for therapeutic intervention. Although recent reports demonstrated the central role of oxygen in the function and differentiation of chondrocytes, little is known of the effect of physiological low oxygen concentrations during the expansion of the cells and whether this alters their chondrogenic capacity. Methods and Materials Initial studies of chondrocyte expansion were performed in mature mice, with cells expanded at either atmospheric oxygen tension (21%) or 5% 02 in monolayer cultures. Chondrogenic differentiation was subsequently assessed via micromass culture. Having determined that oxygen tension influences murine chondrocyte expansion and differentiation, similar studies were conducted using adult human chondrocytes taken from knee arthroplasty off-cuts, with analysis of select genes involved in the chondrogenic program analyzed by q-PCR Results Cellular morphology was improved in hypoxic culture, with a markedly more fibroblastic appearance seen after greater than 2 passages in 21% O2. Micromass cultures maintained in hypoxic conditions demonstrated stronger staining with Alcian blue, indicating stronger expression of cartilaginous glycosaminoglycans. Collagen type II mRNA was twofold higher in monolayer cultures expanded at 5% compared to expansion at 21% O2. Micromass cultures grown at 21% O2 showed up to a twofold increase in the tissue content of glycosaminoglycans when formed with cells expanded at 5% instead of 21% O2. However, no differences in the levels of transcripts and in the staining for collagen type II protein were observed in these micromass cultures. Hypoxia (5% O2) applied during micromass cultures gave rise to tissues with low contents of glycosaminoglycans only. Conclusions In vivo, the chondrocytes are adapted to an avascular hypoxic environment. Accordingly, applying 5% O2 in the expansion phase in the course of cell-based cartilage repair strategies may more closely mimic the normal chondrocyte microenvironment and may result in a repair tissue with higher quality by increasing the content of glycosaminoglycans., Introduction To engineer articular cartilage tissue that more closely resembles the organization of the native tissue, several tissue engineering approaches utilize chondrocytes from the different zones of cartilage. However, controversy remains about the existence of inherent differences between these cell populations. Hence, we investigated zone-related differences between chondrocytes from the superficial, middle and deep zones, during in vitro expansion and subsequent redifferentiation. Methods and Materials Equine articular chondrocytes from the three zones were expanded in monolayer cultures (8 donors). Subsequently, cells were redifferentiated in pellet and alginate bead cultures for up to 4 weeks. GAG and DNA were quantified, and immunohistochemistry was performed to assess expression of various (zonal) markers, including various collagens, COMP and clusterin. Results Cell yield varied between zones, but proliferation rates did not show significant differences. Staining for collagens II, VI and IX, clusterin and COMP was lost after expansion in all cultures, but gradually re-appeared during redifferentiation. Staining for collagen type I was found in all cultures after 4 weeks. Interestingly, COM P and collagen IX were detected mainly in deep and middle zone cultures, whilst clusterin re-appeared in particular in superficial zone cultures. In pellet cultures we observed more intense safranin-O staining in pellets with deep-zone cells, a trend confirmed by a quantitative GAG assay. Conclusions Differences exist indeed between cells from the different zones. Furthermore, differences can be maintained or re-induced after in vitro culture. Appreciation of these zonal differences could lead to important advances in cartilage tissue engineering., Introduction We aimed our study at characterizing the post-expansion cartilage-forming capacity of chondrocytes harvested from the detached fragments of osteochondral lesions of ankle joints (Damaged Ankle Cartilage Fragments, DACF). Chondrocytes derived from normal ankle cartilage (NAC) were used as control cells. Methods and Materials DACF were obtained from 6 patients (mean age: 35years) with osteochondral lesions of the talus, while NAC from 10 autopsies (mean age: 55years). Chondrocytes isolated from the tissues were expanded for two passages and then cultured in Hyaff®-11 meshes (FAB, Italy) for 14 or 28days. Resulting tissues were assessed histologically, biochemically (glycosaminoglycan-GAG-, DNA and collagenI I) and biomechanically. Results DACF contained significant lower amounts of DNA (3.0-fold), GAG (5.3-fold) and collagen II (1.5-fold) and higher amounts of collagen I (6.2-fold) as compared to NAC. Following 14 days of culture in Hyaff®-11, tissues generated by both cell sources were faintly stained for Safranin-O, contained similar amounts of GAG and collagenI I and had similar biomechanical properties. After 28 days of culture, tissues generated by NAC chondrocytes were more intensely stained for Safranin-O and collagenI I, contained higher amounts of GAG (1.9-fold) and collagen II (1.4-fold) and exhibited superior biomechanical properties (1.7-fold and 3.3-fold equilibrium modulus and dynamic pulsatile modulus, respectively). Conclusions We showed for the first time that DACF-chondrocytes have inferior cartilage forming capacity as compared to NAC-chondrocytes, possibly resulting from environmental changes occurring during and after trauma/disease. The study opens some reservations on the use of DACF-derived cells for the repair of ankle cartilage defects, especially in the context of tissue engineering-based approaches., Introduction While intra-articular analgesia is generally effective for pain management during arthroscopic surgery, infusion of amino amides (i.e. bupivacaine) can lead to chondrolysis. The present study evaluates effects of a potential alternative, highly purified capsaicin, on glenohumeral articular cartilage using a rabbit rotator cuff repair model. Methods and Materials Six New Zealand White rabbits underwent a unilateral supraspinatus transection and repair with a single injection of capsaicin into the glenohumeral joint (GHJ). Animals were euthanized at one week and humeral head articular cartilage harvested for biochemical (proteoglycan synthesis and content), cell viability and histological assays. Results Cartilage wet weight, total proteoglycan content (matrix and media combined) and percentage of viable cells was similar (p⇢.05) between treated and untreated shoulders. Chondrocyte proteoglycan synthesis for capsaicin-treated shoulders was 147±45% (p⇠0.05) of that for contralateral, untreated joints. The histopathological score (out of 20) for the shoulders receiving the capsaicin drug was 6.7±1.5 compared to 6.3±1.9 for the unoperated shoulders (p⇢0.3). Conclusions One week following surgery, the cell viability and proteoglycan content of capsaicin-treated shoulders were similar to untreated controls. However, the metabolic activity (proteoglycan synthesis) of the chondrocytes from the treated shoulders was significantly elevated suggesting perhaps a direct effect of the drug or a manifestation of post-surgical inflammatory responses. In contrast to previous reports of bupivacaine infusion in rabbit shoulders, the current results indicate that a single injection of highly purified capsaicin into the GHJ does not induce a deleterious response with regard to matrix metabolism and cell viability of glenohumeral cartilage in the early post-operative period., Introduction Therapies based on in vitro expanded primary cells are more frequently used for tissue regeneration. To proof that the cells stay at the site of implantation, it is necessary to be able to trace the cells in vivo. By iron oxide-labeling the cells can be detected by MRI in live organisms. Methods and Materials Chondrocytes were isolated from biopsies and incubated in growth medium with either 0, 25, 50 or 100 μg/ml very small iron oxide particles (VSOPs) for 90 minutes at 37°C, 5% CO2. Verification of VSOP uptake was performed by Prussian blue staining and the impact of VSOP-labeling on cell proliferation was investigated by cell count after 24, 48 and 72 hours of culture. Furthermore, VSOP-labeled (50 μg/ml) chondrocytes were embedded in alginate beads in three groups with concentrations of 2×106, 5.0×106, or 8.0×106 cells/mL These groups of beads and a negative control of unlabeled cells were placed in growth medium and were scanned with 1.5T MRI using a 5 cm diameter coil. A spin echo sequence with TR=1000 ms, TE=10 ms, FOV=70×70 mm, slice thickness of 3 mm, and a total scan time of 1:56 minutes was used. Results Iron oxide-labeling of human chondrocytes was verified by Prussian blue staining. The proliferation of cells was unaffected by VSOP-labeling. An immediate recognition of the three different cell concentrations was achievable from the images themselves. Quantification of the depression of signal is pending. Conclusions Iron oxide-labeling of chondrocytes was achieved, enabling in vivo detection of cells after implantation., Introduction Platelet-rich plasma (PRP) is an osteoinductive therapeutic approach that is used in treatment of bone and cartilage healing processes. The purpose of this study was to investigate the effect of PRP on human chondrocytes. Methods and Materials Human articular chondrocytes were collected from young patients, single chondrocytes obtained by enzymatic digestion were polled and expanded in vitro for several weeks in Ham's F12 medium plus 10% FBS or in Serum Free Medium or in 10% PL (platelet lysate). In parallel, we performed in vitro pellet culture, to test the capability to maintain chondrogenic potential. Proliferation and differentiation analysis was performed at different time during the in vitro expansion. To evaluate the rate of cell proliferation, at regular intervals the cells were counted. The number of cell duplications was calculated as ratio to starting value. Cell viability was assessed by MTT analysis. To test the ability to maintain cartilage phenotype, chondrocyte expanded in monolayer, was successive cultured in pellet culture. Results Preliminary results, shown that cells proliferation and number of cell doublings were drastically enhanced in cultures supplemented with (PL) compared to cultures performed in FBS or in Serum Free. Histological analysis shown that chondrogenic differentiation was maintained. Biochemical and molecular analyses are in progress to demonstrate that PRP did not markedly affect the chondrogenic potential and the cells remained phenotypically stable. Conclusions PRP may be useful to stimulate the proliferation of human chondrocytes cells. PRP is an effective substitute for FBS to support in vitro expansion of human cells and subsequent tissue-engineering applications., Introduction ACI and MACI® implants have been shown to be effective treatments for the repair of articular cartilage defects. Dell'Accio have reported gene markers that correlated to chondrogenic potential of an ACI product in a nude mouse implantation model1. This study evaluates expression of these molecular markers and a novel marker for their ability to predict chondrogenic potential of MACI® implants. Methods and Materials Gene expression was measured by qPCR in expanded human chondrocytes and chondrocytes that had re-differentiated after long-term culture of the MACI® implant. Human dermal fibroblasts were used as the negative control. The genes monitored were COL2A, FGFR3, BMP2, ALK1 and Hyaline1. Results PCR data indicated that expression of COL2A1, FGFR3, BMP2 and ALK1 did not differ significantly between expanded chondrocytes and fibroblasts. Hyaline1 levels were 121-fold higher in chondrocytes versus fibroblasts. After 28 days of chondrogenic differentiation culture, expression of COL2A1 increased more than 162-fold in chondrocytes, but decreased 30% indermal fibroblasts. These data suggest that the chondrocytes began the process of chondrogenesis while the dermal fibroblasts were non-chondrogenic. Conclusions Previous reports found that expression of COL2A1, FGFR3, BMP2 and ALK1 were predictive of chondrogenesis in a nude mouse intramuscular implantation assay.1 Although we found that COL2A1 levels were much higher in chondrocytes, these results were not statistically significant due to large variation between strains. While the levels of FGFR3, BMP2, and ALK1 were not associated with MACI® implant re-differentiation, a strong association was observed with Hyaline1 expression. The results of this study suggest Hyaline1 as a predictor of MACI product potency., Introduction Children Salter's type III and IV growth plate injuries always induce the skeletal deformity because of bony bridge formation. The purpose of this investigation was to understand the molecular mechanisms of bony bridge formation. Methods and Materials The growth plate injuries of 24 male 6-week-old Swiss Webster mice were generated in the left proximal tibias by drill-hole approach. On days 1, 3, 7, 10, 14, 28 post-surgery, groups of mice (n=4) were killed for specimen collection and further analyses. Results Consisted with the histological changes, both Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay and in situ hybridization experiment using Col2a1 probe showed that there was the sub-injury cartilage region adjacent to the original injury site. The chondrocytes within this region showed the dislocation with the cartilage lacunas and these chondrocytes didn't express Col2a1 mRNA, further confirming they were dead cells. Along with the degradation of sub-injury cartilage, some fibroblast-like cells presented to the cartilaginous region between the sub-injury region and uninjured cartilage. In situ hybridization experiment for Patched 1 (Ptch1), indicator of Indian Hedgehog (IHH) signaling, indicated these fibroblast-like cells could respond to Hh signaling. Conclusions These results suggest that the bony bridge formation involves series of changes of chondrocytes and Ihh signaling may be involved in the formation of the transient perichondrium-like structure between sub-injury cartilage and normal cartilage, and partially contribute to the bony bridge formation. Investigating the underlying cellular and molecular changes after the transphyseal injury will contribute us to explore a prevention treatment in the future clinic., Introduction To investigate the apparent fibroblastic shift in tissue specific DNA methylation patterns observed during chondrocyte monolayer expansion. Methods and Materials The upper and lower zones from full thickness articular cartilage slices were sampled. Chondrocytes were cultured for up to 5 passages in unsupplemented and growth factor supplemented media. Analysis of DNA methylation in articular cartilage, clonal cultures, and polyclonal cultures was performed. Polyclonal cultures were also analyzed for gene expression. Results The DNA methylation patterns of both clonal and polyclonal cultures were observed to shift slightly, but significantly, to a more fibroblastic profile during monolayer expansion in all media. While gene expression levels showed significant variability, no significant differences were observed in the DNA methylation levels between media conditions. High interclonal variability in methylation levels of FGFR1, KRT8, and ROPN1L was observed, while HIF1a, FMOD, and C15orf27 methylation levels were characteristically chondrocytic in most clonal cultures. Analysis of articular cartilage revealed that the upper zone displayed a slightly more fibroblastic methylation pattern than the lower zone, with FMOD, FGFR1, KRT8, and ROPN1L genes showing significant differences between zones. Conclusions Articular cartilage appears to harbor a homogeneous population of HIF1a/FMOD/C15orf27 undermethylated cells, and subpopulations with variable FGFR1, KRT8, and ROPN1L methylation. Chondrocytes taken from the lower zone possessed a more chondrocytic DNA methylation profile than those from the upper zone. The shift in DNA methylation levels during monolayer expansion was neither random nor global, but directed to a more fibroblastic profile in a gene specific manner., Introduction Recent clinical and basic science investigations have revealed chondrotoxicity of local anesthetics, especially those containing epinephrine, administered via intra-articular pain pump. However, exact mechanism of toxicity is unknown. This study evaluates the chondrotoxicity of pH, epinephrine and preservatives found in commonly used local anesthetics. Methods and Materials Human chondrocytes were harvested and cultured in a custom bioreactor designed to mimic metabolism of medication. Pain pumps were used to infuse one of the following medications into the culture system: control media, media titrated to pH 4.5, media with 1:100,000 or 1:200,000 epinephrine only, media with the preservative sodium meta-bisulfte only, 0.25% bupivacaine, 0.25% bupivacaine with epinephrine, 1% lidocaine, 1% lidocaine with epinephrine. Cultures were perfused for 24 and 48 hours, stained with Live/Dead Cell Vitality Assay, examined by fluorescence microscopy, counted, and the percentage of cell death was calculated. Results Cultures containing media titrated to pH 4.5, the preservative sodium meta-bisulfte (found only in anesthetic medications containing epinephrine) and all medications containing epinephrine had high cell death rates compared to controls at all time points (p⇠0.001), while cultures containing 1:100,000 and 1:200,000 epinephrine had no increase death rate. The percentage cell death was not significant for 1% lidocaine (12.5%) and 0.25% bupivacaine (16.5%) at 24 hours, but became significant at 48 hours (61.5% and 55.6%, respectively). Conclusions The marked chondrotoxicity of local anesthetics containing epinephrine appears to be due to the preservative sodium meta-bisulfite and low pH, as these medications are titrated to pH 4.5–5.5 for product stability. 1% lidocaine and 0.25% bupivicaine were chondrotoxic if used for greater than 24 hours. Caution should be exercised when using intra-articular pain pumps, especially for greater than 24 hours., Introduction Chondrocyte culture is a standard technique used for investigation and treatment of articular cartilage defects. Surprisingly, numerical description of cell multiplication during chondrocyte culture has not been documented. Documentation of cell multiplication rate is important to help standardize culture techniques so results can be comparable across different laboratories. The objective of this experiment was to numerically describe chondrocyte counts during cell culture from initial plating until maximal confluence. Methods and Materials Articular cartilage of Outerbridge 0 and 1 was collected from three patients undergoing total knee arthroplasty (age 46–69 years). The cells were isolated and plated in chondrocyte growth medium until confluence (P0). The cells were trypsinized and plated at 20% confluence and allowed to grow until maximal confluence. Images were taken from two standardized points in each plate at daily intervals, cells were manually counted and cell densities calculated. Results Initial cell density (approximately 27 cells/mm2) rose gradually for the first five days. Thereafter, there was an exponential increase in cell density that plateaued at 10 days at approximately 1674 cells/mm2. There was no statistically significant difference in the rate of increase in cell density between the three donors. Conclusions This is the first study that documents the numerical description of chondrocyte multiplication during cell culture and can be used to accurately define levels of confluence so future experiments can be compared more precisely. This data was obtained from articular cartilage retrieved from patients undergoing knee arthroplasty and the rate of replication may not be extrapolated to cells taken from normal knees., Introduction The state-of-the-art in cartilage tissue engineering techniques is autologous chondrocyte transplantation. It employs autologous chondrocytes cultivated in monolayer culture, which enhances proliferation compared to the native cells with a limited division capacity. The disadvantage of this cultivation is the alteration to a fibroblast-like appearance with the time accompanied by an alteration in extracellular matrix. This dedifferentiation process is as well characterized by a dramatic decrease of BMP-7 mRNA transcription. Therefore, we investigated the involvement of BMP-7 in the dedifferentiation process. Methods and Materials To maintain, respectively enhance, the BMP-7 production in human monolayer chondrocytes we chose to transfect a constructed plasmid, encoding the human BMP-7 cDNA controlled by a CMV promoter, into the cells by magnetofection and compared it with the application of recombinant protein. For the determination of differentiation state, the ratio of collagen type II and type I expression was determined. Furthermore, the population multiplication was calculated. Results The results revealed a 10-fold increase of the differentiation index by transfecting 0.5μg plasmid per 100.000 cells and 38-fold with 2.0μg after 14 days monolayer cultivation. The retention of the chondrogenic differentiation was accompanied by a dose-dependent decrease of proliferation, resulting in 68%, respectively 48%, population multiplication in ratio to the control. Recombinant BMP-7 showed different impact on the cells than the overexpressed protein. It accelerated the dedifferentiation process in low concentrations and less in higher compared to the untreated cells. Interestingly, the proliferation was reduced as well in dose dependent manner. Conclusions BMP-7 obviously plays a role in the dedifferentiation process of monolayer chondrocytes., Introduction The durability of cartilage repair tissue depends on good bone-cartilage repair tissue integration, but little is known about what controls integrated repair. During remodeling, osteoclasts attract and promote adhesion of pluripotential stem cells (pro-osteoblasts) to bone. We tested the hypothesis that treatment of microdrilled cartilage defects with chitosan-stabilized clot implant elicits more osteoclasts and a more integrated repair, compared to drilling alone. Methods and Materials Microdrilled bilateral full-thickness cartilage trochlear defects were created in skeletally mature rabbits, and treated or not with chitosan-glycerol phosphate/blood implant. After 1 (N=4), 2 (N=4), or 8 weeks (N=7), histological sections from between, the edge, and through the drill holes of the defect were used to quantify osteoclast density (Tartrate Resistant Acid Phosphatase enzymatic activity) and repair tissue histomorphometric parameters (Safranin O stain). Intact femurs (N=8) served as controls. Results Osteoclasts accumulated at the edges of the drill holes after 1 week, at which time osteoclast activity was stimulated 2.5 to 4-fold by chitosan-GP/blood implants, throughout the subchondral trabecular bone. After 8 weeks of repair, osteoclast density subsided below all defects and was limited to the repairing bone plate. At 8 weeks defects treated with implant, compared to drilling alone, demonstrated more complete trabecular bone repair of the drill holes (p⇠0.05), and a repair tissue that was significantly more integrated with the defect base (∼5% vs ∼37% detached repair, p⇠0.001). Conclusions Chitosan-GP/blood implants elicit an acute influx of osteoclasts following marrow stimulation without inducing bone resorption. Rapid remodeling of drilled subchondral bone was associated with signficantly more repair cartilage-bone integration., Introduction Collagen structure in human osteochondral biopsies was assessed by scanning electron microscopy (SEM). Qualitative scales for orientation and density of collagen in different zones and quantitative measurements of fibre diameter were applied and statistically analysed. Methods and Materials Human osteochondral biopsies (N=10) containing normal, degraded or repair cartilage were embedded in paraffin, sectioned, and post-fixed prior to gold coating and SEM imaging. Images were taken in superficial, transitional, and deep zones of each specimen and were scored twice by 3 readers for collagen orientation and density using an ordinal scale of 0, 1, or 2. Inter-reader and intra-reader reliability were analysed using the Intraclass Correlation Coefficient for agreement, ICC(2,1). Fibre diameters were measured. Results Normal human cartilage displayed the expected zonal dependent collagen orientation with fibre diameters in the range 50–140nm. Degraded tissue revealed less evident vertical orientation in the deep zone. Repair tissue arising from bone marrow frequently contained vertically oriented fibers in the deep zone, tangentially oriented fibres in the superficial zone, and an abundance of thinner fibers of ∼50 nm in diameter. The inter-reader and intra-reader reliability (N=10) were 0.84 and 0.92 for the orientation score and 0.86 and 0.95 for the density score, indicating excellent inter-reader and intra-reader agreement. Conclusions Novel methods to characterize collagen structure in biopsies of cartilage repair tissue have been developed using SEM. Qualitative scoring of zonal dependent collagen orientation and density displayed good reproducibility between readers and will be useful in assessing the quality of tissue produced by different cartilage repair procedures., Introduction Under suitable culture conditions, human bone marrow stem cells (hMSCs) can produce a cartilage-like tissue with a matrix based on type II collagen. To what extent the fibrillar matrix is normal, in terms of the assembly and cross-linking of type II collagen into heterofibrils, is not characterized. This study investigated the ability of hMSCs undergoing chondrogenesis to assemble a type II collagen-based cross-linked network characteristic of developing cartilage. Methods and Materials hMSCs were isolated, expanded, centrifuged into transwell filter units and maintained for 7, 14, 28 days as we have published. Neo-cartilage samples were assayed for collagen and hydroxylysyl pyridinoline (HP) cross-link content with human fetal cartilage as a control. Mass spectrometry identified collagen chains. Results A progressive increase in collagen content from week 1 to week 4 was observed. The collagen content (1% of wet wt.) by week 4 compares well with human fetal cartilage (3% of wet wt. at 15 weeks in utero). High levels of HP cross-links are a characteristic of cartilage collagen. At 1 wk HP cross-links were barely detectable. At 2 weeks the HP content of the neo-cartilage (0.23 moles/mole collagen) approached that of human fetal cartilage (0.23) and was maintained over the next 14 days. Conclusions The neo-cartilage collagen framework showed a high and stable cross-link content by 14 days. Since only a modest wet weight gain between 14 and 28 days was observed despite a 95% increase in proteoglycan content, this strongly suggested that the cross-linked collagen framework restricted proteoglycan-driven swelling and limited further gain in wet weight., Introduction Cartilage Oligomeric Matrix Protein (COMP) is a protein present in the cartilage matrix and is expressed more abundantly in OA cartilage than in healthy cartilage. The present study was designed to investigate the effect of growth factors on COMP deposition and the influence of COMP on collagen biochemistry. Methods and Materials Bovine chondrocytes in alginate beads were cultured with or without 25 ng/ml IGF1, TGFβ2 or FGF2. Human COMP (hCOMP) was overexpressed in bovine chondrocytes using lentiviral transfection. COMP gene expression, COMP protein production, collagen and proteoglycan deposition, and collagen fibril thickness were determined. Results Addition of TGFβ2 resulted in more COMP mRNA and protein than the control condition without growth factors or with addition of IGF1. FGF2 resulted in less and partially degraded COMP. Lentiviral transduction with hCOMP resulted in elevated gene expression of hCOMP and increased COMP levels in the alginate bead and culture medium compared to untransfected cells. Overexpression of COMP did not affect the deposition of collagen, collagen crosslinking, proteoglycan deposition or the mechanical properties. Stimulating COMP production by either TGFβ2 or lentivirus resulted in collagen fibrils with a smaller diameter. Conclusions Taken together, COMP deposition can be modulated in cartilage matrix production by addition of growth factors or by overexpression of COMP. Inducing COMP protein expression resulted in collagen fibrils with a smaller diameter. Since it has been demonstrated that the collagen fibril diameter is associated with mechanical functioning of the matrix, modulating COMP levels could therefore contribute to successful cartilage regeneration strategies., Introduction The aim of this study is to prospectively examine the effect of intraarticular ascorbate injections on the healing process of acute articular osteochondral defects. As the articular cartilage tissue is hypocellular, avascular, alymphatic and aneural; it does not contain mesenchymal stem cells thus has a minimal repair potential. Besides present treatment protocols have a poor prognostic value in the long term outcome of osteochondral defects and are very expensive. Methods and Materials After creating full thickness, 2 mm wide osteochondral defects bilaterally in the knee joints of 48 Sprague Dawley male rats, a 5% concentration of ascorbate solution was administered intraarticularly every 3 days in 3 weeks' time into one joint and the contralateral knee was used as a control with saline solution injected. Articular cartilage regeneration was immunologically evaluated with TSP-1 and COMP elisa assays; histopathologically with TGF-Beta-1 and Aggrecan immunohistochemistry, toluidin-blue and hematoxylene-eosin staining; and biochemically the ration of hydroxyproline/proline and hydroxylysine/lysine has been determined with the HPLC method. For the pathological grading, the modified OARSI system was used. Results When compared to saline injections, the results show that ascorbate has slightly enhanced cartilage healing with TSP-1 (p=0.007), aggrecan (p=0.249) and TGF-Beta-1 (p=0.036) expressions upregulated; HP/P and HL/L (p=0.031) ratios increased and finally COMP (p=0.006) concentrations decreased distinctively. Conclusions In conclusion, ascorbate positively influenced the articular cartilage molecular biology during the healing process whereas it did not affect the pathological grading in the early onset., Introduction IL-1ra blocks IL-1 function and decreases pain in patients with OA (Yang et al., 2008). Autologous protein serum (APS) rich in IL-1ra can be collected from blood within 30 minutes. However, it is unknown if sustained delivery from a carrier increases the residence time of IL-1ra in the joint space, thereby increasing the potential therapeutic effect. The purpose of this study is to determine if a plasma matrix alters the release of IL-1ra. Methods and Materials From 5 consented human donors, 120cc of anticoagulated blood was collected. Platelet-rich plasma (PRP) was prepared using GPS®III disposables (Biomet Biologics). PRP was loaded into modified plasma concentration devices (Plasmax®device, Biomet Biologics) and processed. The output was divided into 4 groups; IL-1ra in concentrated plasma with and without thrombin activation (1000U/ml in 1MCaCl2), or cell-free IL-1ra with and without thrombin activation. I L-1ra was measured using ELISA (R&d Systems) over time. Results Unclotted APS produced an average of 47.1±2.1ng over 24hrs (p=0.34). The cell-free samples produced 33.7±1.5ng without changing over 24hrs(p=0.38). Once clotted, the elution of IL-1ra was slowed, with only 28% being eluted after 10 hours. Release in the cell-free samples was also delayed, but eluted 100% of available IL-1ra after 10 hours. Conclusions The plasma matrix delays IL-1ra release, is biocompatible, resorbable, and autologous, making it an appealing carrier selection. While cell-free samples do contain and elute IL-1ra, more IL-1ra is obtainable in the APS containing cells. Further work will determine if delayed release of I L-1ra will translate into improved pain relief for patients suffering from OA., Introduction Pro-inflammatory cytokines have been linked to cartilage destruction and chondrocyte death in OA. In many cells pro-inflammatory cytokines may cause mitochondrial dysfunction/ apoptosis by increasing reactive oxygen/ nitrogen species. The purpose of present study was to investigate if mitochondrial dysfunction/damage are involved in human chondrocyte responses to IL-1β and TNF-α. Methods and Materials Primary chondrocyte cultures were generated from OA patients undergoing TKA. Cells were treated with IL-1β and TNF-α for 24, 48, 72 hrs. Total DNA was isolated and subjected to quantitative Southern blot analysis to study mitochondrial DNA (mtDNA) damage. ATP levels were measured by ATP bioluminescence kit. Mitochondrial protein levels were evaluated by Western blot analysis. Apoptosis was measured by flow cytometry, DAPI staining, Western blot analysis was performed to study cytochrome c release and activation of caspases 3 and 9. Results Following 24 hr of exposure, both TNF-α and IL-1β induced dose-dependent mtDNA damage. ATP levels were decreased 34%, 57%, 43%, and 66% for the same doses of cytokines compare to untreated controls. Mitochondrially-encoded subunit I of cytochrome oxidase levels were down-regulated following cytokine exposure. Mitochondrial dysfunction correlated with apoptosis appearance. 10 μg of I L-1β did not cause apoptosis after 72 hrs, 50 μg of I L-1β caused 35% apoptosis, 50 μg of TNF-α caused 44% apoptosis, and 100 μg of TNF-α induced 59% apoptosis. Pro-inflammatory cytokine exposure induced cytochrome c release from mitochondria into cytosol and both caspase 3 and 9 cleavage. Conclusions In conclusion, pro-inflammatory cytokines may cause chondrocyte death by induction of mitochondrial dysfunction/ damage, and activation of mitochondrial led apoptosis., Introduction In vitro investigation of chondrocytes in hypoxic environment has been increasingly studied in cartilage research. Quantitative RT-PCR is a powerful approach used in a wide range of scientific methods, with the use of stable reference genes - house keeping genes (HKG). Stable HKGs are one of the hallmarks of reliable results using RT-PCR. The aim of this study was to determine optimal HKGs in human chondrocyte cultures in both normoxia and hypoxia environment. Methods and Materials Cartilage biopsies were obtained from six healthy patients after written consent. After preparation and isolation, the chondrocytes were divided into two groups and grown in 21% and 1% oxygen respectively. RNA was isolated from the cells at baseline, and then from the two groups after 1, 2, and 6 days. We assed the gene expression at these time-points of 8 HKGs: GAPDH, 18s, B2M, RPL13A, beta-actin, RPII, TBP, UBC, and HPRT1. Results The inter-individual coefficient of variance (CV%) between the patients was lowest for HPII (1.7) and UBC (2.1). The intra-individual CV% between the groups and time-points was lowest for HPII (1.2) followed by RPL13A (1.3), B2M (1.4), and HPRT1 (1.5). The total CV% was lowest for RPII (1.7). Commonly used HKGs such as 18s, GAPDH and beta-actin had a total CV% of 5.6, 4.3, and 5.5 respectively. Conclusions We revealed the importance of selecting H KGs according to the study setup. RPII was the most stable in all conditions while commonly used HKGs such as 18s and beta-actin were the most unstable., Introduction To understand repair effects of anti-VEGF combined with fibrin graft interposition with a proximal tibia transphyseal injury murine model, and to assess the reliability of treatment to physeal injury with the compound. Methods and Materials The growth plate injury model was created in 48 skeletally immature rats. Consisted with the histological changes, and in situ hybridization experiment using VEGF-Flt probe were used to study the bony bridge formation, expressing of VEGF and the changes of chondrocytes. The length and metaphyseal-diaphyseal angle of the grafted tibia were compared. Micro-CT assessment and histological staining were used to compare the bony bridge formation under different interventions. Results At injury site, VEGF signaling appeared on day 10 and increased as time went by. VEGF signaling may be involved in the formation of the bony bridge. The deformity angle and medial length of the tibia were significantly different between grafted tibias and non-grafted tibias at 4, 16 and 24 weeks postoperatively (p⇠0.01). There was no significant difference between compound-graft and fat-graft tibias (p⇢.05). There was no significant difference of the Bone Mineral Density (BMD) between anti-VEGF-Flt grafted group or FS combined with anti-VEGF-Flt grafted group and fibrin-graft or fat-graft tibias (p⇢0.05). Conclusions The long-term prevention effects of compound-graft was significantly better than that of others including fibrin-graft and anti-VEGF-Flt graft group, and little better than that of fat-graft group. Fibrin and anti-VEGF-Flt compound graft interposition would play a role in reducing growth disturbance in growth plate injury., Introduction Recombinant human PDGF-BB is both chemotactic and mitogenic for cells of mesenchymal origin (including chondrocytes) and has the potential to enhance cartilage tissue healing in osteochondral defects. Biocompatible scaffolds, combined with growth factors such as rhPDGF-BB, can help to stimulate and guide the regeneration and repair of cartilaginous tissues. Scaffolds composed of collagen have been developed for use in cartilage repair procedures. The current study was performed to evaluate the stability and release of rhPDGF-BB from a regionally specific, collagen/glycosaminoglycan/calcium phosphate plug. Methods and Materials A volume of 450μl rhPDGF-BB (1.0 mg/ml) was combined with the 8.5mm × 8mm plug. The rhPDGF-BB was eluted from the scaffold using elution buffer of varying salt concentrations. Size exclusion high performance liquid chromatography (SEC-HPLC) was performed to evaluate the rhPDGF-BB contained in the eluate. Total rhPDGF-BB released and the binding efficiency to the PDGF-receptor were quantified over a 24 hour time course using an ELISA (PDGF-BB DuoSet, R&d Systems). Results The observed, salt dependent, release profile included an initial bolus release of 51% after ten minutes, followed by a slower phase of release over the remaining 23 hour study period. The cumulative release of rhPDGF-BB was 70–75% after 24 hours. Finally, the PDGF-receptor binding efficiency, as determined by non-linear regression, of the released rhPDGF-BB was equivalent to that observed for the control rhPDGF-BB. Conclusions After 24 hours, a cumulative total of 70–75% of the rhPDGF-BB was recovered from the scaffold and the eluted rhPDGF-BB was biochemically stable. Studies characterizing cellular response are ongoing. Confidential-Review Purposes Only, Introduction The aim of this study was to ascertain whether the addition of thrombin is required to achieve platelet activation and sustained growth factor release in-vitro, when PRP is applied to a collagen based osteochondral scaffold. Methods and Materials Equal combined volumes of test substances were added to collagen/glycosaminoglycan scaffolds (n=3): 500μl PRP; 375μl PRP + 125μl autologous thrombin; 455μl PRP + 45μl bovine thrombin. One ml of DMEM/F12 medium was added to each scaffold and changed completely at 12/24 hours, and 3/10 days, following which release of TGF-β1, PDGF-AB and bFGF were measured using ELISA. Secondly, 500μl of PRP was added to equal sized collagen/ glycosaminoglycan and polylactide co-glycolide scaffolds (n=3) from which only PDGF-AB was assayed. Results A similar cumulative release profile in all growth factors was found over the 10 day period. An increase in growth factor release was seen in the PRP only group at all time points, particularly with PDGF-AB (p⇠0.006). These findings remained apparent when a correction for volume was made (p⇠0.028) suggesting a particular role of collagen in platelet activation. This was shown in the second experiment, in which a significantly increased cumulative volume of PDGF-AB was released from the collagen/glycosaminoglycan scaffold without thrombin activation (p⇠0.04). Conclusions This study shows that collagen is a potent activator of platelets, requiring no further addition of thrombin to achieve satisfactory growth factor release when applied clinically. These results suggest that if PRP is combined with polymer scaffolds, it should be activated with thrombin to achieve optimum growth factor release., Introduction Platelet-rich plasma (PRP) has been reported to stimulate cell proliferation and extracellular matrix synthesis among ligament, tendon and cartilage. We investigated the effect of PRP gel on the metabolism of chondrocytes in alginate beads. Methods and Materials PRP and platelet-poor plasma (PPP) were isolated from Rabbit's blood using platelet concentration system, SymphonyTM (DePuy). Chondrocytes were isolated by enzymatic digestion from the cartilage tissue of rabbits' knee, and cultured in alginate beads at a density of 4×104cell/bead in three different medium PRP and PPP and FBS5% for one week. Concentration of basic growth factors (TGF-β1 and PDGF) were measured using western blotting. DNA, proteoglycan (PG) and collagen content within the beads were measured at day 3, 7. Results Western blotting showed higher concentration of the basic growth factors in the PRP compard with PPP fraction. DNA content at day 3 did not show significant difference among the groups, however at day 7 the DNA content in the PRP group increased significantly. PG content per bead was increased in the PRP group at both time points, however PG per DNA was less in the PRP group at day 7.PRP had no significant on collagen content, however collagen content per DNA was decresead in the PRP group compared with FBS group at both time point. Conclusions PRP has enhancing effect on three-dimensionally cultured chondrocytes concerning cell cloning and total PG production. These results suggests that filling PRP gel into subchondral bone defect followed by chondrocyte implantation could be a new strategy for refractory large osteochondral lesions., Introduction The current study was designed to compare the Mankin (HHGS) and the OARSI Osteoarthritis Cartilage Histopathology Scoring Systems (OARSI) for the purpose of evaluating a new mid-infrared spectroscopic cartilage analysis tool. A good correlation between these scoring systems has been previously demonstrated both between and within scorers (rs⇢.80, p⇠.01). While the HHGS scoring system was reported to be a valid scoring system to differentiate normal versus severe OA cartilage, it is also reported to have low sensitivity to differences in OA severity. The OARSI, although a newer scoring system, was specifically selected due to reported advantages over the HHGS including greater linearity, a wider range of scores for milder cases of OA, and high reliability, reproducibility, and variability. Methods and Materials Punch biopsies and mid-infrared spectra (n=117) were taken from freshly excised tibial plateaus from knee arthroplasty patients. Following histological processing the slides were scored on the both the HHGS scale and OARSI scale by two raters. The scoring results were compared using Pearson's Correlation Coefficient. Results In our set of specimens the proportion of variation in OARSI score that can be accounted for by variation in Mankin Score was 0.49 (Rs = 0.70). Conclusions This good correlation between the scores is expected to form a suitable base for building a predictive model of histology scores from mid-infrared spectra. Further, we expect that greater sensitivity of the OARSI scoring system in the middle-grade OA cases provides a more suitable base for predictive models such as ours for evaluation of promising instruments., Introduction The intervertebral disc (IVD) is a highly avascular structure occupied by specialized nucleus pulposus cells (NPC) that have adapted to survive within an oxygen concentration of between 2 and 5%. Most studies concerning disc biology have utilized in vitro conditions of 21% O2 and are relatively short-term and often use mono-layer culture. Reports concerning the delivery of anabolic/ matrix protective factors to the disc have claimed effective restoration of disc height in vivo and/or increased expression of desirable genes such as aggrecan and collagen II. Nucleus pulposus cells assume a fibroblastic phenotype in monolayer culture-very different from that found in the in vivo setting. Here for the first time we present the effects of long-term hypoxic and normoxic tissue culture of non-chondrodystrophic canine notochordal cells-cells that appear to protect the disc NP from degenerative change. Methods and Materials Non-chondrodystrophic canine notochordal cells were seeded into alginate globules under either hypoxic (3.5% O2) or normoxic (21% O2) conditions using DMEM/F-12 media supplemented with penicillin, streptomycin and fungizone (PSF) and 8% fetal calf seurm (FCS). The cultures were maintained for 5 months following which we used histological, immunohistochemical, scanning electron microscopy and histomorphometric methods to compare differences in extracellular matrix production and viability of the cellular/extracellular matrix. Results Hypoxia induces notochordal cells to produce a highly complex and organized 3D cellular construct rich in aggrecan and collagen II whereas cells cultured under normoxia fail to produce an organized matrix and do not appear viable over long-term tissue culture. Histomorphometric assessment revealed statistically significantly larger cell area under long term hypoxic culture (P=.0001) with the cells retaining an intact cell membrane, nucleus, clear cytoplasm and classic ‘physaliferous’ appearance. Conclusions Hypoxia induces notochordal cells to organize a complex 3D cellular/extracellular matrix without an external scaffold-other than suspension within sodium alginate. These cells produce an extracellular matrix and large construct that shares exactly the same characteristics as the in vivo condition-robust aggrecan and type II collagen production. Normoxic tissue culture conditions lead to a failure of these cells to thrive and a lack of extracellular matrix and significantly smaller cells. It is suggested that future studies of nucleus pulposus and in particular, notochordal cells should be cultured under hypoxia in order to derive meaningful, biologically relevant conclusions concerning possible biological/molecular interventions., Introduction Collagen network structure is the main determinant of cartilage load bearing properties, and can be assessed using Polarized Light Microscopy (PLM). We have developed qualitative and quantitative PLM methods to characterize collagen organization in cartilage biopsies. Methods and Materials Human osteochondral biopsies obtained arthroscopically and equine cartilage were prepared for PLM. Using a newly developed qualitative scoring system, 3 readers rated PLM images on a 0 to 5 ordinal scale describing the range from a totally disorganized tissue (score=0) to ideal hyaline collagen organization (score=5). Inter-reader agreement was analysed using the Intraclass Correlation Coefficient for Agreement (ICC). Quantitative measurements of zonal thicknesses and areas were made by image analysis. Results Normal equine cartilage received perfect hyaline scores of 5, having 2 uniform birefringent zones, representing superficial and deep zones, separated by a non-birefringent transitional zone; proportions of total thickness for deep, transitional and superficial zones were ∼89%, ∼7.5% and ∼3.5%. Normal human cartilage received a score of 4 due to non-uniform zonal interfaces and irregular birefringence patterns; zone proportions were ∼68%, ∼25% and ∼7%. Repair cartilage scores ranged from 0 to 3, reflecting variable levels of collagen orientation achieved with different marrow stimulation procedures. Samples that scored 3 contained a vertically oriented deep zone, evidence of a transitional zone, and a superficial zone that had a predominantly horizontal orientation. The ICC for the qualitative score was 0.89 indicating excellent inter-reader reproducibility. Conclusions A novel method to assess global collagen orientation in osteochondral biopsies has been developed, providing one means to assess cartilage repair tissue quality., Introduction Many cartilage repair procedures rely on subchondral bone marrow to synthesize repair tissue. Since these repair processes also involve bone remodeling and new bone synthesis, it is important to characterize subchondral bone structure in biopsies of cartilage repair. Here we developed a method to measure pore volume density (Vv) and pore surface area density (Sv) in subchondral bone and evaluated reader agreement. Methods and Materials Paraffin sections from ten human osteochondral biopsies (normal, OA, repair) were stained with Safranin-O/Fast-Green. Bioquant Osteo II was used to obtain Sv and Vv in the bone plate and in deeper regions by tracing the contour of reader-defined zones and the bone-pore interfaces. Three readers performed the analysis twice, and Intraclass Correlation Coefficients for Agreement (ICCs) were calculated to assess intra- and inter-reader agreement. Results The thickness of the subchondral bone plate in normal tissue samples ranged from 0.05 to 0.4 mm. Repair tissue could display a thicker remodeled bone plate, in one case up to 2.7mm. Vv (porosity) in these remodeled bone plate was half that of deeper zones (∼0.32 vs ∼0.71), and the range of Sv (pore surface area) was from 4.0 to 8.6 mm-1. Intra-reader ICCs (⇢0.6) were good while inter-reader ICCs (⇠0.6) were fair. Inter-reader differences were mainly related to the definition and delimitation of the remodeled zone and to artifactual structures caused by imperfect section integrity. Conclusions This novel method provides estimates of subchondral bone structure, Sv and Vv, and the thickness of the remodeled zone in human osteochondral biopsies., Introduction Meniscus tear is a common knee injury, which leads to changes in knee load distribution and degenerative arthritis. In such cases, there is a need to protect the cartilage by replacing the menisci. A floating Polycarbonate-Urethane (PCU) meniscal-implant with internal fibers is proposed for pain relief and improvement of pressure distribution. Our goal was to use finite-element (FE) modeling to develop an implant whose ability to distribute pressure is similar to natural meniscus. Methods and Materials The model geometry was based on MR-scans of a cadaveric specimen and analyzed under load-bearing conditions. Several reinforcement configurations were tested. For each configuration, peak/average tibial-plateau (TP) contact pressures, and peak/average von-Mises and tensile stresses were calculated. The model was validated by comparing computational results to experimental TP contact pressures in cadaveric knees. Results Peak and average TP contact pressures across all simulation cases, were 5.5 and 1MPa, respectively. Similarly, PCU peak and average von-Mises stresses were 4.5 and 0.8MPa, respectively. Focusing on the fibers, peak values were predicted in the superior conduit, where fewer fibers could be located due to practical molding reasons. However, in the case of only three conduits containing nine fibers each, peak tensile stress (444MPa) was reduced by ∼20%. Conclusions To conclude, contact pressures developed on the TP cartilage were comparable to those measured under an intact natural meniscus. Safety-wise, strain/stress values in both the PCU and fibers remained within the allowed limits. We believe that the current device will lead to optimal TP pressure distribution, decrease pressure on the cartilage and reduce pain., Introduction When a meniscal lesion occurs, meniscus cells are generally unable to synthesize a valid reparative tissue, which could withstand physiological forces which the meniscus is subjected to. The aim of the study is to evaluate an in vivo model for the meniscal repair. Methods and Materials Swine menisci and articular chondrocytes were harvested from young pigs. Radial slices of menisci were obtained and regularized. Chondrocytes were suspended in fibrinogen and the resulting suspension was placed over a meniscal slice. Immediately after adding thrombin to the cellular suspension, a second slice was placed over the polymerizing cellular hydrogel to form a tri-phasic sandwich. Samples were embedded in acellular fibrin glue in order to provide mechanical protection and to avoid cellular penetration from mouse, and then implanted in subcutaneous tissue of nude mice for 4 weeks. As controls, tri-phasic sandwiches were prepared with acellular fibrin glue between the meniscal samples. All samples were assessed grossly and histologically. Results Experimental samples demonstrated a gross bonding at probing with a pair of forceps while none of control samples showed signs of adhesion between the meniscal slices. Histology analysis demonstrated a continuous hypercellular fibro-cartilaginous tissue at the interface between the two meniscal slices. Moreover, some penetration buds are evident from the cellular fibrin glue to the meniscal tissue. Conclusions The results obtained suggest that cellular fibrin glue has a potential to improve repair of meniscal tears. Further orthotopic in vivo studies are needed to asses the potential for a clinical application of this method., Introduction Arthroscopic partial meniscectomy is a commonly performed procedure to alleviate pain, swelling and mechanical symptoms associated with unstable meniscal tears. Some tears, such as the unstable flap tear and the unstable vertical tear, do not propagate through the vascular zone, and partial meniscectomy is performed. Many chronic meniscal tears, if unstable non-displaced, have pristine cartilage adjacent, and second look arthroscopy after partial meniscectomy reveals varying degrees of chondromalacia in the adjacent cartilage. Methods and Materials In initial testing, eight knees were randomized to two groups. The first group received non-displaced vertical tears of 2 centimeters in length, 3 millimeters from the periphery. The second group received bucket-handle meniscus tears. Knees were loaded in extension with the meniscus intact, with a non-displaced tear, displaced tear (bucket-handle group only) and after partial meniscectomy. The peak contact pressures were compared. Results Initial results showed no significant difference in peak pressures for knees receiving vertical tears between the medial and lateral menisci, or whether the meniscus was intact, torn, or partially meniscectomized. In the bucket-handle tear group, no significant difference was shown between peak pressures in the medial and lateral menisci. However, the peak pressures across the different testing conditions (intact meniscus, non-displaced tear on meniscus, displaced tear on meniscus, partial meniscectomy) were concluded to have a statistically significant difference. Conclusions Partial meniscectomy may negatively alter contact pressures and increase the risk of degenerative arthritis with tears that have not displaced. Clinicians should counsel patients regarding the risk/benefit ratio as it relates to symptom relief versus degenerative arthrosis., Introduction The term insufficiency fracture or spontaneous osteonecrosis of the knee (SONK) is used for patients who have sudden onset of severe knee pain and focal subchondral bone changes. This condition can also occur as an apparent complication of arthroscopy. We hypothesized that this was not a primary disorder of bone or specific to arthroscopy but due to focal force concentration resulting from meniscal deficiencies. Methods and Materials Preoperative MRI studies were reviewed on six subjects with subchondral insufficiency fractures that develop after arthroscopy. Risk factors for secondary osteonecrosis, mensical pathology, body mass index (BMI), bone mineral density (BMD), and demographics were reviewed. These data were also collected on 32 subchondral insufficiency fracture patients with no pre fracture arthroscopy. The two groups were compared. Results None of the five arthroscopy patients had risk factors for osteonecrosis. Three were male and 3 female. Four had posterior horn degeneration and all six had anterior and/or medial meniscal extrusion on MR images. BMI was 25 to 33 except for one 61. Age range was 43 to 74. Two females had BMD one standard deviation above normal for age and one was below. BMD was not available on the males. This population compares with our 32 patient spontaneous onset population. Conclusions Meniscal extrusion appears to be an important risk factor for the development of post arthroscopy subchondral insufficiency fracture formally called spontaneous osteonecrosis of the knee. Hoop stress competency of the meniscus is important in force concentration and is an important consideration for any articular repair procedure., Introduction Geometrical similarity dictates the degree of conformity between the menisci and the condyles, and it has been shown that even very small changes in allograft meniscal implant sizes demonstrate a dramatic change in stress levels. Methods and Materials A set of 12 geometrical parameters, representing typical lengths and widths of the meniscus and joint bone contours were measured in 130 MRI scans of a mixed population of males and females (62±10 yrs., “The Osteoarthritis Initiative” (OAI) database). Mean values of this data were calculated in order to define the most prevalent (‘reference’) implant size in the general population and a novel Poly-Carbonate-Urethane (PCU) meniscal implant was then created by means of an MRI-based reconstruction of femur and tibia geometrical surfaces of a cadaver knee, which corresponded to the reference size definitions. Experimental and computational (finite element modeling) methods were employed to assess the effect of correct matching between a meniscal implant size and a candidate knee on cartilage pressure distributions. Results Both methods confirmed that a meniscus implant performs equally well in distributing joint compressive loads on the tibial plateau surface in a 5% range around the ‘true’ joint space. The meaning of this being that a relatively lenient safety-range exists for the choice of implant by the surgeon. Conclusions The current findings, together with our statistical analysis of the natural distribution of sizes in the general population, imply that an array of nine implant sizes would adequately accommodate the needs of ∼90% of the population, both female and male., Introduction The objective of this study was to assess the in vivo performance of a biodegradable porous polyurethane scaffold. Our hypothesis was that scaffold implantation would promote tissue ingrowth without detrimentally affecting adjacent articular cartilage. Methods and Materials 50 skeletally mature ewes underwent unilateral partial surgical excision of the lateral meniscus. The defect was left unfilled in 20 animals; in the other 30, the defect was filled with an 80% porous aliphatic polyurethane. Animals were evaluated at 3, 6 and 12 months. Outcome measures included histological appearance of the tibial plateau and femoral condyle and histological assessment of the meniscus. Results There was no difference between groups in the percent fill of the meniscal defect. The tissue that filled the empty defect was generally translucent in appearance whereas the tissue fill with the scaffold tended to be dense and fibrous. At 3 months, 38% of scaffold knees showed no damage on the tibia versus 10% of controls. At 6 months, 50% of scaffold knees showed no damage while all the controls had some damage. At 12 months, 10% of scaffold knees showed no damage. On the femur, 75% of scaffold knees showed no damage at 3 months versus 50% of controls; at 12 months 50% of scaffold knees had no damage versus none of the controls. The scaffold was evident histologically at 12 months, infiltrated with cells and surrounded by abundant matrix. Conclusions Implantation of a polyurethane scaffold promoted the ingrowth of dense fibrous tissue into a meniscal defect without adversely affecting adjacent articular cartilage health., Introduction To determine the mRNA expression levels of selected proinflammatory cytokines and matrix metalloproteinases in synovial fluid (SF) cells from osteoarthritic knee joints compared to healthy controls. Methods and Materials Synovial fluid was obtained from 36 patients undergoing total knee arthroplasty due to symptomatic and radiographic evident osteoarthritis (OA) as well as from 10 healthy controls. Expression levels of TNFalpha, IL1beta, MMP1 and MMP3 were assayed among both groups performing realtime qPCR. For subgroup analysis, patients were configured concerning age, gender and BMI. Results All assayed biochemical markers showed significantly higher expression levels among the OA group compared to control, where these markers were not detectable. Furthermore, strong correlation appeared between expression levels of MMP1 and MMP3 among OA patients (r=0,856) while no correlation was found between age, gender or BMI and the expression levels investigated. Conclusions Expression patterns of the examined cytokines and proteinases among SF cells were significantly elevated in OA patients, while no such expression was detected within healthy appearing joints. Consequently, SF cells expressing cytokines and proteinases, known to be crucial in OA pathophysiology, may play a relevant role in the progression of articular cartilage destruction. Considering the fact that SF in an OA-joint comprehends an abnormal amount of bioactive proteins delivering potential detrimental effects to the articular cartilage, temporary clearance, dilution or suppression by means of arthroscopic lavage or disease-modifying medication may represent a therapeutic tool to constitute an interimistic relief or even postpone disease progression due to a decreased inflammative and degrading activity within the human articular environment, Introduction Several studies have shown that chondrocyte apoptosis is positively associated with degree of cartilage matrix damage. On the other hand, SIRT1 (silent information regulator two ortholog 1) has an important effect on regulation of longevity in association with inhibition of apoptosis. Nevertheless, the role of SIRT1 in human cartilage is unknown. To elucidate the role of SIRT1 in the pathogenesis of OA, we examined the effect of SIRT1 inhibition on gene expression changes in chondrocytes. Methods and Materials Human cartilage samples obtained from femoral condyles of patients undergoing TKA and Normal Human Articular Chondrocytes-knee (NHAC-kn) cells (Cambrex) were used as OA chondrocytes and normal human chondrocytes respectively. We investigated whether SIRT1 is up- or down-regulated in OA chondrocytes. We next examined the effect of SIRT1 inhibition by siRNA and chemical inhibitors to investigate whether SIRT1 modulates chondrocyte gene expression associated with OA. Results The expression of SIRT1 was lower in OA chondrocyte than NHAC-kn cells. The inhibition of SIRT1 by siRNA significantly decreased the expression of COLLAGEN2A1 and AGGRECAN and increased the expression of COLLAGEN10A1 compared with the transfection of control siRNA in NHAC-kn cells. Similarly AGGRECAN was significantly decreased and COLLAGEN10A1 and LEPTIN was increased in OA chondrocytes. Consistently, the treatment of chemical inhibitors also increased the expression of LEPTIN and COLLAGEN10A1 and decreased the expression of AGGRECAN. Conclusions Inhibition of SIRT1 induced OA-like chondrocyte gene expression. Our findings suggest that SIRT1 is necessary for preventing chondrocytes from osteoarthritic change and degeneration and that SIRT1 plays an important role in the pathogenesis of OA., Introduction The articular cartilage in Osteoarthritis (OA) is characterized by chondrocyte hypertrophy, apoptosis and calcification. PiT-1, a novel sodium/phosphate cotransporter, has been implicated in this pathologic process. PiT-1 is overexpressed in OA cartilage. Aim: Evaluate if the synovial fluid (SF) from OA patients induce PiT-1 mRNA and protein expression. Methods and Materials ATDC5 cells were cultured with DMEM/F12, FBS 5%, 10 μg/ml human transferrin and 3X10–8 M sodium selenite. These cultures were considered controls and its results were used to normalize the experimental groups results. 10% of SF from patients with OA or healthy donors was added to the cultures. In order to evaluate PiT-1 mRNA and protein expression, Real Time PCR and Western blots were performed at different times: 0, 0.5, 1, 3 and 7 days. Results Real Time PCR: in cultures exposed to 10% of SF from OA patients, PiT-1 mRNA increased 3.4 and 15.9 times compared to the control at day 3 and 7 respectively. Western Blot: in cultures exposed to 10% of SF from OA patients, PiT-1 protein expression increased 1.25 and 2.5 times compared to the control at day 3 and 7 respectively. No changes were detected in cultures exposed to 10% of SF from healthy donors either in mRNA or western blot. Conclusions The SF from patients with OA induces the PiT-1 expression in ATDC5 cells. Because PiT-1 has been implicated in chondrocyte hypertrophy and apoptosis, this cotransporter could be involved in the OA pathogenesis., Introduction Osteoarthritis affects millions of Americans. Optical Coherence Tomography (OCT) is a novel imaging technology that can detect early degenerative cartilage changes prior to surface breakdown, when changes are potentially reversible. Glycosaminoglycan (GAG), a key component of the cartilage matrix, is upregulated during early degeneration. We hypothesized that OCT changes were predictive of GAG content in human cartilage. Methods and Materials Forty-two osteochondral cores were harvested from central and submeniscal regions of human tibial plateaus. Cores were imaged with an OCT scanner and graded as follows: A–obvious birefringence, B–intermediate birefringence, C–no birefringence, D–irregular surface. GAG content was determined using a dimethylmethlene blue assay. Results Cores with OCT grade B-D had a 29% higher GAG content compared to OCT grade A cores (p⇠0.001). The central region had a 30% higher GAG content compared to the submeniscal region (p=0.039). OCT grade had a similar regional variation with a mean grade of 0.74 in the submeniscal region compared to 2.42 in the central region (p⇠0.001). All of the cores with an OCT grade of A were found in the submeniscal region. Conclusions In this study, GAG was elevated in the central weight bearing region, consistent with studies showing regional GAG differences. GAG was also elevated in cores without clear OCT birefringence. As GAG increases during early cartilage repair, this study suggests that loss of OCT-form birefringence is a marker of early cartilage degeneration even when the surface appears normal. Detection at this potentially reversible stage could lead to new treatments to prevent or delay osteoarthritis., Introduction Post-joint injury arthritis affects millions of Americans. Optical Coherence Tomography (OCT) is a nondestructive imaging technology that detects degenerative cartilage changes prior to articular surface breakdown, when potentially reversible changes are occurring. This study tests the hypothesis that OCT can detect acute cartilage changes after impact injury at levels insufficient to produce visible damage. Methods and Materials Forty osteochondral cores were harvested from fresh porcine knees. Cores were scanned with an OCT scanner and then divided into control, low (0.175 J), intermediate (0.35 J) or high-impact groups (0.7 J). Cores underwent impact injury using a computer-controlled impact tower, followed by repeat imaging. OCT image intensities from pre and post-impact images were compared, and the ratio of superficial and deep layer intensity was obtained and analyzed using custom software (Matlab). No change was seen in controls. Results Cartilage prior to impact exhibited a low intensity (dark) superficial layer, followed by a high intensity (bright) deep layer. The intensity of the deep layer decreased after impact. The OCT signal intensity ratio increased by 14% at 0.175J of impact (p⇠0.001), by 17% at 0.35J of impact (p⇠0.001) and by 54% at 0.7J of impact (p=0.02). Conclusions This study shows that OCT can detect acute cartilage changes after impact injury at levels insufficient to cause visible surface damage, supporting the clinical utility of OCT to detect cartilage damage not visualized during standard arthroscopic examination. The ability to identify early changes at potentially reversible stages suggests that OCT could assist in developing treatments to prevent or delay post-traumatic osteoarthritis., Introduction Main consequence of ACL rupture might be development of early osteoarthritis. ACL reconstruction (ACLR) does not prevent OA. To date no one proved that any form of treatment more effectively prevents its development. Some studies demonstrated that ACLR limits the severity of OA, whereas others suggest that it may be even increased by this surgery. The correlation between conservative treatment and development of OA is also unclear. The aim of this study was to compare development of OA during the long-term follow-up in operated and non-operated patients. Methods and Materials 127 patients with an acute isolated total ACL rupture were followed during a 20 year period. In 66 cases ACLR was performed and in 61 cases patients were treated non-operatively. All patients were examined clinically, radiologicaly and evaluated with use of Lysholm and IKDC scores. Radiological assessment was performed according to Kelgren-Lawrence scale. Results According to Lysholm and IKDC objective score operative group (OP) performed better, but difference was not significant (p⇢0.05). Patients of non-operative (NON OP) group demonstrated significantly better results according to IKDC subjective score (p⇠0.05). Osteoarthritic changes were observed in both groups OP (47%), NON OP (51%). In (OP) group 39% patients developed Grade 2 and more of OA, in NON OP (35%) (p⇢0.05). In both groups more severe changes correlated with further injuries. Conclusions Management of ACL rupture may influence development of OA. However, none of methods (operative, non-operative) more effectively prevents OA and limits its severity. ACL reconstruction may in certain circumstances increase both: appearance and severity of OA., Introduction The goal of this project was to determine if Early Intervention Procedures (EIP), such as a Unicompartmental Knee Replacement (UKR), could be expanded, even at the time of TKA. Methods and Materials 87 bone resection samples with predominantly medial wear were obtained after TKA. The proximal tibia, distal femur and posterior femur were photographed and a visual analysis of the lateral cartilage performed using the grading scale described by Weidow et al., 2002. We also obtained Hematoxylin and Eosin stained sections of the lateral tibia and lateral femur and graded them based on the OARSI scale. The operative report was then reviewed for cases determined to have healthy lateral cartilage. Results The visual and histological analyses revealed 49 cases with healthy lateral cartilage. Of these cases, 23 cases presented with severe PF arthritis, 5 cases with a deficient or absent ACL, 1 case with both a deficient ACL and severe PF arthritis, and 20 cases presented with a damaged medial meniscus. Conclusions The remaining cases with healthy lateral cartilage, intact ACL and PCL, and lack of PF arthritis, represented 24% of the 87 cases studied which could have been treated with an EIP. This percent is potentially even higher if patients were treated earlier. These results suggest that EIPs could be used more frequently, and that more in-depth studies are required to investigate further the diagnosis, indications and types of treatment suitable. In addition, the clinical data suggests the meniscus may play a larger role in the development of osteoarthritis than currently thought., Introduction Increasing findings suggest that adipokines which are adipose-derived proteins, can be involved in cartilage destruction during osteoarthritis (OA). However, contradictory data have been found for the effects of adipokines on cultured chondrocytes. The present study investigated in human OA chondrocytes, the effects of phenotypic instability on the expression of leptin, adiponectin and their receptors. We determined also whether their expression is associated with cartilage-specific markers. Methods and Materials The expression of leptin, adiponectin and their receptors (Ob-R, AdipoR1, AdipoR2), as well as collagens type 1, 2A and 2B, aggrecan, Sox9 and MMP-13 was examined by realtime RT-PCR in chondrocytes obtained from patients with OA either directly after cells harvest or after culture in monolayer or in alginate beads. Results Our results showed that leptin and adiponectin are expressed in freshly isolated OA chondrocytes. AdipoR1 and Ob-R are also found while AdipoR2 is barely detected. Interestingly, AdipoR1 is related to aggrecan, collagen type 2A and Sox9. Beside, mRNA levels for adiponectin and leptin are associated with MMP-13. Major changes in the gene expression pattern occurred after culture in monolayer with a shift from the adipokines to their receptors. By contrast, chondrocytes recovered a cartilage-like expression profile of leptin and adiponectin when cultured in alginate beads, but ceased expressing their receptors. Conclusions In conclusion, this study indicates that experimental conditions are determinant for the expression of adipokines and their receptors. Our data provide also further insights for a potential dualistic role of adipokines in both matrix synthesis and cartilage destruction during OA., Introduction The Acute Anterior Cruciate Ligament Rupture (AACLR) has been described as a high risk factor for osteoarthritis (OA) development. Several studies had linked the inflammation secondary to AACLR as responsible for OA onset. Aim: Evaluate if the synovial fluid (SF) from AACLR induce an OA pattern in ATDC5 cells. Methods and Materials Group 1 (control): ATDC5 cells were cultured with DMEM/F12, FBS 5%, 10 μg/ml human transferrin and 3X10–8 M sodium selenite. These cultures were considered controls and its results were used to normalize the experimental groups results. Group 2: 10% of SF from healthy donors was added to the cultures. Group 3: 10% of SF from patients with AACLR was added to the cultures. In order to assess the OA pattern, we evaluated the phenotypic (nodules apparition) and molecular (collagen II and X) changes by optical microscopy and real time PCR respectively at day 3, 7 and 11 of cultures. Significant differences (p⇠0.05) were determinate by T-student. Results Phenotypic changes: in Group 3, nodules apparition was observed at day 11. This was not observed in Group 1 or 2. Molecular changes: Collagen II: at day 3 the mRNA was in Group 2 1,12 (SD: +0,015) and in Group 3 1,27 (SD: +0,053) times (p⇠0.05). Collagen X: at day 11 the mRNA was in Group 2 1,03 (SD: +0,03) and in Group 3 1,07 (SD: +0,06) (p⇠0.05). No significant differences were observed at others evaluated times Conclusions The SF from patients with AACLR induced an OA pattern in ATDC5 cells. These results suggest that the inflammation secondary to AACLR play a role in the pathogenic mechanism., Introduction Impact injury to articular cartilage can lead to posttraumatic osteoarthritis. This study tested the hypotheses that (1) chondrocyte injury can occur post-impact without visible surface damage, and (2) chondrocyte injury patterns vary with impact energy, time after injury, and cartilage thickness. Methods and Materials 160 fresh bovine osteochondral cores were randomly divided into: (1) Control, (2) 0.35J, (3) 0.71J, (4) 1.07J, (5) 1.43J impact groups and subjected to computer controlled impact loading. Following impact, full-thickness sections were prepared and incubated in DMEM/F12 at 37°C. Contiguous sections were harvested 1 and 4 days post-impact for fluorescent viability staining and microscopy. The area of dead and living chondrocytes was quantified using custom image analysis software (VIS) as a percentage of total cartilage area. Results The highest impact energy of 1.43J fractured the cartilage in all cores (n=17). 73% and 68% of the cores remained intact after being subjected to 0.71J/1.07J impacts, respectively. Cores that fractured were thinner (p⇠0.01) than those remaining intact. Cell death increased significantly (p⇠0.05) with increasing impact energy and with greater time post-impact, while controls showed limited death. A progressive increase in dead cells near the bone/cartilage interface and at the surface was consistently observed. Conclusions These data showing progressive chondrocyte death after impact injury show a potential need for using chondroprotective agents immediately post-joint injury as a strategy to delay or prevent the onset of osteoarthritis. Such treatments may be especially important for individuals with thinner articular cartilage such as women who are also known to have overall higher incidence of osteoarthritis., Introduction Mechanical properties of articular cartilage depend on the content, arrangement and interactions of tissue components, i.e. collagen, proteoglycans (PGs) and interstitial water. Potentially, minor changes in the content and arrangement of tissue components may transmit to significant changes in cartilage biomechanics. Quantitative microscopic imaging techniques enable characterization of tissue structure. In magnetic resonance imaging (MRI) of articular cartilage, T1 and T2 relaxation time measurements may be used to highlight the spatial PG content and arrangement of collagen fibrils, respectively. Methods and Materials We have combined quantitative microscopic and biochemical information with composition-based fibril-reinforced poroviscoelastic finite element model (FEM) of articular cartilage, and have evaluated the feasibility of the model to predict, without mechanical testing, the mechanical response of normal and degenerated human articular cartilage. Results The model analysis was successful in unconfined com pression, as the theoretical stress-relaxation curves agreed closely with the experimental tests. Further, the fibril-reinforced poroviscoelastic cartilage model simulated successfully the changes in tissue mechanical properties during maturation of rabbit knee cartilage. By emerging MRI and FEM we could address closely the PG and collagen specific mechanical properties of bovine and human articular cartilage. Conclusions These studies suggest that specific information on tissue composition and structure might enable assessment of cartilage mechanics without mechanical testing. Especially, the quantitative MRI, when combined with FEM, establishes a functional imaging technique that may be more sensitive and more specific to OA changes than the current clinical methods. Furthermore, the functional MRI could provide means to monitor mechanical maturation of repair tissue non-invasively., Introduction Osteoarthritis of the knee has consistently been linked to obesity, defined as a body mass index (BMI) ⇢30 kg/m2. It has been hypothesized that obesity may lead to osteoarthritis through increased joint pressure, accumulated microtrauma, and disruption of normal chondrocyte metabolism. These changes in chondrocyte metabolism have not been thoroughly investigated, and it is the purpose of this study to identify a relationship between BMI and altered chondrocyte metabolism in osteoarthritic tissue. Methods and Materials Osteochondral explants were harvested from the femoral condyles of patients after total knee arthroplasty, and digested at day 1 and 5 post-surgery. Glycosaminoglycan (GAG) content was measured in both the digestion and media with a dimethyl-methylene blue assay and normalized to DNA content using a PicoGreen® assay. Studies have reported GAG's to be a reliable measurement of chondrocyte metabolism and osteoarthritis progression. Results Our results show a significant linear relationship of increasing BMI and increasing GAG content on both day 1 and 5 (P=0.004 and P=0.0087 respectively). In addition, GAG content from obese individuals (BMI ⇢30 kg/m2) was 3 fold higher than non-obese individuals (BMI ⇠30 kg/m2) on day 1 (P⇠0.01), and 2 fold higher on day 5 (P⇠0.01). Conclusions The study results reveal significant relationships between GAG content and BMI in this population of osteoarthritic patients. The significant difference in GAG content between the obese and non-obese patients supports the connection between osteoarthritis and obesity previously reported. Higher patient BMI (⇢30 kg/m2) may be similar to dynamic compression injuries which cause increased GAG synthesis in response to cartilage damage., Introduction The articular cartilage in Osteoarthritis (OA) is characterized by chondrocyte hypertrophy, apoptosis and calcification. These processes are similar to those observed in endochondral ossification. For that reasons, It has been postulated that one possible pathological mechanism in the OA is the articular cartilage return to its embryologic differentiation pattern. Aim: Evaluate if the synovial fluid (SF) from OA patients induce osteogenesis in ATDC5 cells. Methods and Materials ATDC5 cells were cultured with DMEM/F12, FBS 5%, 10 Î1/4g/ml human transferrin and 3X10–8 M sodium selenite. These cultures were considered controls and its results were used to normalize the experimental groups results. 10% of SF from patients with OA or healthy donors was added to the cultures. In order to assess the osteogenesis induction, we evaluated the phenotypic (nodules apparition) and molecular (collagen II and X) changes by optical microscopy and real time PCR respectively. Results Phenotypic changes: in cultures exposed to 10% of SF from OA patients, nodules apparition was observed at day 3. This was not observed in the cultures exposed to SF from healthy donors. Molecular changes: in cultures exposed to 10% of SF from OA patients, collagen II and X mRNA increased 61 and 13,5 times compared to the control at day 3. No changes were detected in cultures exposed to 10% of SF from healthy donors. Conclusions The SF from patients with OA induced osteogenesis in ATDC5 cells. These results suggest that in OA the SF could play a role in the pathogenic mechanism., Introduction Hyaluronic acid (HA) exerts poorly understood chondroprotective effects in osteoarthritis. Human chondrocytes produce reactive species capable of causing cellular dysfunction and death. Evidence indicates that mitochondrial DNA damage plays a role in conditions linked to generation of oxygen free radicals. The purpose of the current study was to evaluate the chondroprotective effects of HA through the preservation of mitochondrial function and amelioration of mitochondria-driven apoptosis. Methods and Materials Primary chondrocyte cultures, generated from cartilage from patients undergoing total knee replacement, were exposed for 30 min to peroxynitrite and hypoxanthine. After 30 min, cells were lysed for dose-response experiments or rinsed and placed in normal culture medium. Prior to treatment, some cells were incubated for 24 h with sodium hyaluronate (100 to 1000 μg/ml), with anti-CD44 antibody, or in a combination of HA and anti-CD44. To mimic oxidative stress, 1o human chondrocytes were preincubated with HA and treated with IL-1β or TNF-α. Following exposure, cells were collected and evaluated for mtDNA repair/damage, ATP synthesis, and apoptosis. Results When primary human chondrocyte cultures were exposed to reactive oxygen or nitrogen generators, mitochondrial DNA damage/ dysfunction and mitochondria-driven apoptosis accumulated. Cytokine treated 1o chondrocytes showed increased levels of mitochondrial DNA damage. Pretreatment with hyaluronic acid caused a decrease of mitochondrial DNA damage, enhanced mitochondrial DNA repair/cell viability, preservation of ATP levels, and apoptosisamelioration. Conclusions The current results demonstrate that enhanced chondrocyte survival and improved mitochondrial function were noted despite oxidative injury. This observation appears to be one therapeutical mechanism for the beneficial actions of hyaluronic acid in osteoarthritis., Introduction The best known cartilage lesion classification was developed by Outerbridge. This classification consists of four grades and is easy to understand and to use for physicians. However, more objective data regarding cartilage lesion and more accurate methods to evaluate the clinical outcomes are required, because new therapies are developed. The aim of this study is to investigate whether human articular cartilage can be evaluated quantitatively by a spectrocolorimeter. Methods and Materials We studied 79 human articular cartilage specimens retrieved from patients who underwent total knee arthroplasty. 79 human articular cartilage specimens were analyzed using a spectrocolorimeter after macroscopic evaluation and the cartilage characteristics on the L*a*b* colorimetric system, the spectral reflectance distribution, and the yellow/red spectral reflectance percentage(Y/R SRP)were examined. Results There were significant difference among four grade in the L*, a* values and Y/R SRP. The spectral reflectance distribution of grade 1 cartilage showed a gradual increase in spectral reflectance ratio along the increase of wavelength. The spectral reflectance curve of grade 2 to 4 cartilage had dip around 580 nm in wavelength. Across all the measurement wavelengths, there was lower reflectance ratio with the progression of cartilage degeneration. Conclusions The present study is the first to clearly demonstrate the relationship between spectrocolorimetric evaluation and the Outerbridge classification of human articular cartilage. The spectrocolorimeter may be a new quantitative evaluation tool for articular cartilage with clinical potential., Introduction We aimed our study at determining whether a pre-culture time of chondrocytes-seeded matrices before their combination with subchondral layers would modulate extent of cartilage differentiation and integration among the two layers of resulting osteochondral (OC) constructs. Methods and Materials Human articular chondrocytes (HAC) isolated from 5 donors (mean age 57years) were expanded in monolayer and then seeded onto collagenI/III membranes (Chondro-Gide®, Geistlich) (70×106cells/cm3) in a fibrinogen solution (Tisseel®, Baxter). The bony scaffolds (Tutobone®, Tutogen) were pre-wetted in a thrombin solution and then combined to the cell-seeded membranes immediately (group-A) or after 3days (group-B) or 14days (group-C) of preculture of the chondral layers. Constructs were cultured with chondrogenic supplements for a total time of 5weeks and assessed histologically (Safranin-O), biochemically (GAG, DNA). Additionally, the mechanical strength of integration was quantitatively assessed using a “90°-peel-off” test. Results Safranin-O positive matrix was limited to the cartilage phase of group-A constructs and extended to the cartilage/bony interface of group-B and group-C constructs. Peak force and total energy of integration in group-A and group-B constructs were significantly higher than group-C constructs (up to 2.5- and 3.2-fold respectively). Biochemical analysis of the delaminated cartilaginous layers after “90°-peel-off” test demonstrated higher DNA and GAG contents in group-B and group-C constructs as compared to group-A constructs (up to 2.3- and 3.1-fold respectively). Conclusions Our study indicates that functional OC grafts can be generated using HAC and scaffolds currently used in clinical practice. Pre-incubation of HAC for 3days in the chondral scaffold allows increasing cartilaginous matrix formation without reducing integration between the two layers., Introduction Osteochondral grafts are being commonly used to repair articular surface defects. The purpose is to achieve the normal architecture of hyaline cartilage with secure and seamless incorporation into recipient sites. However, the details of the incorporation of these grafts have not yet been completely elucidated. The expectation is that graft union would involve cellular proliferation and/ or migration as well as secretion of matrix and fibers into the graft-host cleft. The aim of this study was to observe any changes in graft architecture with time and to determine the sequence of events during graft incorporation into the host bed. Methods and Materials The medial femoral condyle (MFC) of 12 adult New Zealand White rabbits were studied. A cylindrical 4mm diameter and 4mm long osteochondral graft was obtained from the MFC and reinserted into the same site. At weekly intervals, the specimens were fixed in 10% buffered formalin for a week and decalcified in Kristensen's solution for another week. Next, they were dehydrated and critical point dried. Finally, the specimens were mounted and sputter coated with Gold/Palladium before viewing in the Hitachi S-300H scanning electron microscope. Results Cartilage-to-cartilage healing was not observed at any time interval. Where cartilage union appeared to have occurred, this was due primarily to press fit or ‘surface weld’. In some cases, the adjoining graft and host surfaces revealed superficial fractures. There was bony union at the base in all cases and this union had crept up towards the joint surface in the later time intervals. The materials in the cleft between the graft and recipient bed ranged from fibrous to bony elements. The graft surfaces were smooth like the surrounding normal articular cartilage at 1 and 2 weeks but fibrillated at 3 and 4 weeks. Conclusions These results appear to suggest that direct cartilage-to-cartilage healing may not occur following osteochondral grafting. Bone-to-bone healing appears to be universal and rapid and, materials from this source may be responsible for gap healing. The results also raise the possibility that the articular surfaces may deteriorate with time but the reasons are not apparent from this study., Introduction Study purpose: Measurement of precision comcerning angle of insertion and depth accuracy in navigated autologous osteochondral transplantation in comparison to the conventional free hand technique. Methods and Materials Articular surfaces of 6 cadaveric condyles (medial – lateral) were used. Knee referenced by the navigation system. Pins carrying the navigation detectors were positioned to the femur and to the tibia. The grafts were taken from the donor site (measurement I) with the special instrument which carried the navigation detectors. The recipient site was prepared, the navigation detectors were attached to the insertion instrument and the osteochondral grafts were forwarded into the repair site under the control of the navigation system in an angle of 90° to the articular surface (II). The same procedure took place without navigation. The articular surface congruity was measured with the probe (measurement III) Results Angle of recipient plug removal (measurement I) with navigation: 3,27° (SD 2,05°; 0°-9°) deviation of the 90° axis. Conventional technique: 10,73° (SD 4,96°; 2°-17°). Mean difference between navigation and conventional technique was 7,46° (p⇠0.0001). Navigated recipient plug placement (measurement II): mean angle of 3,6° (SD 1,96°; 1°-9°) Conventional technique: mean angle of 10,6° (SD 4,41°; 3°-17°). Significant difference between navigation and conventional technique (p=0,0001). Navigated depth measurements (III):mean depth of 0,25mm (SD 0,19mm; 0mm-0,6mm). conventional technique:0,55mm (SD 0,28mm; 0,2mm −1,1mm). Significant difference for the navigation and conventional technique (p=0,0034). Conclusions Computer navigated assistance in autologous osteochondal transplantation provides more accurate positioning of the grafts and better results concerning the articular surface congruity., Introduction Determine effects of temperature and serum concentration in medium on efficacy of 28-day allograft storage as indicated by chondrocyte viability, especially in the superficial zone, and cartilage matrix content. Methods and Materials Adult goat osteochondral cores (n=65) were freshly isolated or stored (14, 28 days) in MEM at 4°C including 10% FBS or at 37°C including 0%, 2%, or 10% FBS. Cartilage portions were analyzed using Live/Dead® fluorescence assay to determine viability (percentage live) and cell density at the articular surface en face (E) and in the vertical profile (Overall and by layer (Superficial, Middle, Deep)). The remaining cartilage portions were analyzed for sulfated-glycosaminoglycan and collagen. Storage effects were assessed by ANOVA with Tukey post-hoc tests. Results Viability, live and dead cell density varied with storage group (p⇠0.001), without effects on total cell density. After 28 days, 37°C samples had a higher chondrocyte viability than 4°C samples, especially at the articular surface. At 4°C, viability was markedly affected by storage duration (p⇠0.001). After 28 days, viability at the articular surface was reduced by 4°C storage (E:⇠50%, S:⇠20%), but maintained by 37°C storage (E:∼80%, S:∼60–70%) (each p⇠0.001). M viability was higher following 37°C storage (p⇠0.05), whereas D viability was not. With 37°C storage, viability was similar for 0–10% FBS samples. Cartilage thickness, glycosaminoglycan content and collagen content in stored samples had similar levels to fresh controls. Conclusions 37°C storage of osteochondral grafts maintains long-term chondrocyte viability, especially at the articular surface, and may therefore be used to extend acceptable storage duration and improve treatment outcomes., Introduction One of the most important factors for a successful clinical outcome after transplantation of osteochondral allografts is viability of chondrocytes after preservation. In previous studies, several kinds of culture medium have been tested for suitability for preservation of osteochondral allografts. However, Euro-Collins (EC) solution and University of Wisconsin (UW) solution, the standard for cold preservation of most organs have not been tested. Methods and Materials Osteochondral tissues (OCTs) harvested from distal femora of Sprague-Dawley rats were preserved in Dulbecco's modified Eagle's medium (DMEM), saline, EC solution and UW solution for 7 days. Tetrazolium assay was used to estimate relative viable cell number in OCTs. Lactate dehydrogenase (LDH) activity released from damaged cells into supernatants of preservation solutions was measured to estimate cytotoxicity. Fresh OCTs and OCTs preserved for 7 days were evaluated histologically. Results After 7 days' cold preservation, OCTs kept in UW solution had the highest relative viable cell number by the tetrazolium assay and the lowest activity of LDH. In histological evaluation, chondrocyte deformity, such as shrunken cytoplasm and pyknotic nuclei, was observed partially or predominantly in articular cartilage preserved in saline, EC solution and DMEM. In contrast, chondrocyte morphology in articular cartilage preserved in UW solution was relatively unchanged and remained similar to fresh OCTs. Conclusions This study demonstrates that UW solution is better than the other three solutions tested for cold preservation of rat OCTs. If UW solution can protect human osteochondral allograft against damage caused by the cold preservation, the duration of storage of osteochondral allografts may be prolonged., Introduction The purpose of this work is to create an in vitro model of tissue engineered osteochondral composite by combining calcium phosphate scaffold and neocartilaginous tissue produced by isolated swine articular chondrocytes embedded in fibrin glue. Methods and Materials Swine articular chondrocytes were isolated and embedded in fibrin glue. Immediately before gel polymerization, the fibrin glue was placed in contact with the calcium phosphate scaffold. Similar control cylinders were prepared using acellular fibrin glue as chondral element of the composite. The osteochondral composites were left in standard culture conditions and retrieved after 1 and 5 weeks. Samples were macroscopically analyzed and prepared for histological and biomechanical analysis (distraction test). Results Data showed a macroscopic integrity of the osteochondral samples. Histology showed cartilage like tissue maturing within the fibrin glue scaffold, and the presence of GAG between the fibrin glue and the cylinders, infiltrating the scaffold trabeculae in the experimental samples. Control specimens demonstrated inferior biomechanical consistency by gross testing and acellular fibrin glue without infiltration in the calcium phosphate trabeculae. Preliminary distraction test showed first a deformation of the chondral element and then the complete division of the two components. Conclusions The results of this study demostrate that isolated chondrocytes, seeded onto fibrin glue, produce a cartilage-like matrix that integrates with a cylinder of calcium phosphate. This tissue engineered osteochondral composite could represent a valuable model for further in vivo studies on the repair of osteochondral lesions., Introduction Advantages of osteochondral grafting are that the defect is immediately filled with mature hyaline articular cartilage and that stable fixation can be achieved without additional sutures or adhesives. However, a major factor affecting long-term success of osteochondral grafting is poor integration between host and donor cartilage. Methods and Materials Medial and lateral femoral condyles were aseptically harvested from fresh bovine knees. Recipient defects 6mm in diameter were created using an OATS instrument set Osteochondral donor grafts in one of three diameters were harvested: 5.5mm (loose ft), 6.0mm (line-to-line fit) and 6.5mm (tight fit). The 6.5mm donor graft was gently tamped into the recipient site to create a tight press-fit. Ten-mm diameter concentric explants containing the grafted sites were harvested and cultured for 3, 6, or 12 weeks. At each time point, the strength of graft integration with host tissue was assessed by a mechanical tensile test. A full-thickness chondral specimen was cut from the center of each explant, mounted, and tested to failure. Results As expected interface tensile strength increased with time in culture. The tight-fit osteochondral graft design showed the greatest tensile strength after 12 weeks in culture. The greater tensile strength reflects greater chondral integration between the host and graft specimens. Conclusions A tight-fit gap decreases the graft-host interface gap by approximately 21 microns from the current surgical recommendation (line-to-line). We speculate that a tight-fit graft reduces the gap required for matrix production and integration, as well as preventing in-vivo flow of inhibitory molecules such as lubricin, resulting in greater integration., Introduction We evaluated the potential of porous bioactive glass (BG) 13–93 as a subchondral substrate and media supplement for tissue-engineered bi-layered osteochondral (OC) constructs. Methods and Materials In vitro: The effects of BG on the chondrogenic capacity of juvenile bovine chondrocytes (from carpometocarpal (CMC) joints) were studied by assessing biochemical and biomechanical outcomes associated with BG cylinders placed in media of chondrocyte-seeded agarose hydrogel constructs after 28 days of culture and compared to controls 14 days later. In vivo: Rabbit mesenchymal stem cells cultured in monolayer were seeded into poly(ethylene glycol) (PEG) hydrogel and bonded to BG to create tissue-engineered OC constructs. Similar constructs created using allograft bone were used as controls. The OC constructs (3mm × 6mm) were implanted into defects in the medial femoral condyles of rabbit knees and assessed after 12 weeks. Results BG in the culture media resulted in peak values of EY: 743kPa, GAG: 7.6%ww, G*: 2.8MPa and Collagen: 6.2%ww which were each significantly better than controls. These values for BG-treated constructs compare favorably to intact bovine CMC cartilage. The in vivo study showed that bi-layered BG OC constructs histologically appeared to have better integration and significantly greater collagen II expression than allograft bone constructs. Conclusions These data suggest that BG has potential as a media supplement for tissue engineering osteochondral constructs for resurfacing articular cartilage defects and as a subchondral substrate for these grafts. Further studies will be required to optimize the protocol for construct creation and to delineate the long term effects of BG for clinical application., Introduction The development of non-invasive cartilage assessment methodology would permit evaluation of therapeutics without requiring biopsy. Therefore, we evaluated the potential of near infrared (NIR) and mid-infrared (mid-IR) spectroscopy to characterize full-depth matrix changes in the collagen and proteoglycan components of engineered cartilage over time and in response to an anabolic intervention. Methods and Materials Articular bovine chondrocytes were cultured in a collagen type I gel scaffold for 3 or 5 weeks (n = 10). Pulsed low intensity ultrasound was applied to half of the constructs for 20 minutes/day, 5 days per week. A Nicolet Continuum FT-IR Microscope (Thermo Electron Corp) was used to acquire NIR and mid-IR data from the constructs in the spectral region of 800 − 7000 cm-1. Results were compared to biochemical assays for sulfated glycosaminoglycan (sGAG). Results We found that the integrated area of the 850 cm-1 mid-IR peak increased with ultrasound treatment over a time course of 5 weeks, and correlated well with sGAG content (R2 = 0.89). This in agreement with our previous studies that demonstrated correlation of this peak with proteoglycan content. There was no increase in the NIR peak at 4250 cm-1, a spectral feature that arises from type II collagen, with ultrasound treatment, consistent with previous in vitro studies of collagen development under ultrasound intervention. Conclusions These results provide motivation for further development of infrared spectral parameters for full-depth evaluation of cartilage constructs and intact tissues., Introduction We postulate that the biochemical and biomechanical characteristics of articular cartilage are joint and age specific. To verify our hypothesis, we investigated the gene-expression profiles of articular cartilage tissue that was derived from three different joints of differently-aged bovine cows. Methods and Materials Articular cartilage was harvested from the metacarpal (basal region), shoulder (humeral head) and knee (tibial plateau) joints of 4-month-old bovine calves, 15- to 20-month-old (young adult) bovine cows and 3- to 9-year-old (aged) bovine cows within 24 hours of slaughtering. The structural maturity of the tissue was ascertained histologically. The gene-expression profiles of the samples were quantified by a real-time PCR analysis. Results The postnatal development of bovine calves into young adult cows is characterized by a structural evolution of the articular cartilage tissue from an immature (isotropic) to a mature (anisotropic) type. Aging of the animals is not accompanied by any overt change in the structural organization of the cells. In immature articular cartilage, the activity levels of the genes for collagen types IX and X, and Sox9 were similar in each of the three joints. However, in the articular cartilage of young adult and aged animals, the gene-expression profiles of these components were joint specific. Moreover, the gene-activity levels of the collagens were very much lower in the older animals than in the young adults. Conclusions In mature bovine articular cartilage, the gene-activity profiles of key components are joint specific. These differences may be manifested also at the protein-expression level and, consequently, could impact the tissues' biomechanical properties., Introduction The objective of this study was to determine the influence of chondral defect size on defect rim stress concentration, peak rim stress, and load redistribution to adjacent cartilage over the humeral head. Methods and Materials Eight fresh-frozen cadaveric shoulders were mounted at 45° abduction with 5° of external rotation in a materials testing machine. Digital pressure sensors were placed over the humeral head contacting the glenoid. Each intact shoulder was loaded and held while dynamic pressure readings were recorded throughout. Loading was repeated for circular defects (6, 8, 10, 12, 14, 16, 18, 20mm) centered at the contact apex as determined by the initial control load. Results Stress concentration around the rims of defects 10mm and smaller was not demonstrated. For defects 12 mm and greater, distribution of peak pressures followed the rim of the defect with a mean distance of 4.2mm. Load redistribution was observed as the radius to peak value increased above a rim size of 12mm to a mean of 11.8mm compared to 7.9mm for defects 10mm and below. Peak rim pressure did not increase significantly as defects were enlarged from 10mm to 20mm. Conclusions Rim stress concentration was demonstrated for chondral defects 12mm and greater in size. This altered load distribution has important implications relating to long-term integrity of cartilage adjacent to chondral defects in the shoulder. Our understanding of chondral defects in the shoulder, a non-weightbearing joint, is evolving. While the decision to treat is multifactorial, a size threshold of 12mm may serve as a useful guide to clinical decisionmaking., Introduction Epiphyseal cartilage trauma, accidently or caused intra-operatively is known to initiate bone-bridge formation possibly causing full or partial premature physeal closure. The aim of this study was to investigate hypervascularity as one hypothesized underlying mechanism for bone bridge formation using qRT-PCR and 3-Tesla MRI in a living-animal-model. Methods and Materials Male Sprague-Dawley-rats (4 weeks, ∼100g) were subjected to a unilateral transphyseal drilled lesion of 1.2 mm diameter of the proximal tibial physis. Physeal vascularisation and bone bridge formation were investigated by 3-Tesla MRI (n=5; intravenous application of Dimeglumingadopentetat, Gadovist® 0.2ml/100g body weight) while expression profiles of HIF1a, VEGFa, VEGF receptor 1 and 2, and CD 31 were established by qRT-PCR (n=8) on days 1, 3, 7, 14, 28, and 82 post-lesion. Results In the traumatized growth plate, mRNA expression of Hif1a showed a slight upward trend from day 1 on with maximum levels on day 3 while VEGFa, VEGFR1 and 2 showed highest expression levels on day 7. Comparing expression levels of VEGFa, VEGFR1, HIF1a, and CD31 between the lesioned and the contra-lateral physis significant changes were only observed on day 28 (p⇠0.05) giving indirect support for existing bone bridges (observed by 3-Tesla MRI on day 28) as they represent highly vascularised tissue which could further be documented by increased perfusion levels evaluated by 3-Tesla MRI. Conclusions This study gives evidence for HIF1a and VEGF expression to precede angiogenesis and formation of bone bridges at the site of physeal lesion which was clearly documented by qRT-PCR and 3-Tesla MRI., Introduction The objective of this study was to evaluate the mechanical behavior full-thickness osteochondral defects of articular cartilage in the human knee. In addition, change in behavior was measured after a cylindrical lesion was converted to a beveled defect. Also, what part of the lesion size is more critical for diagnosis: the outer or inner diameter? Methods and Materials Ten cadaveric knees were mounted on a load frame. Pressure sensors were placed in the medial and lateral compartments. Each knee was loaded and held. Pressure readings were recorded throughout. Loading was repeated over full-thickness osteochondral defects with vertical walls (6, 8, 10, 12, 14, 16mm). The well-shouldered lesions were then transformed into beveled lesions and loading was repeated. The center of defect to peak pressure distance was calculated. Results An important comparison is the difference in center of defect to peak pressure distance between well-shouldered and beveled defects, using outer diameter as a metric. In the lateral condyle, 4 of 6 test pairs showed increased distance from well-shouldered to beveled lesions, with average distance of 0.2174mm. In the medial condyle, 5 of 6 test pairs showed increased distance from well-shouldered to beveled lesions, with average distance of 0.5231mm. Beveled lesions acted more like well-shouldered lesions of a larger size (10mm beveled acted like 12mm well-shouldered). Conclusions The outer diameter of beveled defects in articular cartilage is a better predictor of pressure distribution on surrounding cartilage than inner diameter. Beveled defects of articular cartilage in the knee should be evaluated and treated based on their outer diameter., Introduction Some preliminar studies concerning cell therapy have been carried-out in animal models to treat the rupture of anterior cruciate ligament (ACL) with two main approaches: a) the use of different fibroblasts or mesenchymal cells for autologous implantation and b) the use of new biomaterials combined with cultured cells. Our purpose was to study the features and behaviour of monolayer cultured fibroblasts isolated from ACL of patients with different ages and type of lesion. Methods and Materials ACL biopsies from 34 patients with ACL rupture. Twenty-eight of 34 patients had acute ACL rupture (elapsed time from rupture to surgery shorter than 1 month), 9 had chronic ACL (elapsed time from rupture to surgery longer than 1 month). Normal ACL samples from 28 patients. The median age of these 62 patients was 32 years (range: 15 – 74 years). Fibroblasts were isolated, the number of cells was estimated and cultured in monolayer. Results The fibroblast culture could be established in 43 cases. First passage was done at the day 15th and the mean growth rate was 21.2 ± 15.5. Negative correlation was observed between the age of the patient and the growth rate. Although without statiscal significance (probably due to the low sample size), the mean growth rate was higher in patients with acute lesion (n=18; 28.3 ± 19.8) followed by normal ACL (n=7; 17.1 ± 8.9) and by those with chronic ACL rupture (n=18; 13.5 ± 9.4). Conclusions The growth rate of ACL fibroblast in culture is higher in young patients with acute ACL rupture., Introduction Mechanical forces play a critical role in nearly all aspects of cell biology. Within synovial joints, they are prerequisite for maintaining cartilage matrix properties whereas excessive mechanical forces leads to a loss of cartilage and the onset of osteoarthritis. How cells sense mechanical stimulation, however, is largely unknown. Our purpose is to develop a new experimental system to study the effects of mechanical loading and the mechanism of mechanotransduction. Methods and Materials We produced a new porous collagen sponge scaffold that has inter-connected pores for 3-D cell culture and a custom-designed and -built apparatus for loading experiments. We isolated human synovial cells and meniscus cells from surgical specimens and incorporated these cells into scaffolds. The 3-D construct was applied to load stimulation and used for the analysis. Firstly, we examined the cell viability, then compared the morphology and the cytoskeleton of cells seeded in monolayer or in 3-D scaffolds. Results Cells in the constructs were viable throughout the culture period of 1 week. The cells were amoeboid in shape in 2-D and spheroid in 3-D culture. Actin was seen to form stress fibers only in 2-D and showed a punctate staining pattern in 3-D culture. The organization of the vimentin meshwork was a more circumferential arrangement in 3-D culture. Conclusions We developed a new 3-D cell culture system in which the morphology and general cytoskeletal disposition of cells are similar to their native condition, i.e. in their natural extracellular matrix. This culture system provides a powerful tool for the study of mechanotransduction and drug screening., Introduction The effect of pulsed electromagnetic fields (PEMFs) on the proliferation and survival of matrix-induced autologous chondrocyte implantation (MACI)-derived cells was studied to ascertain the healing potential of PEMFs. Methods and Materials MACI-derived cells were taken from cartilage biopsies 6 months after surgery and cultured. No dedifferentiation towards the fibro-blastic phenotype occurred, indicating the success of the surgical im plantation. The MACI-derived cultured chondrocytes were exposed to 12 h/day (short term) or 4 h/day (long term) PEMFs exposure (magnetic field intensity, 2 mT; frequency, 75 Hz) and proliferation rate determined by flow cytometric analysis. Results The PEMFs exposure elicited a significant increase of cell number in the SG2M cell cycle phase. Moreover, cells isolated from MACI scaffolds showed the presence of collagen type II, a typical marker of chondrocyte functionality. Conclusions The results show that MACI membranes represent an optimal bioengineering device to support chondrocyte growth and proliferation in surgical implants. The surgical implant of MACI combined with physiotherapy is suggested as a promising approach for a faster and safer treatment of cartilage traumatic lesions., Introduction The purpose was to examine whether a chondrogenic medium and/or mechanical stress is necessary to differentiate human bone marrow stromal cells (hBMSC) into chondrocyte-like cells in a biological osteochondral matrix stimulated in a bioreactor system. Methods and Materials HBMSC were harvested, density centrifugation was performed prior to resuspension and three cell passages. For 7 days a predifferentiation culture was initated by replacing FGF-2 with 100 ng/ml IGF-1 and 5 ng/ml TGF-β2. Afterwards cells were seeded into the biologic hybrid scaffold out of CaReS® (rat collagen I, Arthrokinetics) and Tutobone® (bovine spongiosa, Tutogen Medical) with a concentration of 1×106 cells/ml. The constructs were exposed to a cyclic compression protocol (10 % compression, 0.5 Hz) under continuous perfusion in a mechano-bioreactor for 14, 21 and 28 days. Effects were evaluated using light microscopy and collagen 2, 3, and 10 staining. GAG and DNA were quantified. Biomechanical characterization was conducted using a confined compression quasi-static loading setup. Results GAG quantification showed differences between mechanical and static stimulation after three and four weeks (p⇠0.05). Mechanical tests showed no difference over four weeks, but the mechanical groups were stiffer compared to the static control (p⇠0.05). The quality of tissue was not improved by IGF/TGF in our system focusing histology, biochemistry and mechanical properties of these tissues. Conclusions Media supplements to foster the quality of the tissue showed no progress in our system although it is well known that those are important to induce a chondrogenic phenotype. It could be postulated that those could be left out in such a system., Introduction No medical intervention has yet been demonstrated to effectively restore damaged cartilage in large clinical trials in OA. It could be speculated that study subjects generally had reached a “point of no return” where medical stimulation of cellular repair mechanisms are insufficient to restore the structure damage. To study this, we performed a series of biochemical assessments of bovine cartilage explants undergoing sequential catabolic and anabolic stimulation. Methods and Materials Bovine cartilage explants were cultured in the presence of OSM+TNFα at different duration followed by IGF1. Release of peptide fragments into the supernatant was monitored by MMP and aggrecanase-dependant neo-epitope specific immunoassays. Results Seven days of OSM+TNFα stimulation of bovine cartilage explants induced a massive loss of proteoglycans as observed by histology. Transferring these explants to anabolic stimulation (IGF1) for another 14 days, reversed the loss of proteoglycans. Assaying the supernatants for presence of neo-epitopes demonstrated that (1) OSM+TNFα induced massive release of aggrecan fragments mediated by aggrecanases in the early phase, and changing to anabolic conditions after 7 days did not change this profile; (2) In contrast, aggrecan and type II collagen fragments carrying MMP-dependant neo-epitopes were released only in the late phase, i.e. after day 11; (3) If catabolic stimulation was discontinued after 7 days, MMP-generated fragments was not released into the supernatant. Conclusions These biochemical data strongly suggest that cellular repair mechanism in bovine explants cultures can be at least partly restored after short-term catabolic stimulation, and that biochemical profiling of the supernatants effectively reflect these metabolic changes., Introduction Subchondral bone is important to maintain quality of articular cartilage. The objective of this study is to investigate the importance of subchondral bone remodeling for articular cartilage regeneration. Methods and Materials Fifty male Japanese white rabbits were used. A 6mm cylindrical osteochondral defect was made in left patellar groove. The rabbits were randomly assigned to control, Alendronate (ALN) (0.14mg/kg/week subcutaneously) and ALN-S group (ALN injection for first 8 weeks). Animals were sacrificed at 4, 8, 24 and 52 weeks. The quality of the cartilage was evaluated histologically (Wakitani Score) and with our ultrasound method. Bone morphology was evaluated with micro-CT. Results At 4 weeks, ALN group showed drastically early remodeling of subchondral bone and regeneration of cartilage-like-tissue filling the defect. However, no cartilage-like-tissue was seen in control group. In Wakitani score, 4 weeks ALN group was superior to control group. In 52 weeks, ALN-S group seem to have thicker and more stained cartilage than ALN or control group. However there was no significant deference among the score of three groups. Ultrasound analysis showed the signal intensity of both ALN and ALN-S group was significantly higher than control group in 4, 24 and 52 weeks, suggesting better quality of the cartilage. Micro CT analysis showed the superior remodeling of continuity of subchondral bone and recovery of BV/TV in 8 weeks ALN group. Conclusions Eight weeks injection of ALN had positive effect on subchondral bone remodeling and following cartilage regeneration. The result showed an importance of the early subchondral bone remodeling for cartilage regeneration., Introduction We investigated the potential of periosteal cells to infiltrate PCL nanofiber scaffolds in vivo and subsequently produce cartilage in vitro. Methods and Materials PCL nanofiber scaffolds, with or without chitosan coating were implanted under the periosteum on the medial proximal tibia of six-month old rabbits. TGF-β1 (200 ng) or vehicle was injected into each implant site. After 1, 3, 5 or 7 days, scaffolds were removed, separated from the periosteum, and both the scaffolds and periosteal explants from the implant site were cultured separately for six weeks. Results Cell infiltration was observed in all the scaffolds. Cartilage formation in the uncoated scaffolds increased with duration of implantation (maximum at 7 days). Cells in the uncoated scaffolds implanted for 7 days produced significantly higher levels of both GAG (560 ±412 vs. 228 ±61 μgGAG/μgDNA) and cartilage yield (9 ±11 vs. 0.02 ±0.04%) compared to chitosan-coated scaffolds (p⇠0.007). There was no significant difference in GAG content or cartilage yield between the TGF-β1-injected and vehicle-injected scaffolds. However, significantly more calcium deposition was detected in TGF-β1-injected scaffolds compared to vehicle-injected scaffolds (p⇠0.0001). Cartilage yield from the implant-site periosteum was significantly increased in all the TGF-β1 injected groups compared to vehicle (p⇠0.0014), with the exception of day 1 and 5 of the chitosan-coated scaffolds. Conclusions This study demonstrates that it is possible to seed PCL nanofiber scaffolds with periosteal cells in vivo and subsequently produce engineered cartilage in vitro. These periosteal cell-seeded nanofiber scaffolds may be useful alone or in combination with the periosteal graft for cartilage regeneration., Introduction We've previously reported the development of scaffold free cell delivery system, and showed good regeneration of rabbit osteochondral defect until more than two years by implantation of molded mesenchymal stem cells (MSCs) construct without use of exogenous factors. To build more complex shaped, multi-lineage cell construct without biomaterials, we invented a new simple method to build larger scaffold free constructs by building up cell aggregates one by one. In this pilot study, we examined our new system, named “Bio rapid prototyping method”, to be abed to build scaffold free cell construct. Methods and Materials For initial experiments, we used primary rabbit MSC, which were divided two groups, labeled with Qtracker 655(Invitrogen, US) as red, and 565ã as green, under monolayer condition. The labeled cell aggregates were placed as pre-designed position one by one with fine micro forceps under stereoscopic microscope. After building process, the constructs were cultured under standard condition for 2–3 days. Results We obtained cell constructs as almost expected shape. Snap frozen section showed no necrotic formation in rough histological observation. Conclusions Until now, we tried hMSC, HepG2, or beating cardiomyocyte as cell source for the construct by our method, and showed good results. In this study, the construct are build by our hands under microscope, it takes much hours to build just only 5mm cubic shaped cell only construct. Near future combination of the robotic technology and the bio technology, we may able to build joint cartilage, meniscus, etc, in accordance with patients 3d body data., Introduction The goal of this study was to evaluate ultrasound as a safe, novel imaging modality for the non-invasive monitoring of the healing response of an osteochondral defect that was treated with implantation of a preconditioned bioactive scaffold, and to correlate the ultrasound imaging with histological observation. Methods and Materials Bilateral osteochondral defects were surgically created in the trochlear groove of the knee in fifteen adult male New Zealand white rabbits, under IACUC approval. Twenty-four defects were filled with PLLA scaffolds preconditioned with either IGF-1 or BMP-7, and eight were left as unfilled controls. The knees were then harvested at 3, 6, and 12 weeks post-surgery, and evaluated with histological evaluation, as compared to ultrasound, using FlexScan image processing software. Results Osteochondral defects were clearly identified in each ultrasound performed. Imaging of knees containing an unfilled osteochondral defect demonstrated an empty gap in the trochlear groove, whereas those with a scaffold implant exhibited filling of the defect. Ultrasound images correlate with histologic evaluation in demonstrating a progressive increase in the healing response around osteochondral defects filled with preconditioned bioactive scaffolds for the three, six and twelve week samples. Conclusions Ultrasound was able to distinguish the site and borders of both the osteochondral defect and the implanted scaffold, and was accurate in measuring the dimensions of the osteochondral defect and the scaffold within it. Progressive changes in these measurements allow monitoring of the repair tissue in a temporal fashion. Lateral edge integration in the osteochondral defect was also observed with ultrasonography, correlating with histological results., Introduction The lack of inflammatory response and the feasibility for controlling mechanical, morphological and degradation properties are attractive features of poly (L-lactic acid) (PLLA) for cartilage repair. Our aim was to determine the biocompatibility between Mesenchymal Stromal Cells (MSC) and PLLA and their effect on the mechanical properties of the scaffolds. Methods and Materials MSC were cultured in PLLA films to study adherence and proliferation. We loaded 10×106 and 2×106 MSCs into PLLA scaffolds (1 and 6 mm in thickness), either by injection or aspiration. Scaffolds were maintained in expansion or differentiation media for 21 days. Seeding efficiency and cell distribution and viability were analyzed after the seeding while chondrocyte differentiation was assessed at 21 days. Results Results showed an adhesion of MSC to PLLA films of 27 ± 0,15 and 34 ± 9,0 % of seeded cells at 1 and 4 hours. Proliferation was detected, but ceases at day 10. An efficiency of 80,8 ± 4,1 and 71,5 ± 9,4 % for 1 and 6 mm scaffolds was detected after the seeding. Retaining of cells were more efficient in the 6 mm scaffolds, where cells were poorly spread. Nevertheless, cells homogeneously saturated 1 mm scaffolds. Final results show the production of matrix composed by collagens I, and X, that produced a 2,6-fold increase in the Young modulus. Conclusions We have developed biocompatible PLLA scaffolds that can be efficiently loaded with MSCs. We also show that differentiation of MSCs can be successful in PLLA and that it modify the mechanical properties of the PLLA itself., Introduction Electro-wetting on Di-Electric (EWOD) printing can create droplets of less than 10μm-diameter and work with a variety of hydrogels, cells and growth factors during scaffold fabrication. EWOD has never been used in tissue engineering and can enable control of droplet size and precise deposition of droplets compared to existing micro-droplet technology. Methods and Materials EWOD uses the electrocapillary principle: surface-tension (ST) is a function of electric potential across an interface. The change in liquid-solid ST changes the contact angle at the liquid-solid-gas interfaces. By applying sequential voltages to electrodes under the dielectric layer, four fundamental droplet manipulations can be established: creation, cutting, joining, and transporting. The system will integrate an EWOD array design with a computer-controlled motion system to position the EWOD chip for hydrogel, crosslinker, growth factor and cell printing based on CAD models. Projected built rates are 1cm/min with droplet diameters on the order of 10μm for picoliter-volume droplets. Results Amounts ranging from 0.2–2% (w/v) chitosan were mixed with acetic acid. The maximum viscosity EWOD can handle is 250cP. High-molecular weight chitosan was shown to behave as a non-Newtonian fluid with a non-linear relationship between shear rate and shear stress; Low-molecular weight chitosan is Newtonian with a linear relationship. After EWOD printing, human fetal osteoblast cells were shown to have a success rate of 94%. Conclusions Rheological testing and droplet operations were completed and studied on chitosan hydrogels and crosslinker solutions. EWOD on-chip crosslinking and cell manipulation were demonstrated, which shows the system is capable to make micro array printing for tissue construct., Introduction We analyzed the early structural characteristics and adhesive response of rabbit chondrocytes seeded into uncoated PCL scaffolds (pPCL) or composite PCL scaffolds containing hyaluronan (PCL/ HA), chitosan (PCL/CS), fibrin (PCL/F), and type I collagen (PCL/COL1) and cultured statically. TEM (x7500, 1h after seeding) and anti-CD44 and anti-integrin-β-1 immunofluorescence (1h and 24h after seeding) analyses were performed. Methods and Materials The surface of random chondrocytes (n=7 each) was analyzed using a grid (parallel lines at 1.1765 μm intervals in TEM). The number of lines intersecting the chondrocyte profiles, and the number of intersections with the cell surface contours were recorded in two orthogonal orientations. Roughness, diameters and horizontal-to-vertical ratios were calculated. Results PCL/HA (2.66±0.43) and PCL/COL1 (4.16±1.75) contained significantly fewer “rough” chondrocytes compared to the other scaffolds (p⇠0.05). No significant differences in diameter and horizontal-to-vertical ratio could be found between the different groups. Integrin-β-1 and CD44 were detected in PCL/COL1 scaffolds. In the other hybrids, integrin-β-1 could only be detected 1h after seeding. CD44 immunoreactivity was found in PCL/HA only 24h post-seeding. PCL/ CS, PCL/F and pPCL lacked CD44 immunoreactivity. Chondrocytes in PCL/HA showed the least prominent surface modifications compared to controls, and an increasing CD44 expression. Cells in PCL/COL1 had more prominent yet not significantly different surface modifications, and a constant expression of CD44 and integrin-β-1. Conclusions Previously, we found that PCL/HA and PCL/COL1 supported cartilage ECM synthesis better than PCL/F and PCL/CS. These functional differences may be related to the early structural observations in this study. Future studies will be needed to decipher this relationship., Introduction Collagen gels are weak cell scaffolds. Can photochemically crosslinking collagen gel permit: 1) cell survival; 2) neo-cartilage formation; 3) neo-cartilage integration with the adjacent native cartilage? Methods and Materials Cartilage was collected from swine and healthy human cadaveric tissue and chondrocytes were obtained by enzymatic digestion. Cells (40–60 million/cc) were mixed in 0.8% solubilized rat tail type I collagen liquid. Different concentrations of riboflavin and irradiation doses of blue light were tested to photochemically crosslink collagen I gel resulting in favorable cell survival as determined by live-dead assay. Hydrogels constructs with cells were implanted into nude mice to assess the capacity of neo-cartilage formation in vivo. Cells in gel were also placed into the center of devitalized swine or human articular cartilage (ring model) or between two disks of devitalized swine or human articular cartilage (sandwich model) and implanted into nude mice to assess the capacity for integration and healing after 6, 12 and 18 weeks. Results 0.25mM riboflavin and 40 seconds irradiation permitted high cell viability and minimal gel contraction. Specimens placed in mice showed neo-cartilage formation as evidenced H&E, Toluidine blue, Safranin-O staining, and immunostaining for type II collagen. Neo-cartilage filled native cartilage ring model showed good integration and neo-cartilage between cartilage disks formed tight bonds with existing cartilage. Conclusions Human, as well as swine, chondrocytes have the capacity to form neo-cartilage in this novel crosslinked hydrogel scaffold, and the cartilage can integrate and heal to adjacent native cartilage. Riboflavin and blue light are benign crosslinkers allowing favorable cell survival and neo-cartilage formation., Introduction Biologic scaffolds can augment the repair of tendon defects by enhancing the endogenous reparative response. PDGF-β and IGF-1 are potent mitogens, shown to improve tendon healing in various models. The current study investigates the effects of a tissue engineering method that involves preconditioning biologic scaffolds with gene-enhanced tenocytes transduced to produce either IGF-1 or PDGF-β. Methods and Materials Adult male Sprague-Dawley rats were used to isolate tendons from the rotator cuff, and tendon fibroblasts (RTF's) were transduced with the gene for either PDGF-β or IGF-1, using retroviral vector plasmids. PLLA scaffolds were preconditioned for 3, 6, or 9 weeks by seeding with wild-type RTF's, or RTF's transduced with either growth factor. After lyophilization, wild-type RTF's were then seeded onto either the preconditioned or control scaffolds. Following 3 or 7 days in culture, samples were pulse-labeled with H Proline and H Thymidine to assess collagen and DNA synthesis. Results Our results demonstrate that the cells grown on scaffolds that were preconditioned with RTF's had greater DNA and collagen synthesis than scaffolds that were not preconditioned. Scaffolds preconditioned for longer durations demonstrated significant increases in DNA synthesis over untreated controls. Similar trends were seen for collagen synthesis, with increases compared to control scaffolds for all preconditioning treatments after both 3 and 7 days of culture. Conclusions This study demonstrated improved collagen and DNA synthesis in scaffolds preconditioned with RTF's. These findings may potentially be attributed to the increased levels of proteins released by both growth factor-transduced RTF's and wild-type RTF's, as directly related to the duration of preconditioning., Introduction The purpose of this study was to compare the histologic appearance of chondral repair tissue at 18 months following treatment with TruFit® plugs or a standard microfracture technique in an ovine model. Methods and Materials Single, 5.1 mm diameter osteochondral or full-thickness chondral defects were created in the medial femoral condyle of 12 adult sheep. The osteochondral defects were repaired with a 5.3 mm diameter X 5 mm long, press-fit, TruFit® plug (n=6) and the full-thickness chondral defects were treated with a standard microfracture technique (n=6). The histologic appearance of the repair tissue was evaluated in each group at 18 months using a modified O'Driscoll scoring system and compared using a t-test. Results At 18 months, both groups showed evidence of organized fibrocartilage repair tissue filling the defects. The repair surfaces were smooth, congruent, and there was excellent lateral integration to the adjacent hyaline cartilage in both groups. Both groups demonstrated thickening of the subchondral bone immediately beneath the repair tissue with the lower phase of the TruFit® plug being replaced with cancellous bone. There was no statistical difference (p⇢0.05) in the quality of the fibrocartilagenous repair tissue between the TruFit®and microfracture groups. Conclusions In this model, both TruFit® and microfracture treatment of focal chondral defects produced a smooth, congruent, fibrocartilagenous repair tissue that was histologically similar at 18 months. The results of this study suggest that, in isolated chondral lesions, the histologic quality of the cartilage repair tissue produced following treatment by either TruFit® plugs or microfracture at 18 months is the same., Introduction New developed interventions for cartilage regeneration, with the application of 3D-scaffolds, require a further evaluation of the surgical technique. This study compared different scaffold fixation techniques in a human cadaver knee model. Methods and Materials A 2cm2 cartilage defect was created at the weight-bearing portion of the medial and lateral condyles of 20 human cadaver knees. The fixation techniques, Tissuecol® (Baxter, Utrecht), transosseous fixation, SmartNail® (ConMed Breda) and continuous cartlilage sutures (Vicryl®6.0, Ethicon, Amersfoort), were randomly assigned to the defects to implant a custom-printed porous PEGT/ PBT 1000/70/30 scaffold. Subsequently, the knees were closed and subjected to a loaded (35N) continuous-passive-motion protocol at vertical orientation. After 60 and 150 cycles the knees were reopened and the implantation sites photographed for fixation evaluation, focusing on area-coverage, outline-detachment and scaffold integrity. After 150 cycles an endpoint fixation test was performed. Differences in quality of fixation technique were analyzed by a non-parametric Kruskal-Wallis test followed by a multiple comparison test and Bonferroni correction. Results Individual evaluation of the fixation techniques revealed marginal differences for area-coverage and outline-detachment after 60 and 150 cycles. The Tissuecol® scored superior for scaffold integrity compared to transosseous (p⇠0.05) and cartilage sutures (p=0.01). Endpoint fixation was highest for the cartilage sutures whereas Tissuecol® showed a weak final fixation strength (p=0.01). Conclusions This study showed that the quality of 3D-matrix fixation is a combination of scaffold properties and mechanical stability of the fixation technique applied. Special attention devoted to scaffold properties, in relation to the fixation technique, will result in an improvement of scaffold fixation., Introduction We have been developing a new tissue-engineering technique involving scaffold-free tissue engineerd construct (TEC) bio-synthesized from synovium-derived mesenchymal stem cells (MSCs). In the present study, we investigated the frictional properties of TEC-repaired mature cartilage-like tissue. Methods and Materials Synovium-derived mesenchymal stem cells (MSCs) were cultured through 4 to 7 passages. After an addition of ascorbic acid 2-phosphate, the cells were allowed to undergo active contraction for 8 hours to develop TEC. A round-shaped, cartilaginous defect was created in the medial condyle of the femur of 12 month-old mature pigs. A TEC mass was allografted with the TEC. Six months after surgery, a cylindrically-shaped repaired specimen was extracted and subjected to a reciprocating friction test. The surface of the specimen was rubbed against a glass at the speed of 20 mm/s with the load of 70, 140 and 280 kPa. Results Laser microscopic observation indicated that the surface roughness of the TEC-repaired cartilage was slightly smaller than that of normal cartilage. The repaired tissue was covered by adhesive substance having fewer tubercles of micro size than normal cartilage. Immediately and 60s after the application of compressive force, the coefficient of friction of the TEC-repaired cartilage was slightly decreased with the increase of load. Conclusions The coefficient of friction was decreased closes to normal cartilage in the mature group. This implies that the restoration of lubrication properties is enhanced in the TEC-repaired mature tissue in accordance with the low roughness and adhesive substances at the surface. (Supported, in part, by NEDO(106001904-0) & MEXT(BERC)), Introduction In vitro expansion of hMSCs is important in cartilage tissue engineering. Our hypothesis was nanotopography enhances hMSC chondrogenic differentiation leading to additional cell sources for cartilage tissue engineering. Methods and Materials Novel nanotopographies with randomly distributed 11 nm high islands were fabricated by polymer demixing of polystyrene and polybromostyrene. Control surfaces were flat polystyrene. RT-PCR quantification of aggrecan and Collagen II mRNA was completed after hMSCs were cultured 21 days on nanotopographic surfaces in chondrogenic differentiation media(10ng/mL TGFβ1 and 10 ng/mL BMP6) or on nanotopographic surfaces in proliferation media. ANOVA and post hoc Tukey-Kramer Multiple Comparisons Test were completed. Results Steady-state levels of Collagen II mRNA were significantly increased in hMSCs cultured on 11 nm high surfaces in differentiation media(p⇠0.0001 versus hMSCs cultured on flat surfaces in differentiation or proliferation media and 11 nm high surfaces in proliferation media; n=3). Steady-state levels of aggrecan mRNA were significantly increased in hMSCs cultured on 11 nm high surfaces in differentiation media (p⇠0.001 verses hMSCs cultured on fat surfaces in differentiation or proliferation media but not 11 nm high surfaces in proliferation media; n=3). Conclusions hMSCs cultured on 11 nm high nanotopagraphy and in differentiation media containing TGFβ-1 and BMP6 expressed significantly higher levels of collagen II and aggrecan compared to hMSCs cultured in differentiation media on fat surfaces. Collagen II and aggrecan are phenotypic markers of early chondrocytic differentiation suggesting that nanotopography has a direct influence on stem cell differentiation toward the chondrocyte phenotype. Thus, nanotopographic surfaces represent a novel enabling technology for cartilage tissue engineering., Introduction Articular cartilage shows poor intrinsic repair, leading to progressive joint damage. Therapies like marrow stimulation or tissue engineering of cartilage depend on chondrogenesis of progenitor cells. However, chondrogenesis needs to take place in a diseased joint. We postulate that catabolic factors in diseased joints will inhibit chondrogenesis of progenitor cells. Methods and Materials We examined the effect of interleukin-1 (IL-1) and tumour necrosis factor alpha (TNFα) on human mesenchymal stem cells (hMSC) undergoing chondrogenesis. Osteoarthritic (OA) synovium derived conditioned medium was added to hMSC undergoing chondrogenesis. Chondrogenesis was examined by determining mRNA levels of chondrocyte specific matrix molecules. Proteoglycan deposition was analyzed by safranin O staining of histological sections. Results Chondrogenesis was completely abolished on day 14 by adding IL-1 from day 0, 3, and 7. The expression of type II and X collagen and aggrecan mRNA was blocked completely. Addition on day 10 resulted in only partial inhibition of chondrogenesis. TNFa showed similar effects but appeared to be less potent than IL-1. OA-synovium conditioned medium strongly inhibited chondrogenesis of hMSC. However, blocking IL-1 or TNFα did not inhibit completely prevent the inhibitory activity of synovial fluid on chondrogenesis. These results show that in general chondrogenesis of hMSC is inhibited by factors produced by OA synovium. Conclusions Chondrogenesis of hMSC is blocked by single catabolic factors as well as by factors produced by OA synovium. Our findings have major implications for therapies that depend on repair by mesenchymal stem cells. Successful cartilage regeneration will fail if the catabolic environment of a damaged joint is not altered., Introduction Mesenchymal stem cells are developed for cell-based regenerative therapies. Synovium-derived stromal cells(SSCs) are promising as a source for cartilage regeneration. In vivo and ex vivo procedures required for gene therapy expose cells to considerable oxidative stress. Selenium has been shown to be an essential trace element with antioxidant properties. We hypothesized that selenium can improves stem cell potency via an increase in the proliferation and self-renewal potency of SSCs. We explored the ability of selenium that increase the proliferation of SSCs with the upregulation of stemness gene expression and the effect of selenium on the acquisition of active migration ability in SSCs pretreated with selenium. Methods and Materials Isolated SSCs were analyzed by flow cytometry. To investigate the effect of selenium on the proliferation of SSCs, we performed a MTS assay and BrdU incorporation assay. The expression of stemness genes and cell migration related genes was analyzed by RT-PCR. The wound healing and cell transwell migration assay were performed. Results The SSCs in each population were negative for CD14, CD31, CD34, CD45 and positive for CD44, CD90, CD105. The number of BrdU-labeled cells was increased in response to 5 ng/ml selenium, which is consistent with the observed increase in the MTS assay. Selenium treatment exerted apparent effects on the overexpression of stemness genes (Oct4, Nanog, c-Myc, Rex1, and Klf4) and migration-associated factors. Selenium-treated cells significantly increased the migration efficiencies in a time dependent manner, as compared to control SSCs. Conclusions We demonstrated that selenium increase proliferation, migration and stemness genes expression in synovium-derived stromal cells., Introduction There is great potential for the use of cell-based therapies for repairing cartilage lesions caused by osteoarthritis. Bone marrow stromal cells (BMSCs) are a heterogeneous population of multipotent stem cells which can be differentiated to generate a three-dimensional hyaline cartilage using a defined combination of growth factors. However, there are currently no markers available for identifying those BMSCs which have the greatest chondrogenic capacity. Methods and Materials A sterile FACS sorter was used to deliver single BMSCs into individual wells of 96-well plates and each clone was proliferated in the presence of FGF, seeded onto PGA scaffolds and differentiated to produce cartilage using a tissue engineering approach. The quality of the cartilage produced by each individual clone was measured using biochemical assays for collagen types I and II and proteoglycan. Results Several BMSC clones were expanded sufficiently, without signs of senescence, to use in cartilage tissue engineering. Of those, 2 clones produced a macroscopically good tissue engineered cartilage (mean wet weight 16.8mg) with proteoglycan and type II collagen comprising 29.0% and 6.1% of the dry weight respectively. Cartilage from the other 5 clones had a mean wet weight of only 1.9mg and contained significantly lower amounts of proteoglycan and type II collagen. Conclusions We have demonstrated for the first time that different clonal populations of BMSCs have different chondrogenic capacities. We are currently using gene array technology to identify unique cell surface markers present on highly chondrogenic clones. Such markers would be used to identify those BMSCs which would be most effective for cartilage regeneration., Introduction A current challenge in mesenchymal stem cell (MSC)-based cartilage repair is to solve the donor and tissue-dependent variability of MSC cultures and to prevent chondrogenic cells from terminal differentiation like in the growth plate. The aim of this study was to select the best source for MSC which could promise stable cartilage formation in the absence of hypertrophy and ectopic in vivo mineralization. We hypothesized that MSC from synovium (SMSC) are superior to bone-marrow- (BMSC) and adipose-tissue-derived MSC (ATSC) since they are derived from a joint tissue. Methods and Materials MSC were characterized by flow cytometry. MSC pellets were cultured under chondrogenic conditions and differentiation was evaluated by histology, gene expression analysis, and determination of alkaline phosphatase activity (ALP). After chondrogenic induction, pellets were transplanted subcutaneously into SCID mice. Results BMSC, ATSC, and SMSC revealed similar COL2A1/COL10A1 mRNA levels after chondrogenic induction and were positive for collagen-type-X. BMSC and ATSC showed significantly higher ALP activity than SMSC, in which low ALP-activity before transplantation correlated with marginal calcification of explants. At elevated MMP mRNA levels, some transplants had specifically lost their collagen-type II, but not proteoglycan in vivo, or were fully degraded. Conclusions In conclusion, lower donor-dependent ALP activation and reduced mineralization of SMSC-derived heterotopic transplants surprisingly did not lead to a more stable ectopic cartilage but correlated with specific degradation of collagen-type II, fibrous dedifferentation or complete degeneration of spheroids. This emphasizes that beside appropriate induction of differentiation, locking of MSC in the desired differentiation state is a major challenge for MSC-based repair strategies., Introduction By their limited availability and their rapid dedifferentiation during expansion, chondrocytes seem not the best candidates for cartilage tissue engineering, but other autologous cell sources are actually promising. The aim of this work was to study the influence of growth factors or culture supplements (hyaluronate) (1) on the differentiation of human mesenchymal stem cells (MSC) towards chondrocytes in 3D collagen sponge culture and (2) on the extracellular matrix synthesis profile. Methods and Materials MSCs were isolated from human bone marrow and expanded in monolayer. After passage 3, MSCs were seeded into collagen sponges and cultured for 28 days in vitro with different mediums containing ITS (Insulin Transferin Selenium), FBS (fetal bovine serum), +/- hyaluronate, and TGFβ1 followed or not by BMP-2. Extracellular matrix (ECM) production and chondrogenic differentiation of MSCs during 3D culture were evaluated at D28. Chondrogenic gene expression (Coll2, Coll1, Coll3, Coll10, Aggrecan, Versican, COMP, Sox9, Osteocalcin, Alkaline Phosphatase) were investigated by real-time quantitative RT-PCR. Newly synthesized ECM was assessed histologically and immunohistochemically (Coll1 and Coll2). Results Hyaluronate alone did not promote chondrocyte differentiation of MSCs. TGFβ1 alone or associated to hyaluronate or/and sequential BMP-2 provoked chondrocyte differentiation of MSCs and an ancillary important ECM production rich in proteoglycans and Coll2. On the other hand, the association of hyaluroniate with TGFβ1 and sequential BMP-2 showed similar results associated with small calcium-rich deposits characteristic of an osteoblastic differentiation. Conclusions Chondrogenic differentiation of MSCs seeded in collagen sponges is promoted by the sequential contact with TGFβ1 switched by BMP2 thus leading to local Coll2 synthesis., Introduction Recently, there have been several reports using various SPIO nanoparticles to label stem and other cells for tracking their distribution or accumulation at the target lesion. For magnetic targeting of labeled cells, it is important to determine the effect that the labeling procedure may have on the differentiation capacity of cells. In this study, two FDA-approved agents, Resovist® and protamine sulfate (PS) were used to label human synovium-derived cells and chondrocytes. We examined the effect of labeling on differentiation capacity of cells. Methods and Materials Cells were isolated by sequential digestion and labeled for 24h with 500μg/ml Resovist® and 5μg/ml PS. Chondrogenic differentiation was induced using a pellet culture. The synovium-derived cell pellets were cultured in chemically defined chondrogenic medium with 10ng/ml TGF-β1 and 100ng/ml BMP-2 and the chondrocyte pellets were cultured in standard culture medium. The synthesized GAG was determined by binding to DMB dye. Pellets were stained with prussian blue (PB) for iron detection and with Safranin-O for proteoglycan detection. The expression of collagen I and II was detected by immunofluorescence staining. The cartilage-specific ECM genes expression was analyzed by RT-PCR. Results Synovium-derived cell pellets was larger than chondrocyte pellets without regard to labeling. The GAG production did not show a siginificant difference between the labeled and unlabeled cell pellets. Chondrogenic differentiation in labeled and unlabeled synovium-derived cell pellet cultures over 4 weeks exhibited chondrocyte-like cells embedded in a proteoglycan-rich ECM, as evident in the safranin-O staining. PB staining confirmed the presence of iron-oxide particles within the labeled cells. Chondrocyte pellets also showed positive staining. Positive immunofluorescent staining for collagen II was shown in the ECM of both groups. RT-PCR revealed no difference between labeled and unlabeled pellets of both cells. Conclusions Resovist® and PS were used to effectively label human synovium-derived cells and chondrocytes with no adverse effects on chondrogenic differentiation and phenotypic expression. Magnetic labeling may be applicable for targeting cells for cartilage repair., Introduction Mesenchymal stem cells (MSC) are promising for the treatment of articular cartilage defects; however, common protocols for in vitro chondrogenesis induce typical features of hypertrophic chondrocytes reminiscent of endochondral bone formation. We here analysed the early healing response in experimental full-thickness cartilage defects, asking whether MSC can differentiate into chondrocytes in an orthotopic environment, without the induction of hypertrophic markers. Methods and Materials Cartilage defects in knees of minipigs were covered with a collagen-type I/III membrane, and half of them received transplantation of expanded autologous MSC. At 1, 3 and 8 weeks, morphological and molecular aspects of repair were assessed. Results At 1 and 3 weeks after surgery only marginal Col2A1 and no AGC expression were detectable in both groups. At 8 weeks Col2A1 and AGC levels had significantly increased. The orthotopic environment triggered a spatially organized repair tissue with upper fibrous, intermediate chondrogenic, and low layer hypertrophic differentiation of cells and a trend to more safranin-O and collagen type II-positive samples after MSC transplantation at 8 weeks. Compared to in vitro chondrogenesis, Col10A1 and MMP13 mRNA levels were suppressed. Conclusions These data indicate that, as opposed to in vitro chondrogenic induction of MSC, the in vivo signalling molecules and biomechanical stimuli provide an appropriate environment for progenitor cells to differentiate into collagen type X-negative chondrocytes. Thus, until better in vitro induction protocols become available for chondrogenesis of MSC, their pre-differentiation prior to transplantation may be unnecessary., Introduction Cell therapy approaches for cartilage repair rely on isolated chondrocytes which lose their chondrogenic potential during expansion. Mesenchymal stem cells (MSC) represent an alternative if their default pathway towards osteogenesis can be prevented. In this study, mixed pellet cultures containing different ratios of chondrocytes and MSC were prepared to examine possible cellular crosstalk leading towards chondrogenesis. Methods and Materials Chondrocytes from human articular cartilage expanded for 8 days in monolayer culture and human bone marrow MSC were labeled with PKH 67 and 26, respectively. Cells were mixed at different ratios and chondrogenesis was induced in pellet cultures in serum free medium with and without TGF-β1 and dexamethasone. Proteoglycan deposition, collagen II, collagen X synthesis and S100 protein were assessed via histology and immunohistochemistry. DNA/GAG content was quantified using CyQuant kit and DMMB assay. Results Fluorescence of labeled cells indicated an equal distribution throughout cocultured pellets. 100% chondrocyte pellets produced neocartilage rich in proteoglycans, collagen II, and had high expression of S100. 100% MSC pellets did not produce any chondrogenic specific proteins. In 50–75% MSC pellets proteoglycan and collagen II were detected and most of cells expressed S100 protein. Proteoglycan production was confirmed via GAG/DNA measurements. Collagen X, indicative of osteogenic differentiation, was detected only in 100% MSC pellets, indicating that presence of only 25% chondrocytes stabilized chondrogenic phenotype of MSC. Conclusions Detection of cartilage matrix proteins in cocultured chondrocytes and MSC pellets incubated in absence of external chondrogenic factors suggests crosstalk from chondrocytes to MSC inducing chondrogenesis and preventing further differentiation of MSC towards osteogenesis., Introduction Apoptosis is defined as programmed cellular death. This biological phenomenon has been described by some authors, when studying the articular cartilage after a traumatic event. It observes also, that some mediators (Interleukin-1, Nitrous Oxide and TNF-α), when in contact with chondrocytes, they are capable to perpetuate the effect of programmed death, culminating in osteoarthritis. On the other hand, apoptosis can be interrupted by substances such: diacerhein, glucosamine and osteogenic protein-1 (OP-1). Purpose: to evaluate if the injection of intra-articular platelet-rich plasma (PRP) can reduce impact-induced chondrocyte apoptosis. Methods and Materials A double-blind study was developed with four knees of two adult rabbits. Each knee was injured 3 times with 1 kg weight released from the top of a cylinder of one-meter length. Subsequently, 1ml PRP was injected in the right knees and 1ml of normal saline in the left knees. The animals were euthanized ten days after the intervention. All cartilage was removed from the 4 knees and prepared for analysis in electronic microscopy (EM). The individuals involved in the preparation and analysis of the grids were blind to the experiment. Seven to ten meshes of EM were obtained for each knee. The sum of all apoptotic cells in each mesh was registered. Results The PRP-injected knees summed apoptosis 47,62% (50/105) and 48,36% (59/122), respectively. NS-injected showed 56.67% (17/30) and 70.40% (88/125) of apoptosis. PRP injected knees had statistically significant less apoptosis 48.02% than NS injected 67.74% (p Conclusions Immediately post-traumatic intra-articular injection of PRP reduces impact-induced chondrocyte apoptosis in rabbits., Introduction Mesenchymal stem cells (MSC) are a promising source for cell-based tissue engineering of cartilage. The purpose of this study was to evaluate the chondrogenic potential of MSC in two different 3-D cultures, a scaffold-free high-density diffusion-culture (DC) using 106 cells as well as a pellet-culture (PC) using 2,5 × 105 cells, where optimal combinations of growth factors were identified. Methods and Materials MSC were expanded in medium containing FGF-2 and cultured under serum-free conditions in DC or PC. Except for control cultures the medium contained TGF-β3 and IGF-1, TGF-β3 and FGF-2 or TGF-β3. Cultures were stained with toluidine blue and safranin-O and evaluated by immunohistochemical staining for type II collagen. Quantitative real time reverse transcriptase polymerase chain reaction (qRT-PCR) was performed for cartilage markers. Results Except for controls all cultures showed positive staining for proteoglycan and type II collagen. Type II collagen, aggrecan and SOX-9 mRNA were detected in all differentiated cultures with higher amount in PC than DC. The highest expression of type II collagen and aggrecan was found in PC with TGF-β3 and either IGF-1 or FGF-2. An increased type X collagen expression could be shown whenever FGF-2 was added. Conclusions The study shows that an effective chondrogenic differentiation can be obtained in DC. Collagen type II expression and aggrecan content of DC showed results which do not rank far behind PC. Therefore DC, which show an appropriate graft size and cartilage like structure, might be an interesting tool to be used for clinical cartilage repair in regenerative medicine., Introduction Differentiation of BM-MSCs into adult chondrocytes represents a complex physiological process. Full characterization of each individual stage of chondrogenesis through CD cell surface antigens has failed to provide a reliable and reproducible tool. The physiological micro-environment of chondrocytes is hypoxic, which triggers over-expression of SLC2A proteins (GLUTs) in their membranes. This could provide a new generation of functional and morphological markers of chondrogenesis. Methods and Materials BM-MSCs were cultured and differentiated along with adult chondrocytes in hypoxic (3% O2 tension) and normoxic (20% O2) conditions. These cell populations were screened for the presence of the 12 GLUT genes and for the variation of their transcription and translation during chondrogenesis. Functional characterization of the GLUTs expressed in the cellular membrane was carried out using radio-isotope labeled hexose fluxes according to the substrate specificity and kinetic properties particular to each SLC2A isoforms. Results The functional genotype and phenotype of the adult chondrocyte and hypoxic BM-MSCs comprised a mosaic expression of SLC2A transporters. Moreover, similarities in functional “GLUT signature” between BM-MSCs cultured in hypoxic conditions and adult chondrocytes were identified. Investigation of the uptake of a panel of five individual sugars (glucose, fructose, 2-deoxy-glucose, 3-orthomethyl-glucose, and galactose) in these cellular populations, in the presence and absence of a GLUT1-specific inhibitor, showed that SLC2A class II transporters (GLUT 5, 7, 9, 11) play a more important role in chondrogenesis compared to the ubiquitously-expressed GLUT1. Conclusions Identification of the chondrogenic BM-MSC “GLUT signature” will enable selection of those chondrocyte precursors that have the best potential for producing viable articular cartilage., Introduction We Isolated Peripheral blood mononuclear cells and stromal adipose tissue cells from adult horses. Adult stem cell (ASC) are undifferentiated cells commonly present in functional tissues able to renew themselves, when injured, can develop mature functional tissues. We used the surface specific markers CD34, CD45, CD47, CD14, CD117, CD73 and CD90 to identify MSC by flow cytometry analyses. The aim of this study was to investigate the possibility to isolate pluripotent precursor cells from equine peripheral blood and fat tissue, to compare the surface antigen groups Methods and Materials We obtained mononuclear cells from horses by Ficoll gradient centrifugation of peripheral blood. We also isolated fibroblastic cells from fat tissue, incubated the two samples with monoclonal antibodies for flow cytometry analysis to determine if in harvested cells were found specific precursor cells surface antigens. With the positive cells we conducted plasticity trials to evaluate the aptitude of the precursors isolated to generate different tissues. We stimulated our both cells colonies to generate cartilage, glial cells and fat tissue. Results Peripheral blood precursors and stromal cells from adipose tissue were harvested from horses. The presence of specific stem cell precursor surface markers were proved using antibodies synthesized to recognize human surface markers From the horse blood we got a CD34+, CD90+, CD 73+, CD45-, CD14-, CD117- phenotype and the fat cells CD90+, CD117+, CD14-, CD47-, surface markers were lost at the end of the cloning phase Conclusions Indicates cross antigenicity and let us presume proteic sequences homology between horse and human receptors., Introduction For healing articular cartilage damage, synovium-derived cells has been studied as a useful cell source because the cell has several advantages. Our previous study showed that co-treatment of TGFβ-1, BMP-2 and dexamethasone promoted chondrogenesis of synovium-derived cells. To analyze underlying detail mechanisms of the promoted chondrogenesis, we conducted microarray analysis at each time point of chondrogenic process. Methods and Materials 1) Human synovium-derived cells were isolated from knee joint and cultured in DMEM with 10% FBS. 2) Passage 2 synovium-derived cells were centrifuged to make pellets and then cultured with chondrogenic medium containing dexamethasone, BMP-2 and TGF-β1. 3) Total RNA from the pellets were analyzed by RT-PCR and microarray. Illumina's Sentrix HumanRef-8 Expression BeadChips were used in this experiment. 4) GAG amount in pellets were determined by DMB analysis. 5) Pellet specimens were subjected to Safranin-O staining for detection of proteoglycan. Results The protein level of proteoglycan and GAG and the RNA level of type 1,2,10-collagen, SOX-9, aggrecan were increased during chondrogenesis. In microarray analysis, genes showing minimal variation were excluded(p-value⇠0.0001, 5204 genes). We could divide all 5204 genes into 19 clusters showing similar patterns of gene level change. Among the genes showing increased expressions, genes relating organ development (especially relating extracellular matrix) and glycolate metabolism were remarkable. Among the genes showing decreased expressions, genes relating cell proliferation and cell death were notable. Conclusions In this study, we could analyze the expression changes in several genes during chondrogenesis of synovium-derived cells. Further experiment will follow to enlighten detail mechanism of these genes on chondrogenesis., Introduction Human embryonic stem (hES) cells are pluripotent cells and have the capability to differentiate to various cell types. The hES cells are suggested as the ultimate source for cell based therapies and may represent an alternative cell source for the treatment of cartilage defects. Differentiation of hES cells to chondrogenesis by direct co-culture is the purpose of this study. Methods and Materials Here we studied how co-culture with human articular chondrocytes affects the expansion potential, morphology, expression of surface markers and differentiation abilities of hES cells. Undifferentiated hES cells were co-cultured with neonatal or adult articular chondrocytes in a pellet mass system. Results Our results demonstrate that unlike undifferentiated hES cells co-cultured hES cells could be massively expanded on plastic with a morphology and expression of surface markers similar to mesenchymal stem cells. Co-culture further resulted in a more homogenous pellet and significantly increased cartilage matrix production. Co-cultured cells also formed colonies in agarose suspension culture demonstrating differentiation towards chondroprogenitor cells, while no colonies were detected in the hES cells. Co-culture also resulted in a significantly decreased osteogenic potential. Conclusions Our results confirm the potential of the culture micro-environment to influence hES cell morphology, expansion potential, and differentiation abilities over several population doublings. These hES cells share some qualities that are characteristic for chondrocytes. So far all results point out that direct co-culture could have a strong chondrogenic effect on undifferentiated hES cells. This direct co-culture model can be useful for studying early chondrogenesis, Introduction Hypertrophy of transplanted chondrogenic cells is thought to limit cell-based cartilage repair, as it leads to apoptosis and ossification. The present study compares gene transfer of the transcription factor SOX-9 and BMP-2 as agents of chondrogenesis in human MSCs maintained as pellet cultures Methods and Materials Cultures of human marrow-MSCs were infected with 5 × 102 particles/cell of Ad.SOX-9, or Ad.BMP-2, seeded into aggregates and cultured for three weeks in serum-free medium. Untransduced cells or cultures transduced with marker genes (GFP/ Luc) served as controls. Expression of SOX-9 was determined by western blot and BMP-2 by ELISA, and aggregates were analyzed histologically, immunohistochemically, biochemically and by real time RT-PCR for chondrogenesis and hypertrophy. Results Levels of SOX-9 or BMP-2 (35–55 ng/mL) in the media were initially high and declined thereafter. SOX-9 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs as judged by lacuna formation, strong staining for proteoglycans and collagen type II, increased levels of GAG synthesis, and expression of mRNAs associated with the chondrocyte phenotype. However, BMP-2 modified aggregates showed a strong tendency to progress towards hypertrophy, as judged by expression of alkaline phosphatase, immunohistochemical staining for type X collagen protein, and lacunar size. Conclusions SOX-9 and BMP-2 were strongly effective in provoking chondrogenesis by primary human MSCs in pellet culture. However, chondrogenesis triggered by SOX-9 gene transfer showed less evidence of hypertrophic differentiation than that triggered by the BMP-2 cDNA, suggesting that SOX-9 may be a more suitable for use in cartilage repair., Introduction To determine whether the implantation of alginate beads containing human mature allogenic chondrocytes is feasible and safe for the treatment of symptomatic cartilage defects in the knee. Methods and Materials A biodegradable, alginate-based biocompatible scaffold containing human mature allogenic chondrocytes was used for the treatment of chondral and osteochondral lesions in the knee. Twenty-one patients were clinically prospectively evaluated with use of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and a Visual Analogue Scale (VAS) for pain preoperatively and at 3, 6, 9 and 12 months of follow-up. Of the 21 patients, 13 had consented to the taking of a biopsy for investigative purposes from the area of implantation at 12 months of follow-up, allowing histological assessment of the repair tissue. Results A statistically significant clinical improvement became apparent after 6 months and patients continued to improve during the 12 months of follow-up. Adverse reactions to the alginate/fibrin matrix seeded with the allogenic cartilage cells were not observed. Histological analysis of the biopsy specimens rated the repair tissue as hyaline-like in 15.3 %, as mixed tissue in 46.2 %, as fibrocartilage in 30.8% and as fibrous in 7.7%. Conclusions The results of this short term pilot study show that the alginate-based scaffold containing human mature allogenic chondrocytes is feasible and safe for the treatment of symptomatic cartilage defects of the knee. The described technique provides clinical and histological outcomes equal to those of other cartilage repair techniques., Introduction Hallux rigidus is characterized by limited range of motion at the first metatarsophalangeal joint (I MTPJ) with pain and disability. Treatment options in severe hallux rigidus are arthrodesis or prosthetic replacement, although significative drawbacks of these techniques are reported. Bipolar fresh osteochondral allograft have been successfully used in the treatment of arthritis of the ankle joint and demonstrated to be able to provide new joint surfaces with cartilage able to survive the transplantation and satisfactory clinical results. Aim of this study was to present the applicability of bipolar fresh allograft to the I MTPJ and to describe the results. Methods and Materials 3 patients (4 feet), mean age 49yrs, affected by hallux rigidus grade III, underwent fresh bipolar osteochondral allograft of the MTPJ. Patients evaluation included clinical and X-Rays evaluation at different established follow-up (up to 3 yrs). Results Before surgery patients complained pain and no range of motion at the I MTPJ. Mean AOFAS score was 48±6, while it was 80±4 at follow-up (p⇠0.005). Good consolidation of the graft, with satisfying clinical result in term of stable pain free join with a good range of motion was resumed in 2 patients (one bilateral) at follow up. One graft non-union occurred as post-operative complication and required revision. Conclusions The presented technique demonstrated a new possible application of fresh osteochondral allograft. Although the number of cases is still exiguous this report may offer an interesting opportunity for further research and application in the controversial topic of grade III hallux rigidus treatment., Introduction DeNovo® NT Natural Tissue Graft Purpose: To evaluate the clinical application of particulated allograft cartilage. Preclinical animal studies demonstrated the potential for particulated articular cartilage to fill focal articular cartilage lesions of the knee with hyaline-like cartilage. Methods and Materials A prospective clinical case study of DeNovo NT Graft (particulated human juvenile cartilage allograft) was initiated in 2007 after obtaining the local IRB approval. This is a preliminary report on the first 7 patients who were treated with DeNovo NT Graft. A major inclusion criterion was the presence of one or two persistently symptomatic untreated focal contained femoral articular cartilage lesions of a normally aligned stable knee with intact menisci. Results During 2007- 2008, 7 patients were enrolled (1 female and 6 males) with ages ranging from 20 to 49 with a mean age of 35. The 7 patients have current follow up of between 1 month and 1 year. The small subject number does not allow for statistical analysis, but improvements are noted from the baseline using the KOOS score (and KOOS subscales), IKDC subjective and health assessments, Marx and VAS. Conclusions The early MRI evaluations show filling of the defect and reveal no transplant dislodgment. Patient enrollment will continue and outcomes will be monitored until all patients reach 2-year follow up. This initial report of DeNovo NT Graft demonstrates early clinical improvement in a small cohort., Introduction The present study was designed to evaluate the implantation of alginate beads containing human mature allogenic chondrocytes for the treatment of symptomatic cartilage defects of the knee. MRI was used for the morphological analysis of cartilage repair. The correlation between MRI findings and clinical outcome was also studied. Methods and Materials A alginate-based scaffold containing human mature allogenic chondrocytes was used for the treatment of symptomatic (osteo)chondral lesions in the knee. Twenty-one patients were prospectively evaluated with use of the WOMAC and VAS for pain preoperatively and at 3, 6, 9 and 12 months of follow-up. Of the 21 patients, 12 had consented to follow the postoperative MRI evaluation protocol. MRI data were analyzed based on the original MOCART and modified MOCART scoring system. The correlation between the clinical outcome and MRI findings was evaluated. Results A statistically significant clinical improvement became apparent after 6 months and patients continued to improve during the 12 months of follow-up. One of the two MRI scoring systems that were used, showed a statistically significant deterioration of the repair tissue at one year of follow-up. We did not find a consistent correlation between the MRI criteria and the clinical results. Conclusions Two MOCART-based scoring systems were used in a longitudinal fashion and allowed a practical and morphological evaluation of the repair tissue. However, the correlation between clinical outcome and MRI findings was poor. The promising short-term clinical outcome of the allogenic chondrocytes/alginate beads implantation was not confirmed by the short-term MRI findings., Introduction Limitations of fresh osteochondral (FOC) allograft transplantation are, scarce supply and short shelf-life. A new cartilage repair technology with off-the-shelf, processed OC grafts (Chondrofix®, a proprietary product of Zimmer Orthobiologics) has been developed and evaluated in an equine model. Methods and Materials OC plugs harvested from equine femoral condyles were machined into either mushroom (MOC) or cylindrical (COC) shape and then processed before implantation. With IACUC approval, four defects were created in the medial trochlea in 6 horses and randomly repaired with MOC, COC, FOC or left untreated. All horses were allowed load-bearing immediately after surgery and underwent controlled strenuous exercise on a high-speed treadmill starting at 4 months postoperatively. Three animals were euthanized at 9 months and the remaining 3 at 18 months post-operatively. Arthroscopic examinations were performed at 3, 6 and 9 months for all animals and at 12 and 18 months for those 3 animals sacrificed at 18 months. Repair tissue was evaluated by MRI, histology, and immunohistochemistry. Results Arthroscopically, all MOC and COC retained smooth surfaces and were stiff when probed. MOC maintained the surface level better with the surrounding native cartilage than COC, which could be improved with a better clearance ft. Both MOC and COC performed better histologically than FOC treatments. Subchondral bone cysts were observed for FOC, but not for MOC and COC. Type II collagen and aggrecan were labeled in all repair tissue. Conclusions Durable cartilage repair was achieved by implantation of both MOC and COC grafts after 18 months, even with immediate load-bearing of the grafts., Introduction Osteochondral lesions of the ankle result from rotational injuries of the ankle in athletes. The purpose of this study is to evaluate the results of arthroscopic treatment of ankle osteochondral lesions and to define the clinical and arthroscopic factors influencing prognosis. Methods and Materials From 1993 to 2002 a total of 108 patients underwent 132 arthroscopic procedures for diagnosis and treatment of osteochondral lesions of the ankle. The evaluations included a pre and postoperative clinical scoring, radiographic, CT and MRI of the ankle. Operative techniques included: microfructures technique, fixation of the lesions using polylactic acid “Biofix” rods, diagnostic arthroscopy followed by cartilage lesions shaving, retrograde drilling of the lesion to the subchondral bone (12 patients) bone graft filling of subchondral cysts and osteochondral autografts. Results Traumatic etiology of the lesions was found to be associated with postero-medial Talar lesions. Significant clinical and radiographic improvements comparing pre and post operative CT and clinical score. Clinical improvements where found to correlate directly with CT grading. Tibial and Talar “kissing lesions” correlated with poor pre operative clinical score. Lesions with sclerosis and cyst on CT, appeared to have less clinical improvement with surgery. Conclusions Ankle x-rays and CT plays a limited role in planing the intra-operative procedure. Findings like sclerosis and subchondral cyst carry less favorable prognosis. Arthroscopy is a valuable tool for evaluation and treatment of ankle osteochondral lesions. The operative technique should be selected according to arthroscopic findings and the surgeon should be prepared to tailor the different types of treatment to each lesion., Introduction We report on the arthroscopic MACI technique for the treatment of chondral defects of the ankle. This bioscaffold can be implanted into the defect using exclusively fibrin glue, allowing the possibility to perform the procedure in articular sites, in which putting stitches for the periosteal patch is impossible. Methods and Materials Five patients suffering from large (⇢1.5cm2) cartilage lesions of the ankle, underwent MACI procedure. The implantation procedure was performed through traditional arthroscopic portals. After the debridment, the membranes were introduced through a cannula and spreaded onto the lesions by a probe. Keeping the correct graft orientation, the seeded membranes were then fixed with fibrin glue at optimized thrombin dilution, temporarily excluding water flow. Clinical outcomes were assessed by ICRS evaluation package. The graft integration was evaluated by MRI at 6 and 12 months after surgery. Results Filling of the defect was shown 12 months after surgery by MRI, which showed in 4 cases out of 5, a hyaline-like cartilage signal. Improvement was found subjectively and in ankle function levels at 12 months after the operation. At 18 months after implantation a biopsy was arthroscopically obtained from one patient. It showed smooth articular surface, hyaline-like matrix cartilage, cluster cell distribution and a nearly normal tide-mark. Conclusions Clinical improvement and hyaline-like appearance of the repair tissue suggest that arthroscopic MACI implantation is an effective technique for the treatment of large lesions of the articular cartilage of the talus. The arthroscopic approach allowed an optimal view of the lesion, avoiding arthrotomy or malleolar osteotomy., Introduction The aim of this study was to explore the feasibility of T2 mapping after matrix associated autologous chondrocyte implantation with Hyalograft C in the ankle joint. Methods and Materials Ten patients (25.5 ± 5.3 years) were included in the study. Mean defect size was 1,31 ± 0,37 (0,8–2) cm2 and the mean follow up period was 4.1 ± 1,8 (1.5–7.25) years. All defects were singular and located on the talar dome. MR examinations were performed on a 3T MR unit with a flexible coil using a multi-echo spin-echo technique. T2 maps were obtained using a pixel wise, mono-exponential non-negative least squares (NNLS) fit analysis. Region of Interest (ROI) analysis was carried out for the repair site and reference region of intact cartilage for comparison. T2 of the repair sites and of the cartilage reference regions was statistically analysed with a double-tailed, unpaired t-test. Results Global T2 values of the repair tissue (mean 30 ms, ± SD 8) were significantly lower (p = 0.03) compared to global T2 values of native cartilage (36 ms, ± 9). Conclusions High resolution capabilities at 3.0T make T2 mapping feasible in the ankle and demonstrated a slight difference in collagen and water content between Hyalograft C repair tissue and healthy hyaline cartilage., Introduction Studies have shown encouraging early results of Autologous Chondrocyte Implantation (ACI) in the ankle. We present a case series with clinical results up to 10 years. Methods and Materials 23 patients who had chondral or osteochondral defects of the talar dome underwent treatment with ACI between June 1998 and September 2005. Chondrocyte harvest was taken from the ipsilateral knee. Patients were evaluated prospectively using the Mazur ankle score and Lysholm knee score pre-operatively and at yearly intervals. Arthroscopic and histological evaluation was performed at 1 year. Results 16 males and 7 females were treated with a mean age of 37 years (17–61 years.) Median follow-up was 6 years (3–10 years.) Mean pre-operative Mazur ankle score was 54.0. At 1 year, 2 years and last follow-up, mean ankle score was 63.7, 68.0 and 66.5. Mean preoperative Lysholm knee score was 92.3. At 1 year, 2 years and last follow-up, mean knee score was 87.1, 85.4 and 89.4. Conclusions The good early clinical results of ACI in the ankle are maintained at longer term follow-up. Harvesting chondrocytes from the ipsilateral knee may lead to a reduction in knee score at early follow-up, but in general this recovers towards baseline in longer follow-up. Currently we are taking chondrocyte harvest from the ipsilateral ankle in order to eliminate any potential morbidity in the knee., Introduction The treatment algorithm for osteochondral lesions of the talus (OLT) remains controversial. We present a prospective case series of 42 patients with OLTs surgically treated between 2000–2006 using a new algorithm based on the ICRS or international cartilage repair society classification of cartilage defects and the size of the defect. Methods and Materials A total of 42 patients with an average age of 34.9 years (SD 12.1) who had failed previous non-operative or operative intervention were prospectively assigned into 3 groups (based on the size and the depth of the lesion) to receive either drilling (N=25; antegrade or retrograde), Single plug osteochondral autograft (N=8) or mosaicplasty / allograft reconstruction (N=9). Approximately 54.8% (23/42) were female, and 78% (32/42) reported antecedent trauma. Mean follow-up was 18 months and median duration of symptoms was 24 months. Results There was a statistically significant improvement (p ⇠ 0.0001, Wilcoxon Signed Rank test) in American Orthopaedic Foot and Ankle Society scores (average 24.8 points), Visual Analog Scale (VAS) Function scores (average 3.51 points), and decrease in VAS Pain scores (average 4.49 points) after surgery regardless of the procedure. 78.9% reported satisfaction with their treatment. Age, BMI (body mass index), duration of symptoms and length of follow-up did not correlate with the outcomes of post VAS Pain, VAS function, or AOFAS. Conclusions Using a combination of drilling, autograft plugs and allograft is successful in treating talus osteochondral lesions. Our treatment algorithm based on the ICRS staging of cartilage defects produced results comparable to published data., Introduction Qualitative evaluation of postoperative outcome in cartilage repair techniques is an issue due to morbidity of bioptic second look. T2 mapping is becoming increasingly popular in the knee, providing information about the histological and biochemical contents of healthy or reparative tissue. Nevertheless, little is known about its applicability to the ankle due to thinner cartilage layer. Aim of this study was to investigate the validity of T2 mapping in ankle cartilage characterization. Methods and Materials 20 healthy volunteers and 30 patients affected by osteochondral lesions of the talus and previously treated by different cartilage repair techniques, were evaluated by T2 mapping. Reparative procedures included microfractures, Autologous Chondrocyte Implantation (open or arthroscopic) and Bone Marrow Derived Cell's Transplantation. DPFSE with or without fat suppression, T2FSE with or without fat suppression, 3D SPGR and T2-Mapping using a dedicated phased array coil and 1.5 T MR scanner were used as MRI acquisition protocol. MRI results were correlated with clinical score (AOFAS) in the cases who received a cartilage reconstruction treatment. Results A statistically significant correlation (p⇠0.05) was shown between MRI and clinical results. A reduced mean T2 value, suggestive for fibrocartilage features, was shown at repair sites in microfractures, whereas no significative differences with healthy hyaline cartilage mean T2 value, were found in other repair techniques with good to excellent clinical score. Conclusions T2 mapping demonstrated to be adequate in characterizing cartilage tissue in the ankle. Further studies are required to implement a tool which may over time be a valuable noninvasive alternative to bioptic evaluation., Introduction Synthetic osteochondral grafts offer advantages over other treatments for osteochondral defects as they are single procedures with no donor site morbidity. Tru-fit™ is a biphasic osteochondral biodegradable graft consisting of a matrix of calcium triphosphate and the cartilaginous substitute of poly DL-lactide-co-glycolide. Methods and Materials Five patients with traumatic osteochondral lesions of the talus and one patient with a lesion of the distal tibia were treated with Tru-fit™ osteochondral plugs. All had failed previous arthroscopic debridement and microfracture and had a cystic bone defect beneath the fibrocartilage layer. Four patients required medial maleolar osteotomy for access. The superficial fibrous cartilage was removed from the defect and cores of subchondral bone were made to accommodate the Tru-fit™ plug. The patients were kept non weight bearing for 6 weeks but mobilised their ankles from day 2. Results At 12 months the postoperative AOFAS, AOS and SF36 scores had significantly improved and all patients were satisfied. There were no surgical complications but one patient injured his peroneal tendons playing football at 9 months post operatively. All patients returned to work and sports. Follow up MRI scans demonstrate good incorporation of the plugs with reduced surrounding bone marrow oedema but continue to show high signal within the plugs themselves. T-2 cartilage mapping demonstrates incorporation of cartilage but at 1 year the signal remains different from the surrounding hyaline cartilage Conclusions The preliminary clinical and radiological results of this procedure are encouraging with excellent improvement in symptoms but more patients and longer follow up are required before firm conclusions can be drawn., Introduction Microfracture is a frequently used technique for the repair of cartilage defects. Here, we introduce a cell-free cartilage implant used for covering of defects pre-treated with microfracture (Mfx) that attracts mesenchymal progenitors (MSC), improves defect filling and leads to a more hyaline-like repair tissue compared to Mfx. Methods and Materials The implant based on a resorbable polyglycolic acid scaffold, hyaluronan and serum attracts MSC as evaluated using a 96-well chemotaxis assay. Cartilage repair tissue formation was shown in high-density MSC cultures after stimulation with hyaluronan and in an ovine joint defect model by gene expression analysis and immunohistochemistry. Clinically, the cell-free implant was used for treating a traumatic and a degenerative articular cartilage defect. Cartilage repair was evaluated by MRI at 12 month after implantation. Results Human serum from whole blood significantly recruits MSC. Gene expression analysis showed that hyaluronan supports the induction of chondrogenic marker genes. In the ovine model, at 6 months, the repair tissue formed by the cell-free implant was hyaline-like and showed a cartilage matrix rich in type II collagen. Clinically, at 12 months, the traumatic and the degenerative defect showed a good to excellent defect filling with a hyperintense repair tissue signal and smooth peripheral integration. Conclusions Covering of cartilage defects with the cell-free implant after Mfx treatment improves repair tissue formation by guiding MSC into the defect and supporting chondrogenic differentiation. First clinical results suggest that defect covering with a resorbable polymer-based implant may be a good treatment option in microfracture., Introduction From 2004 we used MACI for treating focal chondral defects of the knee. We reviewed the two-year outcomes with clinical evaluation, second-look arthroscopy and core biopsies. Methods and Materials From March 2004 to August 2006, 132 consecutive knees with focal chondral defects (grade III or IV by modified Outerbridge) were treated with MACI technique. An initial arthroscopy was carried out. After one month we implanted cells with a parapatellar arthrotomy approach. A standardized postoperative rehabilitation protocol was carried out. We reviewed 43 patients with 3 yrs follow-up with clinical evaluation, ICRS and IKDC evaluation form. A second-look arthroscopy and core biopsies were performed in 8 of them (12 months follow-up). Results Improvement in mean subjective IKDC score from pre-operative (36.5) to 12 months (57.2) post-MACI were observed. Knee function levels also improved (88 % ICRS III/IV to 64.2 % I/II) from pre-operative period to 12 months post-implantation. Objective pre-operatively IKDC score of A were observed in 2%, score B in 87% and score C in 11%. This improve to 93% score B and 7% score A at 36 months post-implantation. Second-look arthroscopy carried out in 8 knees demonstrated all grafts to be normal / nearly normal based on the International Cartilage Repair Society (ICRS), and core biopsies demonstrated 3 (43%) grafts to have hyaline / hyaline-like tissue, 4 (57%) hyaline-like tissue / fibrocartilage; Conclusions Improvement in clinical, second-look arthroscopy and core biopsies indicate that the knees treated with MACI had improved function and focal defects resurfaced., Introduction Both, matrix-assisted chondrocyte transplantation (MACT) and osteochondral autograft transplantation (OCT), are applied for treatment of articular cartilage defects. While previous clinical studies have compared the outcome between these procedures, there is no information investigating the respective ultrastructural composition using T2 mapping comparing cartilage T2 values of the repair tissue (RT). Methods and Materials A total of 18 patients that underwent MACT or OCT for treatment of cartilage defects at the knee joint (9 MACT, 9 OCT) were matched for gender (1 female, 8 male pairs), age (33.8), BMI (28.3), defect localization, and postoperative interval (41.6 months). T2 assessment was accomplished by T2 maps, while the clinical evaluation included the Lysholm and Cincinnati knee scores, a visual analogue scale for pain (VAS), the Tegner activity scale, and the Short Form-36. Results Global T2 values of healthy cartilage (HC) were similar among groups, while T2 values of the RT following MACT (46.8 ms, SD 8.6) were significantly lower when compared to RT T2 values after OCT (55.5 ms, SD 6.7) (P ⇠ 0.05). MACT values were also lower in comparison to HC (52.5 ms, SD 7.9) within MACT patients (P ⇠ 0.05), while OCT values were higher compared to HC (49.9 ms, SD 5.1) within OCT patients (P ⇠ 0.05). Only the Lysholm score revealed a significant difference in the clinical outcome (MACT 77.0, OCT 66.8; P ⇠ 0.05). Conclusions These findings indicate that MACT and OCT result in a different ultrastructural outcome, which is only partially represented by the clinical picture., Introduction Various approaches have been proposed to treat articular cartilage lesions; nevertheless opinions on indications and clinical efficacy of these techniques are still controversial. In this prospective clinical study our goal is to evaluate second generation ACI technique for treatment of femoral condyles cartilage lesions at a medium term follow up. MRI has become the method of choice for non-invasive follow-up of patients after cartilage repair surgery. Methods and Materials Prospective evaluation of 40 cases with full-thickness knee chondral lesions (mean value 2,5 cm2) treated with arthroscopic Autologous Chondrocyte Implantation technique. In 23 patients associated procedures were performed, whereas 15 patients had undergone previous surgery. At 5 years follow up ICRS form, Tegner were used for clinical evaluation. All the patients were also evaluated with MRI and analyzed with MOCART score. Results ICRS objective evaluation showed 90% of patients rated good and excellent results at 5 years follow up and the subjective score (mean value 83) demonstrated a marked improvement from preoperative to 5 years of follow up. Also the Sport activity level evaluated with Tegner score showed a statistical significant improvement at 5 years follow up (5). The mean total MOCART score obtained at 5 years was 73,5, with a significant correlation with the clinical outcome. Conclusions This arthroscopic surgery appears to be a valid solution for treatment of grade III-IV cartilage defects. MRI is reliable, reproducibile and accurate tool for assessing cartilage repair tissue., Introduction The objectives of this study was to evaluate the clinical outcome and the safety of BIOSEED-C in consecutive patients treated for symptomatic articular cartilage defects of the knee joint at different times −2weeks, 1, 3, 6, 12, 24 months-post implantation Methods and Materials 50 patients −57% males, 14.3% females with mean age 35.18 years-were followed for 24 months. The defects were localized on femoral condyles (62%), patella (28%) and troclea (10%); 52 % had simple lesions, 31% complex and 17 % salvage procedure. Mean defect size was 4.6 in simple, 4.8 in complex, 9.3cm2 in salvage lesions; 56% of defects were on the femoral condyle, 4.4% in patella, 20 % in tibia plate and 20% in the troclea. The clinical outcome was determined with standard scores (ICRS-IKDC) and 1.5T high resolution magnetic resonance imaging (MRI) by the MOCART score. Results IKDC objective scoring system at 24 months showed a normal or nearly normal knee in 86.2%.; IKDC subjective knee evaluation was 84%; In MRI evaluation 24 months after implantation a complete filling of the defect was found in 78% and a complete integration of the border zone to the adjacent cartilage in 83% with intact subchondral lamina and bone present in 85%. No product-specific adverse events were recorded. We recorded no graft failures, but three cases of delamination have been noticed. Conclusions Based on the results obtained, we conclude that BIOSEED-C® is a successful and safe therapeutic option for the treatment of cartilage lesions of the knee., Introduction Osteonecrosis of the knee is a serious potential complication of corticosteroid therapy with limited treatment options in young, active patients. We report on outcomes of fresh osteochondral allografting for high-grade steroid-associated osteonecrosis of the femoral condyles. Methods and Materials Between 1984–2006, osteochondral allografting was performed in 19 patients (25 knees) with symptomatic modified Ficat stage 3/4 osteonecrosis secondary to corticosteroid therapy; 13 females, 6 males with mean age of 27.8 years (range16–68). 5 had bilateral surgery (total 22 knees). 16 knees had unicondylar (12 lateral, 7 medial), 6 bicondylar lesions. 11 (44%) had previous surgery (average 1.5, range 1–5). Clinical evaluation included modified D'Aubigne-Postel (18-point), IKDC pain, IKDC function, Knee Society (KS) function scores. Subjective outcome measures included questionnaires evaluating pain, function, and satisfaction. Results Mean graft surface area was 11.0 cm2 (range 5.3–19.0). 15/25 (60%) required additional bone grafting. Mean follow-up was 67.1 months (range 25–235). 5 patients had further surgery, 24/25 had avoided arthroplasty, graft survival rate was 88% (22/25), 16 (73%) of which were considered successful (18-point score ≥ 15). Mean score improved from 11.0 to 15.7 (p⇠001), mean IKDC pain score from 7.3 to 3.0 (p⇠0.05), mean IKDC function score from 3.5 to 8.1 (p⇠0.05), mean KS function score from 58.6 to 90.0 (p⇠0.005). 10 patients completed questionnaires; all reported improved pain, function and overall satisfaction. Conclusions Fresh osteochondral allografting led to significantly improved pain and function, and avoided arthroplasty, in a majority of patients. Osteochondral allografting is a reasonable salvage option or management strategy for osteonecrosis of the femoral condyle., Introduction Despite the improvement in the treatment procedures, articular cartilage lesions remain affected by long recovery time that preclude the athlete competition for long time. The rehabilitative challenge is to optimize the achievement of the goals without jeopardizing the graft vitality. In this prospective comparative study we evaluated the results in a group of athletes treated with an intensive rehabilitation protocol after second generation arthroscopic autologous chondrocyte implantation (Hyalograft C). Methods and Materials 31 competitive male athletes with grade III-IV cartilaginous lesions of the medial or lateral femoral condyle or trochlea were evaluated at 1, 2 and 5 years follow up. We also compared the results obtained to a homogeneous group of 34 non-sportive patients. All the 31 sportive patients followed the intensive rehabilitation protocol, but 11 were also treated with isokinetic approach and rehabilitation on field. Results When comparing the 2 groups, a higher improvement in the athletes group was achieved at 5 years follow up (p=0.037) in the EQ-VAS and in the IKDC subjective evaluation either at 12 months and at 5 years of follow up (p=0.001, p=0.002, respectively). Analyzing the resumption of sports activity, 80,6% of the athletes returned to previous activity level in 12.4 months (s.d. 1.6); patients treated with isokinetic approach and rehabilitation on field had faster recover and an even earlier return to competition (10.6 months, s.d. 2.0). Conclusions For optimal results, ACI rehabilitation needs to not only follow but also to facilitate the process of graft maturation. A proper rehabilitation may safely allow a faster return to competition and influence positively also the clinical outcome at medium term follow up., Introduction The chondrocyte's cells culture provides within few weeks autologous cartilage in 3 dimensions, in carrier fleeces of 6cm 2 with 2mm thickness. The carrier fleeces are resistant with a certain degree of elasticity. We can cut them and fix them on the sub-chondral bone. Methods and Materials This technique has been applied for over 5 years in the arthrosis of the knee on 20 patients in 34 sites. In 11 cases kissing lesions were treated. The technique used is the one of BioSeedC: preparation of the graft bed and a press-fit fixation of the graft with trans-bone pulley threads, auto-blocked in the channels by knots. In 5 cases a patella re-alignment was performed and in 8 cases a HTO. 1 case of astragalo-tibial graft has been realised. 2 meniscus grafts have been associated, one at the same time and the other one deferred. Results A MRI control has been systematically performed around the 8th month allowing the demonstration in all cases of good growth and fixation of the graft. arthroscopic controls have shown a good cartilage; one biopsy of the grafted zone has histologically shown a normal hyaline cartilage. The clinical score was clearly improved from the 6th month except in 2 cases. A good result in the ankle is encouraging, avoiding the subastragalar arthrodesis. Conclusions The middle term results of the autologous 3D chondrocyte graft “BioSeed -C” in the arthrosis of the knee are encouraging as they show a good cartilage growth and fixation of the graft., Introduction The Cartilage Autograft Implantation System (CAIS) is being developed as an alternative articular cartilage surgical treatment, which provides a chondrocyte-based repair in a single surgical procedure. The CAIS technique first uses a proprietary device for the preparation of morselized, autologous cartilage uniformly dispersed on a 3-dimensional scaffold. This construct is then fixed in the articular cartilage defect. Preclinical studies show that the embedded chondrocytes migrate onto the scaffold and form hyaline-like matrix. Methods and Materials Two prospective, randomized pilot clinical studies are underway: one in the US and the other in Europe. Both studies were designed to assess safety and initial performance of CAIS. Twenty-nine (29) patients at 5 investigative sites were randomized in a 2:1 schema (CAIS:microfracture) in the US and 24 patients at 5 enrolling sites in Europe. Subjects were clinically evaluated, including MRI, and asked to complete clinical outcome questionnaires. Short-term follow up occurred at 1 & 3 weeks and 2, 3, 6, and 12 months post-surgery with long-term follow-up scheduled every 6 months up to 4 years. Results Preliminary data from both studies indicate that CAIS is safe at 18 months in the US study and 6 months in the European study. The CAIS device has demonstrated safety and improvement over baseline in treated subjects. Safety and clinical outcome results will be reported through 18 months from the US pilot Study and 6 months from the European Pilot Study. Conclusions The instrumentation enabled the successful preparation and fixation of a minced autologous cartilage tissue-loaded implant in a single intraoperative setting., Introduction The purpose of this study was to compare the outcomes of microfracture (MF) and ACT Bioseed-C (C) procedures for the treatment of the articular cartilage defects of the knee joint in active professional athletes. Methods and Materials Between the 2004 and 2006, a total of 49 professional athletes with a mean age of 25.8 years (14 to 36) and with a symptomatic focal lesion of the articular cartilage in the knee underwent microfracture or ACT-BIOSEED-C procedure. There were 20 athletes in MF group and 29 athletes in C group. The mean follow-up was 2 years (range 6ms to 4 years). Patients were evaluated by ICRS, MOCART, KOOS scoring. Follow up examination have been made after six month, and every year after surgery. Results The ICRS functional and objective assessment showed that69% after microfracture had excellent or good results compared with91% after ACT (C). In 1, 2, 3 and 4 years after the operationsthe ICRS did not showed statistically significant differences betweenthe groups (p=0.453). 58% of athletes following MF and 64% athletesfollowing C returned to sports activities at the pre-injury level atan average of 7.5 months after the operations (NS), but only 37%after MF compared with 46% after C procedure maintained sportsactivities at the pre-injury level after 4 years. Conclusions At 4 years follow-up, our prospective clinical study in athletes has shown significant worst outcome of the microfracture vs ACT for the repair of articular cartilage defects in the knee., Introduction Bipolar chondral lesions are generally considered a contraindication to cartilage repair techniques, leaving few treatment options besides arthroplasty. We report on outcomes of fresh osteochondral allografting as a salvage option for bipolar cartilage injuries of the knee. Methods and Materials Between 1984–2006, fresh osteochondral allografting was performed in 52 patients (54 knees) with bipolar chondral lesions of the knee. Clinical evaluation included an 18-point scale, post-operative Knee Society function score, and reoperation. Subjective outcome measures included patient questionnaires evaluating pain, function and satisfaction. Results 25 males, 29 females had a mean age of 39 years (range 15–66). 39 lesions involved the tibiofemoral, 15 the patellofemoral joints. Patients had an average of 3.4 previous surgeries (range 1–8). Mean allograft area was 18.2 cm2. Mean follow-up was 78 months (range 24–236), 7 patients were lost to follow-up. 33 patients had further knee surgery; 4 had repeat allografts, 15 underwent conversion to arthroplasty (2 partial and 13 total) at a mean of 41 months (range 6–119). 89% of the surviving grafts were considered successful (18-point score ≥ 15). Mean 18-point score improved from 11.9 to 16.2 (p⇠0.001). Mean post-operative Knee Society function score was 82 (range 45–100). 24 patients completed questionnaires; 88% were satisfied, 92% reported less pain, and 96% reported improved function. Conclusions Fresh osteochondral allografting successfully avoided arthroplasty in 72% of knees and resulted in significantly improved function, reduced pain, and high patient satisfaction. Fresh osteochondral allografts are a reasonable salvage option in the challenging patient population presenting with symptomatic bipolar cartilage lesions of the knee., Introduction The purpose of this study is clinical evaluation of a novel II generation autologous chondrocyte implant, BioCart™II for cartilage repair. Methods and Materials BioCart™II is a matrix-assisted, fibrin hyaluronic acid-based implant containing autologous chondrocytes propagated with a unique growth factor to maintain their chondrogenic potential. The scaffold enabling a three dimensional distribution of the cells. 20 implantations in 18 patients aged 17–50, with cartilage lesions 15 in the knee and 3 in the ankle, diagnosed by CT MRI and/or arthroscopy underwent biopsy from the intercondylar notch and from the distal margin of the talus respectively. Two to three weeks later, BioCart™II was implanted through a small longitudinal parapatellar incision or a longitudinal antero-medial incision of the ankle. Deep lesions were treated with two layers. Rehabilitation included 3 weeks of non-weight bearing and CPM, followed by 3 weeks of pwb. Full activities were resumed at 4–6 months and follow-up was 10–36 months. Results At diagnosis all patients scored under 4 points in the subjective ICRS questionnaire improving to over 6 post operation. The IKDC score improved from grades C and D before the operation to A or B post operation. Second look arthroscopy and biopsy on two patients, showed excellent coverage, full integration and new hyaline-type cartilage. Post operative MRI on 10 patients showed good integration of the graft with bone edema in some. Conclusions BioCart™II is safe, effective and user-friendly both for the patient and the surgeon. The short time from biopsy to implantation and good to excellent clinical outcome further encourage the continued use of this technique and product., Introduction Hyaluronan was introduced successfully in 1998 to transplant cultured chondrocytes in cartilage defects: this study presents the first ten years clinical result using the Hyalograft technique Methods and Materials 290 patients were treated with Hyalograft C in the knee joint in the last decade (1998–2008): 72.4% were male; 27.6 % had simple lesions, 31% complex and 41.4 % salvage procedure. Mean defect size was 4.9 in simple, 4.8 in complex, 9.3cm2 in salvage lesions. 55.6% cases showed defects on the femoral condyle, 4.4% had patellar defects, 20 % tibial coin lesion and 20% had a troclear defect. Clinical follow up was performed at 1,3,5 and 7/10 years using the ICRS-IKDC scores as well as MOCART score. EuroQol EQ-5D evaluated the patient quality of life. 2nd look biopsy have been made in 26 pts. Results At 7-years follow-up subjective IKDC mean Score passed from preop. value of 34.3 to 79.3; 84% of patients experienced a significative improvement in knee function and symptoms: better results have been evaluated in simple (100%) and in salvage lesions (81.8%); 91 % of treated knee classified as normal or nearly normal. None Hyalograft-C related adverse event have been found; we had found 30 graft failures due to graft delamination, insufficient regenerative cartilage, disturbed fusion; 11 patients resorted to TKA due to progressing osteoarthritis. Conclusions Hyalograft-C is a useful treatment option for cartilage defects with good outcome that is actually comparable to other ACT. In patients with osteoarthritis critical indication should take place., Introduction The aim of this study was to develop a technique to decellularize a porcine cartilage-bone construct with a view to using this as a biological scaffold for transplantation into human osteochondral defect. Methods and Materials Decellularization was based on a modification of the basic technique of Booth et al (2002). Cartilage-bone matrix (n=9) were decellularized by exposing the tissue to freeze-thaw cycles. This was followed by subjecting the tissues to hypotonic (10mM tris-HCl, pH8.0) buffer, ionic detergent (0.1% [w/v] sodium dodecyl sulfate [SDS]), in the presence of protease inhibitors. This was followed by incubation in nuclease solution containing DNase and RNase. Fresh and decellularized cartilage were compared histologically using hematoxylin and eosin, sirius red and alcian blue staining. Immunohistochemistry staining for galactose-α-1,3-galactose (α-gal), collagen I, II and VI was performed for fresh and decellularised samples. The biochemical composition of fresh and decellularised cartilage was examined. This included DNA, hydroxyproline and DMB sulphated sugar assays. The biphasic property of fresh and decellularized cartilage was determined using a pin on plate indentation test. Results H&E staining revealed absence of visible whole cells. Analysis of DNA revealed the absence of genomic DNA in comparison to fresh tissues. The decellularisation process had minimal effect on the collagen content of the cartilage. Nevertheless there was a significant loss of GAG following decellularization. The biomechanical testing showed a significant change in comparison to the fresh cartilage. Conclusions In conclusion, this study has generated data on the production of an acellular cartilage bone matrix scaffold for use in osteochondral defect repair., Introduction To assess P-ACI for articular cartilage lesions of the knee, a study was carried out on patients with minimum 5 year follow up with clinical assessment and second look arthroscopy when indicated. Methods and Materials Between October 2000 and August 2003 the author carried out P-ACI on 116 patients, 120 knees, 168 lesions included in this study. There were 88 single, 34 double and four triple lesions. Lesion aetiology was traumatic in 93, degenerative in 65 and OCD in 17. Previous surgery was frequent, predominantly arthroscopic debridement (78), meniscal surgery (65), arthroscopic microfracture (19) and ACL reconstruction (13). Results Results were assessed using the ICRS Cartilage Repair Evaluation Package 2000. Significant improvement was seen in Activity Level, Objective Knee Examination and SF-36 Physical and Mental Component Scores. IKDC subjective assessment improved by average 29.6 points. “Second look” arthroscopy was carried out on 79 knees with 109 lesions at average 26 months predominantly for graft hypertrophy/extrusion presenting as painless mechanical symptoms. “Third look” arthroscopy occurred in 37 knees with 55 lesions at average 44.4 months from index implantation. “Fourth look” arthroscopy occurred in 10 knees with 14 lesions at average 59 months from index implantation. Four for advancing degenerative change requiring TKR. ICRS VCRA was predominantly recorded Grade II at subsequent arthroscopy. Conclusions This study demonstrates good clinical outcome and satisfactory repair when assessed arthroscopically. Subsequent arthroscopic surgery was frequently required, predominantly related to the periosteal patch in the first year, after which progressive degenerative change became more significant., Introduction The matrix-associated autologous chondrocyte transplantation (MACT) for articular cartilage defect healing has been clinically applied with a variety of 3D-scaffold types. The present study shows the differences between four different MACT-matrices (scaffolds). Methods and Materials Four transplant-types (Biogide(R), CaReS(R), Novocart(R) and Hyalograft(R) C) were analysed at the time of transplantation (t0) and after four further weeks (t = 4w) of in vitro culture. Histology, electron microscopy and molecular biology were used to evaluate the cell distribution, phenotype of the chondrocytes (differentiated or fibroblast-like), cell-scaffold interactions, cell vitality and metabolic activity. Results In dependence of the architecture of the scaffold type, chondrocytes showed very different distribution patterns at t0; differences were also evident between the patients. In the course of four weeks of prolonged cultivation the cells proliferated and changed their distribution. Chondrocytes showed high vitality and metabolic activity in all transplant materials, especially at the 4w stage. Chondrocyte gene expression indicated a tendency of increased collagen type II synthesis after four weeks of in vitro culture. The sites of differentiation varied according to the carrier materials but were generally located in matrix-rich cell accumulations. Conclusions The scaffold architecture influenced the cell distribution while seeding as well as the movement of the cells on the scaffold, and has therefore influence on the cell distribution in the defect. The increase in collagen type II seems to be initiated by the 3D-conditions in matrix-rich cell accumulations. The tendency towards differentiation in vitro presents an indication for successful cartilage regeneration in vivo., Introduction Histological features of successful autologous chondrocyte implantation(ACI) have been widely reported, but the repair characteristics of revised and clinically failed grafts are not well understood. Methods and Materials We examined 39 cases of revised MACI and patients received total knee replacement after MACI for the mechanical properties, histology and the matrix profiles of repair biopsies. Results Histologically, the majority of these biopsies (n=39) were hyaline-like (HLC) and fibrocartilage (FC) in both the revised (30% and 38% respectively) and failed (34% and 22% respectively) cases. Compositionally, more revised cases were positive for aggrecan, collagens VI and IX, and Ki67 compared to failed cases, but not quantitatively different (P⇢0.05). More HLC biopsies were positive for aggrecan and collagen II (compared to the FC group), with diffuse and often colocalized matrix distribution. The majority of H LC biopsies stained positive for Sox-9, whereas FC cases were negative. Most (75%) FC biopsies were positive for Ki-67, compared to the HLC group with 25%. MMP-13 was negative in all biopsies. Qualitatively, reduced collagen II and IX, and increased Ki67 production was noted in FC biopsies (P⇠0.05). An intact repair site showed FC with 30% greater stiffness in the inferior portion compared to the superior, with an associated proteoglycan content increase. Conclusions Revised and failed biopsies display predominantly hyaline-like and fibrocartilage in repair type, are histologically dissimilar to healthy cartilage, but do not differ in composition. Hyaline-like repairs show lower proliferation but improved matrix to fibrocartilage repairs. Our study furthers knowledge into failed and revised cartilage repair by MACI., Introduction The purpose of this study was to assess outcomes of autologous chondrocyte implantation (ACI) in the patellofemoral (PF) joint and determine a treatment algorithm for tibial tuberosity realignment and appropriate articular cartilage treatment. Methods and Materials Prospectively collected data of sixty-three patients who underwent an ACI of the PF joint between 2002 and 2006 were retrospectively reviewed. The primary endpoint was survivorship of ACI. Secondary endpoints included validated outcome scores and subsequent surgical procedures. Results Fifty-two (83%) patients with a mean age of 31.8 years were followed up at an average 4 years (minimum, 2 years). The mean lesion size was 4.19 cm2 and was located on the trochlea in over 50% of cases. Mean improvement in the preoperative to postoperative scores was significant (p0.05). Patients undergoing anterior medialization (AMZ) fared better than those without realignment. Fifty percent of patients with ACI needed a subsequent procedure. There were 5 clinical failures (9%), which were defined as progression to arthroplasty or conversion to osteochondral allograft transplantation. Conclusions ACI of the PF joint provides a significant improvement in symptoms and function in patients with or without a history of a failed prior cartilage procedure. Combined ACI with AMZ improves outcomes more than ACI alone. ACI of the PF joint is associated with a high re-operation rate and an appropriate surgical plan is necessary to successfully treat these patients., Introduction Cartilage lesions represent a significant clinical problem because of their limited ability to regenerate. Recent advances in our understanding of the functions of mesenchymal stem cells (MSC) have shown its chondrogenic potential. The use of autologous MSC represent an improvement on the currently available techniques for cartilage transplantation avoiding the first surgery for cartilage biopsy and chondrocyte cultivation. Methods and Materials We followed prospectively five patients with Gr.III or Gr. IV chondral lesions treated with standard arthroscopic approach and implanted with concentrated MSC from the iliac crest with or without the use of a scaffold. All patients followed the same specific rehabilitation program after MSC implantation. IKDC, KOOS, Lysholm and Tegner were collected at pre-op and every 6 months post-operatively. Results Five patients with a mean age of 46.6 have been treated in our institution in the last 2 years with this technique for grade 3 and 4 cartilage knee lesions. At a mean follow-up of 24 months, patient showed improvements in all scores. Mean pre-op values were IKDC subjective 53.74, KOOS Scores: P=83/S=87.2/ADL=86.6/SP54/ QOL= 44; Lysholm 74 and Tegner 4.5; at final follow-up mean scores were IKDC subjective 73.0, KOOS Scores: P=89.7/S=90.7/ADL=97/SP=71.7/QOL=75; Lysholm 85 and Tegner 6. No adverse reaction or post-op complication were noted in all patients. Conclusions This preliminary report shows that Mesenchymal Stem Cell Implantaion can be a viable technique in the treatment of Gr. III and Gr. IV chondral lesions., Introduction Retropatellar cartilage defects are difficult to treat. A regeneration of pristine hyaline cartilage is desirable but yet impossible. The AMIC-procedure (Autologous Matrixinduced Chondrogenesis) is an option to faciliate regeneration of substitute cartilage. These case reports document the successful cartilage regeneration in patients with chondral and osteochondral retropatellar defects where other treatment options have failed. The aim of this case control study is to evaluate the value of the AMIC-procedure in retropatellar cartilage defects. The operative procedures are introduced. Methods and Materials A case control study introduces 2 cases in which retropatellar AMIC was performed. One was a chondral, one an osteochondral defect. The follow up is 12 months. Pain reduction evaluating the Visual Analogue Scale (VAS) and range of motion of the affected joint were evaluated. Results In both cases a pain reduction in the VAS from 10 to 2 could be achieved. The Range of motion improved in flexion from 90 to 130 degrees. X ray and MRI document almost complete regeneration of bone and cartilage in both cases. Conclusions The AMIC-procedure was successfully carried out in these patients where other options had failed. Matrixinduced chondrogenesis may be an alternative in retropatellar chondral defects which are otherwise difficult to treat. Further studies are encouraged., Introduction Ideal treatment of osteochondral lesions of the talus (OLT) is still controversial. Aim of this study is to review the 10 years follow-up clinical and MRI results of open field Autologous Chondrocyte Implantation in the treatment of OLT. Methods and Materials From December 1997 to November 1999, 10 patients, age 25.8 ± 6.4 years, affected by OLT, received open field Autologous Chondrocyte Implantation. The mean size of the lesions was 3.1 cm2 (2.2 − 4.3). All patients were evaluated clinically (AOFAS score), radiographically and by MRI pre-operatively and at established intervals up to a mean follow-up of 119 ± 6.5 months. At the final follow-up MRI T2 mapping evaluation was performed. A bioptic sample was harvested in 5 cases during hardware removal 12 months after implantation. Results Before surgery the mean score was 37.9 ± 17.8 points, at 24 months it was 93.9 ± 8.5, while at final follow-up it was 92.7 ± 9.9 (p⇠0.0005). Bioptic samples showed cartilaginous features at various degrees of remodelling, positivity for collagen type II and for proteoglycans expression. No degenerative changes of the joint at follow-up were found radiographically. MRI showed well-modelled restoration of the articular surface. The regenerated cartilage showed a mean T2 mapping value of 46 msec with no significant difference compared to that of normal hyaline cartilage. Conclusions The clinical and histological results have confirmed the validity of the technique utilized with a durability of the results over time. T2 mapping was adequate in detecting the quality of the regenerated tissue coherently with the bioptic results., Introduction To compare the chondrogenic potential of mesenchymal stem cells (MSCs) from bone marrow or adipose tissue to cartilage-derived chondrocytes in pursuit of an alternative source for cell therapy for cartilage regeneration. The pluripotential nature of stem cells is widely considered to make them the ideal choice for any cell therapy. Autologous chondrocyte implantation, has shown much promise but remains a two step procedure requiring the harvest of cartilage followed by culture in vitro and subsequent implantation. This procedure has also not yet been indicated for osteoarthritis (a common cartilage pathology). Autologous or allogeneic stem cells could, theoretically, provide a single step “off the shelf” solution for regeneration of cartilage defects. Our study, (30 subjects), was designed to compare the chondrogenic potential of chondrocytes taken from healthy or osteoarthritic cartilage, with that of MSCs prepared from bone marrow of the same subject. MSCs isolated from human adipose tissue were also compared Methods and Materials Chondrocytes isolated from different areas of joints affected with osteoarthritis were compared to bone marrow MSCs from the same subject undergoing total knee replacement. Cells were isolated and expanded under optimized culture conditions including autologous serum, with or without a fibroblast growth factor (FGF) variant. Proliferative capacity, chondrogenic potential and proteoglycan production were analyzed. Results Our results indicate a clear advantage for FGF-directed cartilage derived chondrocytes, even from osteoarthritic joints over the MSCs from either source. Conclusions Chondrocytes under optimized conditions maintain a higher chondrogenic potential than MSCs and could serve as a legitimate source for cartilage repair in osteoarthritic patients., Introduction Arthroscopic debridement represents a relatively simple and effective treatment in the management of glenohumeral degenerative joint disease. The purpose of this study was to review the outcomes of patients who have undergone arthroscopic debridement procedures for isolated degenerative joint disease. Methods and Materials Patients that had an arthroscopic debridement performed by one of four surgeons at a single institution were retrospectively identified. All patients were confirmed to have significant (at least grade 3) articular damage from the operative report. Any patient with adhesive capsulitis or a concomitant rotator cuff or SLAP repair was excluded. Preoperative simple shoulder test (SST) and American Shoulder Elbow Score (ASES) scores were compared against postoperative outcomes with at least a 1 year follow-up. Postoperative Constant scores and range of motion were also recorded. Results Ninety patients were retrospectively identified and 72 (80%) were available for follow up. The average follow up was 27 months (12 − 90 months). Of the 72 patients, 17 (23.6%) went on to total shoulder replacement at an average of 10.2 months. In patients that did not progress to arthroplasty, SST scores improved from a mean of 6.0 preoperatively to 9.0 postoperatively (P ⇠0.001), ASES improved from 52.0 to 76.2 (P ⇠0.001). The average postoperative constant score was 72.4, and 80% of the patients stated that they were satisfied with the surgery and would do it again. Conclusions Arthroscopic debridement of the glenohumeral joint is an option that has the potential to delay more extensive procedures and provide a significant amount of pain relief., Introduction Autologous Chondrocyte Implantation in patellofemoral compartment has unpredictable outcome. Controversy arises when patellofemoral ulcer is a part of the tricompartmental disease. We conducted a study to correlate radiological findings with clinical presentations in patients with patella subluxation and knee pain. Methods and Materials A prospective study was conducted on patients presenting with knee pain or clinical subluxation of the patella. All patients underwent plain knee anteroposterior, lateral and skyline radiographs. Unenhanced axial ct scans of both knees performed at 0, 10o and 20o of flexion. Sulcus and Laurin's patellofemoral angles were measured. Tricompartmental knee arthritis was evaluated on radiographs. Results A total of 62 patients (124 knees) were included in this study. Patients were divided into 3 age groups; Group 1: 18 and below, Group 2: 19 to 40 and Group 3: above 40. Average age was 14.3, 24.9 and 48.9 respectively. Mean values of angles were calculated at 00, 100 and 200 of flexion. In Group 1, 83.3% had patella subluxation on CT. None had any radiological features of degeneration. In Group 2, 52.4% had patella subluxation on CT while 9.52% had degenerative changes. In Group 3, 47.8% had patella subluxation on CT, 56.5% had degenerative changes and 21.7% had cartilage ulcers. Conclusions In younger patients, patella subluxation was associated with anatomical abnormaltities such as trochlear dysplasia, while in older patients, it was part of the tricompartmental disease., Introduction Analysis of hyaline cartilage of the ankle joint is technically demanding, due to the high congruency and thin cartilage layer; therefore, biochemical MR techniques have been developed for quantitative analysis of articular cartilage. The purpose of the present study was to assess T2 and T2* mapping as well as Diffusion-Weighted-Imaging (DWI) for evaluation of cartilage and cartilage repair tissue. Methods and Materials Ten healthy volunteers (mean 32.4 years) and twelve patients after MACI of the ankle joint were included in the study. Morphological imaging was performed in order to identify the cartilage repair tissue. Biochemical imaging was performed at 3T by using a 2D multi-echo spin-echo sequence for T2 mapping, a 3D gradient-echo for T2*-mapping and a 3D partially balanced, steady-state gradient-echo pulse sequence with and without diffusion weighting for DWI. T2, T2*, and DWI values were assessed using a Region-of-interest (ROI) analysis. Results Quantitative assessment of hyaline cartilage of healthy volunteers (T2: 51.1±4,6ms; T2*: 16,6±3,7ms; DWI (diffusion constant): 1.27±0.16) and healthy seen control cartilage sites in patients after MACI (T2: 48.4±7.5ms; T2*: 16.1±4.1ms; DWI: 1.28±0.17) showed no significance difference (p=0.05). Within the cartilage repair tissue T2 and T2* values showed similar results (T2: 49.3±6.6ms (p=0.767); T2*: 16.9±4.2ms (p=0.505)), whereas DWI showed a significant increase (1.49±0.32 (p=0.039)). Conclusions High-resolution T2, T2*, and DWI techniques were performed in-vivo in a clinical feasible scan time, thus demonstrating new possibilities in the evaluation of articular cartilage on the ankle. Valuable parameters for the characterization of cartilage repair tissue could be gained., Introduction Better MRI imaging has improved the noninvasive evaluation of cartilage, however, arthroscopy is still considered the gold standard. Anecdotal evidence suggests that transplanted lesion size is commonly larger than expected by preoperative imaging. While not an issue with 1st generation ACI, where cells can be diluted to accommodate for larger lesions, 2nd generation procedures utilize membranes with size limitations. In light of these developments, we conducted a study comparing size estimates based on MRI and arthroscopy with final defect size at the time of ACI. Methods and Materials Patients were included if they had undergone MRI, arthroscopy and ACI within 18 months to avoid confounding by defect progression. A radiologist measured all defects on MRI; sizes recorded during arthroscopy and implantation were obtained from surgical notes, and compared with paired t-tests. Results 38 patients were enrolled with 53 defects of the condyles and trochlea; defect size averaged 4.5 cm2. Defect locations demonstrated significant trends towards larger sizes between MRI, arthroscopy and ACI. Final sizes were larger than the MRI estimates by 88% (p=0.02) and 95% (p=0.03) for the medial and lateral femoral condyles, and by 50% (p⇠0.001) for the trochlea. Conclusions Our study compared the size of cartilage defects measured by a cartilage radiologist based on high-resolution MRI, and compared this with surgical measurements by an experienced surgeon. Even in this best-case scenario, final defect sizes were significantly larger than those estimated preoperatively. Our findings can be used as a guideline when ordering biologic implants for cartilage repair, especially when using size sensitive membrane technologies., Introduction Aim of this study is the investigation of lower limbs biomechanics before and after meniscectomy. Methods and Materials Materials and methods: motion analysis of the knee before surgery, six months and one year after was performed on ten patients that underwent partial medial menisectomy. Ten healthy volunteers acted as a control group Data were acquired by means of Vicon motion analysis system Results In gait patterns investigation, joint kinematics does not show significant modifications before and 6 months after surgery, 12 months after surgery hip and knee show a greater flexion. The dynamic analysis showed alterations in sagittal moment. Before surgery the knee flexion moment is reduced. After partial meniscectomy the knee flexion moment increases in both the limbs. In squatting investigation, main focus was on repeatability. Before surgery high inter subjects variability affects knee joint angle; while after surgery high variability affects also hip and ankle. Conclusions After meniscectomy, gait and squatting patterns are still altered. Before surgery, the joint mechanical structure is not highly altered and modifications are mainly due to pain avoidance schemas; after partial meniscectomy, pain disappears and the new joint behaviours are probably caused by the new mechanical asset and/or proprioceptive mechanisms., Introduction Femoro-Acetabular Impingement (FAI) is a frequent clinical pathology in the young adult hip. Frequently, athletes are severely handicapped by this disease. FAI is due to mechanical abnormalities of the proximal femur or the acetabulum resulting in rapid focal destruction of acetabular cartilage, most frequently in the antero superior quandrant of the acetabulum. The purpose of this study was to simulate the mechanical effects of a loaded focal acetabular cartilage defect in function of cartilage thickness and ICRS-Classification in a Finite Element-Model. Methods and Materials In the Finite Element-Model. The following assumptions were made: - a rectangular chondral defect: 30mm × 10mm × 2mm in size - femoral head diameter: 50mm - cartilage defect depth: 2mm - ICRS Grades: o, I, II - Cartilage Elasticity Modulus: 18.8 MPa (Gradeo), 11.1 MPa (Grade I), 10.5 MPa (Grade II) -Specialattention was made to see if the femoral head touches the subchodral bone of the acetabulum in this size of chondral defect. Results The simulation shows different mechanical patterns in normal (healthy) cartilage (ICRS o) and degenerative cartilage (ICRS I, II). At 2500 N and 5000 N loading (Gait, Running), it is interesting to note that no femoral head contact with the subchondral bone of the acetabulum has been seen in any of the defects. At 20 000 N the femoral head hits the subchondral bone of the acetabular chondral defect. Conclusions In average weight patients a 3cm2 acetabular chondral defect cannot be considered as a severe lesion. However in heavy weight althletes these lesions have serious clinical implications., Introduction Previous clinical literature has shown that intraarticular (IA) injection of hyaluronic products are safe and effective for the treatment of hip osteoarthritis (OA), but the available casuistry is still numerically limited showing also a short follow up. Aim of the study: to assess the safety and efficacy of administering Jointex® (Chiesi SpA 800–1.200 KD, 8 mg/ml) on painful hip OA in a large cohort of patients for a long follow-up. Methods and Materials Adult, outpatients suffering from symptomatic hip OA (Kellgren & Lawrence Grade 1, 2 or 3) were injected with one syringe of 4 ml (2 vials) of Jointex® under ultrasound guidance, repeated after six months and when clinically necessary an adjunctive injection was performed. Patients were assessed at baseline and at every three months to 12 months for Lequesne index, pain (evaluated by VAS) and NSAID consumption (number of days patients assumed NSAID in the last month). Results 229 patients were enrolled, 99 M and 125 F, mean age 63. 62 had bilateral hip OA. 90 patients were followed up for 12 months, 415 injections were performed. No local or systemic adverse events were reported. A statistically significant reduction (p⇠0.05) was observed at all time points versus baseline for all assessment criteria: Lequesne index, pain VAS, NSAID consumption. Conclusions The data from our cohort study seem to demonstrate the long-term efficacy and safety of intra-articular ultrasound guided treatment with Jointex® in symptomatic hip osteoarthritis. They do, however, need to be confirmed by further data to be collected after longer follow-up times., Introduction Autologous chondrocyte implantation (ACI) has been used most commonly as a treatment for cartilage defects in the knee and there are few studies of its use in other joints. There is only one published report of its use in the hip. Is there a role for ACI of the hip? Methods and Materials We describe 14 consecutive patients studied prospectively with chondral or osteochondral lesions of the femoral head that underwent ACI and were prospectively reviewed with a follow up of 5 years (mean 30 months). 3 patients presented with Perthes and five with AVN. Six had chondral loss following trauma and one presented with an area of bone loss in a hip with congenital dysplasia. Defect size was a mean 6.2 cm2. Pre-operatively hip function was assessed by the patient using the Harris Hip Score and MRI. Post-operatively these were repeated at 1 year and hip scores repeated annually. Hip arthroscopy and cartilage biopsy provided cells for culture in a GMP laboratory where passage numbers were limited to two. Three weeks later by open surgery, all unstable cartilage was excised, the base was debrided or excised and bone graft applied, and suture of a membrane of periosteum or collagen membrane over the defect undertaken. A mean 5.2 million chondrocytes were inserted beneath this patch following a test of the seal. Results Ten of the fourteen patients improved at one year, with a mean rise in Harris Hip score from 57 to 63 points. Five patients underwent arthroscopic examination at one year and in four there was evidence of good integration of the new cartilage. In one patient arthroscopy was difficult due to previous trauma. One patient developed AVN as a post-operative complication following a posterior approach. Four patients have progressed to hip replacement or resurfacing but it is of note that all these patients had cyst formation pre-operatively. Conclusions The short-term results of ACI for osteochondral lesions of the hip suggest that if good early results are obtained they are observed to continue out to 5 years. There is a high failure rate in those with pre-operative cyst formation in the hip., Introduction Hemi-arthroplasty is a more conservative option used for elderly patients who have suffered femur neck fractures and femoral head osteonecrosis. This study investigated the friction and wear of cartilage in the natural acetabulum articulating against a metallic femoral head under constant load with different clearances. We hypothesise that the outcome of hemi-arthroplasty could be improved if the clearance is optimised. Methods and Materials Porcine acetabulums were dissected from 12-month old animal's hip joints and mounted in PMMA at 450 with respect to the loading, and then a silicon rubber replica was made to assess dimensions. The cobalt chrome alloy head diameters were 34, 35, 36, or 37mm with 0.008um roughness (Ra). Clearances were small (≤0.7mm), medium (⇢0.7 & ≤1.4mm), and large (−41.4mm). Two-hour 400 N constant load friction tests were conducted using a pendulum motion friction simulator with a flexion-extension motion of ±15° in 25% bovine serum. The friction coefficient (mefff) was measured throughout testing and wear damaged assessed visually at the end of the test and classed as zero, mild and severe. Results The friction coefficient (the friction factor at o° position) was not significantly different in testing with different clearances, and it was approximately 0.27 following 7200 cycles (range±0.025). However, if wear and friction were correlated: the friction coefficient of zero-wear group (mefff=0.20) was significantly lower (T test p=0.0037) than both mild and severe wear groups (mefff =0.28). Conclusions The friction of hemi-arthroplasty under constant load was dependent on both the clearance and the acetabulum geometry, cartilage is damage led to elevated friction., Introduction There is a controversy in the literature concerning the impact of full thickness cartilage lesions in ACL-injured knees. Methods and Materials Hypothesis: A full-thickness cartilage lesion at the time of the ACL-reconstruction does not influence the knee-function measured by KOOS. Study Design: Cross-sectional study. Methods: Of the 4849 primary ACL-surgery cases in the Norwegian National Knee Ligament Registry per 12.12.2007, thirty patients met the following inclusion criteria: A full thickness cartilage lesion (International Cartilage Repair Society (ICRS) grade 3 and 4), age less than 40 years, no associated pathology or meniscus injury and less than one year between the knee-injury and the ACL-reconstruction. To each of the 30 patients in this study group, two controls with no cartilage lesion were matched from the registry. Preoperatively all patients completed the Knee Injury and Osteoarthritis Outcome Score (KOOS). Peroperatively the surgeon graded the cartilage injury according to ICRS, and also recorded the location and size of the lesion. Results There was no significant difference between the groups for any of the five subscales according to KOOS-score. A cartilage lesion was located in the medial compartment in 67 % of the cases, in lateral compartment 20 % of the cases and in the patello-femoral joint 13 % of the cases. Conclusions The combination of a full thickness cartilage lesion and an ACL-rupture did not result in an inferior knee function at the time of the ACL-reconstruction as measured by KOOS., Introduction Cartilage defects in the knee comprise a socioeconomic burden as it may cause lifelong nonfatal disability at young age. Studies have found defects in up to 60 % of knee arthroscopies. In clinical trials the majority of these patients are excluded. This study investigates whether patients included in RCT represent a selected group and if this might causes bias. Methods and Materials Randomized clinical trials (RCT-level 1) on cartilage repair studies were identified and analyzed in order to define common inclusion criteria. These were applied to all patients (n=70) referred to a cartilage repair center during seven months in 2008. Lysholm knee score, occurrence of previous surgery and localization of cartilage defects were noted. Ratio of previous cartilage repair was 45/70 patients. Medial age was 37 years (range 15–51 years) and we included 22 women and 48 men. Results Common inclusion criteria were; single femoral condyle lesion, age range 18–40 years and size of lesion range 3.2–4.0 cm2. 5/70 patients matched the RCT. Previous cartilage repair stated lower Lysholm score 53 (±13.7) in comparison to no previous cartilage repair 69 (±13.7), p-value ⇠ 0.001. Other parameters such as age, localization and size of defect showed no significant difference. Conclusions Cartilage injuries of the knee represent a heterogenic patient group. Even RCTs show considerable variation regarding inclusion criteria, which might persuade selection bias. New clinical trials should document patients eligible for cartilage repair and flowchart of patients not included, to better evaluate treatment of cartilage defects in the knee., Introduction This study presents an assessment of cartilage repair with the Medicarb implant using post-operative long-term arthroscopic imaging. Methods and Materials A total of 128 knees treated by carbon fiber resurfacing rods for grade 3 and 4 articular cartilage lesions were assessed arthroscopically, with an average follow-up of 22.6 months (9- 48 mos.). Mean age of patients was 37.4 years (18- 60 yrs.) Cartilage repair scores were determined by an independent observer, using the ICRS scale for cartilage repair assessment. Results Mean repair assessment scores using the carbon rods were: 10.5/12 for medial femoral condyle repairs, 9.76/12 for lateral femoral condyle repairs, 9.9/12 for trochlea repairs and 9.4/12 for patella repairs. Grade I and II scores were recorded for 95.1% of medial femoral condyle repairs; 76.0% of lateral femoral condyle repairs; 86.1% of the trochlea repairs, and 89.7% of patella repairs. Patellar resurfacing was combined with a realignment and anteriorization procedure in 77 knees. A grade of I and II repairs were seen with rods on the patella in 96.7%, when combined with a mechanical correction, compared to 66.0%, when used on the patella without a combination procedure. There was no difference in repair scores in patients who were ⇠ 40 yr-old nor ⇢40 yr-old. Conclusions Results from the study show that carbon fiber resurfacing is an effective method of treatment. Results are improved by correction of knee deformity in conjunction with treating the defect. Evidence of recovered synovium was also observed as an additional benefit., Introduction In cases where the subchondral bone is exposed in the mirror area, there is no consensus about the effectiveness of mosaicplasty. The objective of this study is to examine whether the treatment of the femoral osteochondral lesions with autologous osteochondral transplantation even in the setting of exposed subchondral tibial bone in the same joint compartment is successful. Methods and Materials From August 2001 to July 2007, there were 17 cases 18 knees who were treated in the above methods. The mean age at surgery was 60.0 years, and the mean follow-up period was 36.5 months (range 16 to 86 months). There were 6 cases in osteonecrosis and 11 cases 12 knees in osteoarthritis. Eleven patients had second-look arthroscopy at about 11 months after mosaicplasty. We assessed IKDC subjective score, the area of the lesion and exposing subchondral bone of tibia, the ability of straight sitting in Japanese style and IKDC evaluation at second-look. Results The mean area of the lesion and exposing subchondral bone were 562 mm2 and 496 mm2. The mean preoperative IKDC subjective score was 41.0, and follow-up one was 80.5. There was significant difference. However, there were only 5 cases who had no symptoms. In preoperative period, there was one patient who could sit straightly, but 6 patients in the follow-up period. There were no significant differences in the other points. Conclusions There were some problems to leave minor symptoms and not to know long-term results, but this operative technique was successful in short-term., Introduction We present our experience of a microfractured defect covered with a collagen matrix (Chondrogide, Geistlich Biomaterials, Switzerland)c alled Autologous Matrix-Induced Chondrogenesis (AMIC) originally described by Behrens combined with discharching osteotomies. The idea behind is to maintain the stem cells that are mobilised through bleeding in the defect are maintained under the matrix to help to form a regenerate. Methods and Materials From August 2003 to July 2007 a number of 56 patients have been treated by AMIC in Fribourg by the senior author. Patients were treated for chondral and osteochondral lesions (OCD)at the knee joint and for OCD of the talus. 38 patients (40 knee joints) with a minimum follow-up of 1 year (ø follow-up 2.5 years, range 1–4 years) underwent retrospective analysis using clinical scores (ICRS, Lysholm). 17 patients had MRI which was analysed by Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) score. 11 second look arthoscopies were reviewed using the ICRS Cartilage Repair Assessment and Oswestry Arthroscopy Score. 5 biopsies have been examined histologically. Results 23 men and 17 women with a mean age of 36 years (range 14 − 64) underwent the AMIC procedure. Patients were treated for OCD (11), femoropatellar (20) and posttraumatic (9) lesions. Defects were located on the medial femoral condyle (16), lateral femoral condyle (3) and femoropatellar defects (21) with a mean size of 3.87cm2 (range 0.72 − 12). ICRS and Lysholm scores improved especially for OCD and femoropatellar lesions. MRI showed 18% complete fillings and 24% by hypertrophy; complete integration to the border zone was observed in 47%, but surfaces were only intact in 12%. Arthroscopically we found good fillings with some hypertrophies, but repair tissue was somewhat softer. ICRS Arthroscopy Score showed a mean of 9/12 and Oswestry a mean of 6/10 points. Histologically all biopsies showed fibrocartilage with some hyaline-like elements. Conclusions AMIC improved the clinical outcome and decreased pain in the cartilage defects treated in this study. Arthroscopy showed good fillings with some hypertrophies. In the MRI the AMIC zone was well integrated to the border zone but filling was mostly incomplete and surfaces damaged. Histology showed fibrocartilage with some hyaline-like elements. Especially in OCD and femoropatellar patients, but less in the purely cartilagineous lesions of the femoral condyle the AMIC procedure is an interesting, one step technique., Introduction The aim of our study was to prospectively assess the biochemical properties of cartilage repair tissue after matrix-associated autologous chondrocyte transplantation (MACT) with Hyalograft C® via delayed Gadolinium Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC) and T2 mapping over a period of one year. Methods and Materials Ten patients after MACT were examined at two different times of follow-up with a delay of one year. The mean age was 32.2 ± (SD) 11.0 years and the mean defect size was 3.9 ± 1.9 cm2. The mean follow-up period after MACT surgery was 4.4 ± 2.0 range 2 to 7 years. Besides dGEMRIC and T2 mapping, clinical scores were assessed. Results The clinical scores did not change significantly from the first to the second evaluation. δR1 (1/sec) was 1.06 ± 0.58 and 0.84 ± 0.41 in repair tissue and 0.56 ± 0.27 and 0.51 ± 0.23 in healthy reference cartilage. The relative ΔR1 (rΔR1; ratio between RT and healthy cartilage) for repair tissue were 1.89 and 1.65 respectively. The T2 values were 43.8 ± 8.4 ms and 45.0 ± 8.7 ms for RT and 45.5 ± 4.5 ms and 44.9 ± 5.9 ms for healthy cartilage tissue. Conclusions We found both techniques to indicate that the properties of the RT were more similar to native cartilage at the second MRI visit. The ΔR1 decreased, which suggests a slight increase in the glycosaminoglycane concentration of repair cartilage compared to healthy cartilage. Hyalograft repair tissue apparently remained stable at mid-term follow up in our subjects., Introduction Purpose: The aim of this presentation is to demonstrate the presence of mesenquimal stem cells in the knee joint area after the microfractures technique and to demonstrate the regeneration of hyaline cartilage lesion grade IV treated by mechanical shaving and microfracture technique plus intrachondral allograft insertion of growth factor delivered by plasma rich in platelets. Methods and Materials During the knee video arthroscopy, a mechanical shaving was performed until the bone layer; after this, a microfracture technique was performed in multiple sites of the lesion, just after of the microfracture technique the bone marrow was exposed; the isotonic solution was removed and 10 mL delivered bone marrow fluid was collected for steam cells search by immunohistochemistry techniques; the activated plasma rich growth factor (PRGF) was placed into the chondral lesion environment. Another knee video arthroscopy was performed for histological evaluation of chondral tissue four months later. Results Mesenquimal stem cells were detected in the bone marrow fluid collected. Four months after the procedure, a recuperation of hyaline cartilage was observed at macroscopic level. The histological analyses of the hyaline cartilage demonstrated a homogeneous stroma colored by eosin and the presence of chondrocytes in multiple points of biopsy with more than one nucleus suggesting the regeneration of the cartilage. Conclusions This is a technique that presents promising results. In the present case, the patient returned to the practice of physical activity in four months after the first surgical procedure, presenting a good evolution and avoiding others procedures and/or complications as osteoarthrosis., Introduction Anterior cruciate ligament (ACL) rupture has been associated with early occurrence of osteoarthritis, which was originally believed to be due to instability derived from ACL insufficiency. However, some studies suggested that the initial injury to the articular cartilage may also play a role in the development of osteoarthritis. The aim of this study is to evaluate the relationship between acute ACL rupture and cartilage lesion by use of quantitative magnetic resonance (MR) imaging technique, T2 mapping. Methods and Materials 50 knees of 50 patients (23 women and 27 men, mean age 27.3±5.7 years) with acute ACL injury were studied with a 1.5 Tesla MR imaging system. T2 mapping in the coronal plane were performed (1500 msec TR, 8 TEs of 12.4–99.2 msec, 140×140 mm field of view, 3.0-mm slice thickness, and 384×384 matrix), and T2 of the cartilage at medial and lateral condyle was measured. The relationship between T2 of the cartilage and the presence or absence of bone bruise was investigated. In 33 patients, operative findings of cartilage were evaluated and compared with the MRI findings. Results Of the 50 patients, 24 had a bone bruise at lateral femoral condyle. In the patients without bone bruise, there was no significant difference in the mean T2 between lateral femoral condyle and medial femoral condyle, 32.3 ms and 31.5 ms, respectively. The mean T2 of cartilage with bone bruise was significantly higher than that without bone bruise, 38.1 and 32.6 ms, respectively. The operative findings of cartilage were well correlate with the MRI findings. Conclusions It is known that T2 value increases with the loss of collagen anisotropy and increase in water content observed in the damaged or deteriorated cartilage. Thus the presence of bone bruise at the femoral condyle after acute ACL rupture was thought to indicate the presence of cartilage deterioration at that site. Deterioration of articular cartilage associate with bone bruise should be taken into account in clinical treatment for acute knee injury., Introduction Fixation of an osteochondral fragment to an osteochondral defect has often been performed in the treatment of patients with osteochondritis dissecans (OCD). However, the effect of this procedure remains controversial. The purpose of this study was to evaluate the histologic changes of the internally fixed osteochondral fragments in the knee. Methods and Materials Ten knees of 10 patients with OCD of the knee underwent open reduction and internal fixation of the unstable fragments, and histologic changes before and after internal fixation were evaluated. The patient age ranged from 11 to 22 years (mean 15 years). The procedure was performed either with bioabsorbable pins only, or with a combination of an autologous osteochondral plug and bioabsorbable pins. A needle biopsy was done at the time of fixation, and the time of second-look arthroscopy at a mean of 7.8 months (6 to 9 months) after surgery. Results The biopsy specimens at the second-look arthroscopy improved significantly in the histologic grading score compared with the specimens before fixation. In the specimens at the second-look arthroscopy, extracellular matrix was stained more densely than at the time of fixation, especially in the middle to deep layer. Conclusions Comparison of the biopsy samples obtained from lesions before and after fixation enabled us to confirm the improvement of histologic findings of fixed sites., Introduction Autologous chondrocyte implantation (ACI) is widely used to treat symptomatic articular cartilage defects of the knee. On the other hand, the fibrin matrix Autologous chondrocyte implantation (ACI) is a new tissue-engineering technique for the treatment of deep cartilage defects, in which autologous chondrocytes are seeded on a three-dimensional scaffold provided by a gel type fibrin matrix. Methods and Materials The clinical results after ACI with fibrin gel using sequential patient evaluation are reported 30 patients diagnosed with chondral defect of the knee from June 2005 to May 2007, (mean age, 36.6years). Clinical and functional various score systems for the knee, Magnetic resonance imaging (MRI) evaluation, the International Cartilage Repair Society (ICRS) Cartilage Repair Assessment was performed at 12 months postoperatively. Diagnostic histological examination of the graft was performed in only 10 patients. The biopsy was stained with hematoxylin and eosin, Masson's trichrome, and safranin-O, and immunostained with Col type I, II antibodies. Results Clinical and functional statuses and the ICRS showed significant improvement after surgery (p⇠0.01). The mean scores of Henderson classification (MRI evaluation) significantly improved from 7.86 ± 2.12 points to 14.31 ± 1.45 points (p=0.001) and no significant graft-associated complications were encountered. The fibrin matrix ACI showed hyaline-like cartilage or mixed hyaline-like and fibrocartilage in 67.9 % of biopsies. Conclusions Fibrin matrix ACI offers the advantages of technical simplicity, minimal invasiveness, a short operating time, and easier access to difficult sites than classical ACI. Based on the findings of this clinical pilot study, fibrin matrix ACI offers a reliable means for the treatment of articular cartilage defects of the knee., Introduction The incidence of childhood obesity is rapidly increasing worldwide. Especially in industrialized nations the obesity epidemic is predominantly evident. Overweight and obesity are afflictions, which lead significantly to an increased risk of high blood pressure, high cholesterol values, diabetes, hepatic steatosis, sleep apnoea, asthma, and joint problems and in general to a poor health status as well. Methods and Materials 20 morbidly obese patients, BMI higher 99.5 percentile, mean BMI 39.3 kg/m2, mean age 14.3 years (9–19 years), who were suffering from chronic knee pain have been included in the study. Radiographic investigations and a MRI scan (1.0 Tesla) of the painful knees have been performed in a special open MRI tool. Results All patients presented a lesion of the cartilage at least in one region of the knee, although retropatellar cartilage lesions have been found in 19 knees. 10 cartilage lesions grade I, and 4 grade 2 have been watched narrowly after MRI scan in the lateral compartment of the knee, whereas the medial compartment showed in 8 cases a grade I, in 13 cases a grade II and even in 2 cases a grade III defect of the cartilage. Even two adipose young patients showed changes in the sense of an incipient osteoarthritis. Conclusions It is concluded, that a couple of morbidly adipose children and teenagers show pathological changes of their cartilage. Whether obesity is responsible by itself for the development of the pattern of diseases or other factors are causally involved will show further research., Introduction Autologous Matrix Induced Chondrogenesis (AMIC®) combines microfracturing with application of a cell-free collagen scaffold. No cultured chondrocytes are necessary. It was established for focal cartilage repair in the knee as a cost-effective one-step procedure. Methods and Materials 38 focal chondral/osteochondral defects (ICRS III-IV°) of the femoral condyle, trochlea and/or patella in 35 patients (27 male, 8 female, mean age 35.8 (18–52) years) were treated by standardized microfracturing and application of a cell-free collagen type-I/III scaffold (Chondro-Gide®, Geistlich Biomaterials). The mean defect size was 3.8 (1.0–10.8 cm2). The results were evaluated prospectively by functional outcome scores, subjective clinical ratings and MRI with an average follow-up of 30.4 months (range 24–54 months). Results Significant improvements were seen in the Cincinnati-Score (50.4 to 87.3) as well as in the Lysholm-Score (59.6 to 88.6, each p⇠0.001). Pain on a 10-point VAS decreased significantly from 6.2 to 1.8 while subjective knee function improved from a mean of 4.6 to 7.5. In 5 revision cases at 4–26 months the repair tissue revealed reasonable results with regards to surface formation, filling and integration. The MRI follow-ups showed an adequate filling of the defect, no prolonged effusion occured. 85% (30/35) of the patients were satisfied with the functional results (ICRS I°+II°). Conclusions The AMIC® procedure is a minimal invasive effective one-step therapy for focal chondral or osteochondral cartilage defects in the knee. It was shown to provide stable results at mid-term follow-ups., Introduction Many treatment options for full-thickness cartilage defect repair in the knee show good short-term clinical outcomes. The mid-term clinical scores of Autologous matrix-induced chondrogenesis (AMIC®) show a “Plateau Effect” after 3 years. Methods and Materials AMIC® enhances the microfracture technique by stabilizing the blood clot with a collagen type I/III matrix (Chondro-Gide®, Geistlich, Switzerland). Mesenchymal stem cells are activated by microfracturing and the matrix is glued onto the defect with a partially autologous fibrin glue. A retrospective study was carried out to investigate the objective and subjective clinical outcome over a period of up to 4 years post-operatively. 49 patients (mean age 36±10 years) with focal chondral defects of the knee joint (22 retro patellar, 23 femoral condyle, 4 trochlea) were treated with AMIC®. The average defect size was 3.6 cm2 (2–8 cm2). The patients were followed-up clinically with MRI and different knee scores. 30 patients were included into the 2-year, 9 into 3-year and two patients into 4-year evaluation. Results The scores showed significant improvement (p=⇠0.05) in all postoperative evaluations except after 4 years compared to preoperative value. There was no significant improvement or decline within postoperative values at 1, 2 and 3 years. Both 4-year results showed a decline compared to the 3-year postoperative value but higher values than the pre-operative score. Conclusions The results indicate that AMIC is a treatment option for local cartilage defects in the knee which obtains good mid-term results. It shows a “plateau-effect” of the clinical scores after 2 years with no further improvement of subjective outcome., Introduction Subtotal menisectomy is associated with early degeneration of the knee. Meniscus transplant is one approach to this problem. Purpose: This is an evaluation of ten-year clinical outcomes of twenty-two meniscal allografts. Methods and Materials Methods: Records of twenty-one patients with twenty-two meniscal allografts were reviewed. Maximum follow-up time is 13.25 years and the minimum is 9.85 years. Lysholm scores were recorded for pre-op, best ever and present level of function. Patients were followed in clinic or by telephone interview by a person independent of the care team. Joint space on x-rays was evaluated using the non-operative side as a control if asymptomatic. Results Lysholm scores were available for nineteen patients. Comparison of Lysholm scores showed significant improvement. Two patients were not significantly improved. Three patients progressed to unicompartmental arthroplasty at an ten years post transplant. Two have poor Lysholm scores and fourteen have good to excellent scores. X-ray data were available for fourteen of the nineteen patients. Difference in joint space in the fourteen was 1.2 mm. Six patients had second looks. One meniscus failed primarily and was successfully reimplanted. There were 3 retears and 3 had increasing chondromalacia. The majority continue to do well. Workers comp did equally well. Conclusions Meniscal allograft prolongs the functional life of meniscus minus knee by being chondroprotective. Degree of chondromalacia at the time of implant is prognostic of longevity. The three patients who went on to unicompartmental arthroplasty all returned to work and had long periods of improvement before failure, Introduction Osteochondritis dissecans (OD) often negatively affects the articular surface of the knee. There is not yet an optimal treatment intervention defined due to the limited amount of prospective research available. The main limitation of previous studies is heterogeneity of the cartilage defects. We evaluated the medium-term outcome of osteochondral autograft transplantation (OATS) in homogeneous OD-lesions. Methods and Materials We evaluated 7 male patients (mean age 33.4) with 8 classical OD lesions at the lateral boundary of the medial femoral condyle. An average of 3.6 osteochondral plugs was used. Patients were evaluated preoperative, at 6 months and 1 year postoperative. The ICRS-evaluation package, International Knee Documentation Committee (IKDC) and Knee injury and Osteoarthritis Outcome Score (KOOS) questionnaires were used for clinical evaluation. Prospective follow-up MRI was performed using a semi-quantitative scoring system. Results The IKDC subjective score improved significantly from preoperative to 1-year follow-up (p=0.001). The KOOS evaluation showed a significant improvement on all subscales; pain (p=0.002), symptoms (p=0.003), activities-of-daily-living (p=0.019), sport and recreation (p=0.014) and quality-of-life (p=0.001). MRI showes good surface congruency, no edema or protuberance of the cylinders, good similarity of cartilage thickness and a non-complete osseous integration. No correlation could be found between MRI findings, percent of coverage and the patients' satisfaction. Conclusions OATS remains a valid treatment option in selected cartilage defects. A subgroup of osteochondral defects on the lateral border of the medial femoral condyle improve significantly following OATS. Future research should focus on identifying the appropriate choice of treatment in well described cartilage lesions, instead promoting one superior technique., Introduction Purpose: To evaluate the incidence and risk factors for knee cartilage and ligament injury in elite college football players Introduction Each year at the NFL combine, approximately 330 of the nation's best collegiate football players are invited to display their skills and physique. In this environment, MRI is used liberally to evaluate any worrisome history or physical findings. The low threshold for ordering MRI is this select group of athletes offers an outstanding opportunity to examine chondral injury patterns. Methods and Materials Methods: From 2005–2007, all players entering the NFL combine were screened for knee injuries(N=980 players). Height, weight, and BMI measurements were taken at the start of the Combine. MRI and plain film findings were recorded into a database along with position, height, weight, BMI, and surgical history. Results During the 3-yr period reviewed, a total of 980 players were available for analysis. The total number of chondral injuries was 199 (20.3%) of all players. 82 players(8.4%) had medial compartment chondral injuries. 104 players (10.61%) had lateral compartment chondral injuries, 118 players (12.04%) had patellofemoral compartment chondral damage, and 79 players (8.06%) had chondral injuries in more than one compartment. Players with a weight greater than 222.5 lbs, or a BMI greater than 30.5 had a significantly higher risk or cartilage injury. Height of the players was not a significant risk factor. Conclusions Discussion: This study is the first of its kind, highlighting the high prevalence (20.3%)of chondral injuries in the N FL recruitment classes of the past 3 years., Introduction We determined if meniscus tear type correlated with function and activity levels two years after meniscectomy. Methods and Materials 206 patients underwent partial medial meniscectomy and 117 underwent partial lateral meniscectomy (average age=49, range, 18 to 80). At index surgery, type of meniscus tear was recorded. Tears were designated bucket handle/ vertical longitudinal (BV), flap/radial (FR), complex (C), or horizontal (H). Patients undergoing microfracture or ACL reconstruction were excluded. Patients were followed for a minimum of two years (average=4.6 yrs) after meniscectomy. Patients completed questionnaires including Lysholm and Tegner scores to assess function and activity. Results For medial meniscus, there was significant correlation between tear type and patient age. BV (n=35) group was significantly younger than the FR (n=65) and C (n=193) groups (p⇠0.01). BV group had significantly higher Lysholm (88) scores versus FR (79) and C (78) (p⇠0.01). BV group also had higher Tegner activity levels (5.4) than C (4.6) (p=0.04). For lateral meniscus, BV (n=15) and FR (n=37) were significantly younger than complex (n=45). FR had higher Lysholm and Tegner scores, but there were no significant differences. 28% of BV medial meniscus tears required further surgery while less than 15% of other types of medial or lateral tears required further surgery. Average time to second surgery was 2.4 years. Conclusions In this series, BV medial meniscus tears had better function and activity at least two years post-meniscectomy, perhaps due to younger age. This group also required more reoperations the first two years after index meniscectomy. Tear type did not influence outcomes after lateral meniscectomy., Introduction Meniscus suture repairs have a high rate of repeat surgery. We determined what factors lead to failure of meniscus suture repair. Failure was defined as repeat surgery within 2 years of initial repair. Methods and Materials 283 meniscus suture repairs were performed by one surgeon. Average age was 31 years (range, 18–71) with 177 males and 106 females. All repairs used an inside-out suture technique. 137 had ACL reconstruction and meniscus repair (93 concurrent reconstructions and 44 two-staged reconstructions). 181 medial menisci and 102 lateral menisci were repaired. Of medial repairs, 80% were posterior third of the meniscus, 11% middle third, 1% anterior third, and 8% extended to all areas. Of lateral meniscus repairs, 49% were posterior third, 26% middle third, 22% anterior third, and 3% extended to all areas. Results Twenty-eight (28) patients (10%) required repeat surgery on the repaired meniscus within 2 years and were considered failures. Average time to repeat meniscus surgery was 12 months (range, 2.5–24 months). There were no differences based on age, gender or location. Thirteen percent (13%) of medial repairs and 4% of lateral repairs failed (p=0.012). Medial repairs failed significantly earlier (5.6 months) than lateral repairs (12.9 months) (p=0.001). For patients who had ACL reconstruction and meniscus repair, factors associated with failure included age (failed age=22; non-failure age=29; p=0.013), and concurrent ACL reconstruction (two-staged repair failures=2%; concurrent repair failures=11%; p=0.04). Conclusions Failure of index meniscus suture repair within two years is more likely to occur in medial repairs and in young patients with concurrent ACL reconstruction., Introduction We determined anatomic location of partial medial meniscectomy (PMM), then correlated location with function and activity two years post-meniscectomy. We hypothesized that posterior third loss would decrease function and activity more than loss of middle or anterior thirds. Methods and Materials 120 patients (18–60 years) underwent PMM as controls in a device study. There were 74 acute (no prior PMM) and 46 chronic (1–3 prior PMM) patients. At index surgery, location of meniscus removed was documented and categorized as posterior (A), middle (B), or anterior (C) third. Patients were followed clinically for ⇢2 years after meniscectomy. Patients completed validated questionnaires including Lysholm and Tegner scores for function and activity. Results 17 patients had isolated A meniscectomies, 83 had combined AB thirds, 10 had isolated C, and 10 had combined ABC PMM. Patients with isolated A meniscectomies had significantly lower Lysholm scores (78) versus all other patients (88) (p=0.01) and significantly lower Tegner scores (3.5) versus all other patients (4.5) (p=0.03) two years after PMM. Based on actual measurements, isolated posterior PMM group averaged 41% meniscus removed, well below the 50% loss threshold previously reported as predictive of decreased function and activity. Conclusions Function and activity levels were significantly decreased two years after surgery in patients who had isolated posterior third PMM versus all other patients with meniscus loss in other anatomic locations. Decreased function and activity occurred even though ⇠50% meniscus was removed. We confirm the importance of preserving the posterior medial meniscus. Potential positive benefits of replacing or regrowing lost meniscus tissue are supported., Introduction The purpose of this study is to evaluate the outcome of the microfracture procedure in treating full-thickness cartilage lesions of the knee in a large patient sample using a random-effects model for longitudinal data analysis. Methods and Materials 350 patients (avg. age=47.6 year(range, 12–76), 65% male; 55% female) who underwent the microfracture procedure by a single surgeon were identified for analysis. Subjects who underwent concomitant ligament or meniscus surgery were excluded. Data were analyzed with a random-effects model for longitudinal analysis. Outcome variables were Lysholm Score(LYS) and the Tegner Activity Scale(TAS). Independent variables included gender, age, degenerative versus traumatic lesion, and years since surgery(YSS). Average follow-up was 4.3 yrs(range 1 to 12). Results For gender, there was no significant difference in the trajectory of the plots over time. [LYS-squared=4954+435(YSS)–45(yrs-squared)+408(gender)–10.6(yrs*gender)+1.3(yrs-squared*gender); p=0.88] [TAS=3.73–0.001(YSS)-0.002(yrs-squared)+0.7(gender)+0 .035(yrs*gender)+0.0006(yrs-squared*gender)p=0.99]. Although there is a trend favoring the outcome of traumatic over degenerative lesions there was no significant difference in the trajectory of the plots. [LYS-squared=5539–3.4(age)+456(YSS)–47(yrs-squared)–333(djd)-85.6(years*djd)+8.8(yrs-squared*djd) p=0.328] [TAS=6.0–0.03(age)-0.04(YSS)+0.002(yrs-squared)–0.539(djd)+0.123(years*djd)–0.011(yrs-squared*djd) p=0.272] Subjects were divided into two age groups, ⇠= 45 years and ⇢45 years. Subjects with traumatic lesions demonstrated a significant difference in the trajectory of LYS scores over time by age group. [LYS_squared=5194+607(YSS)-58.2(YSS squared)+598(age)–336(YSS*age)+24.5(YSS*age) p=0.0375] Conclusions We found no significant differences in the trajectory of outcome over time between genders, or between degenerative/ traumatic chondral lesions. We did identify age-dependent differences in outcome over time. Subjects with traumatic lesions demonstrated a significant difference in the trajectory of Lysholm scores over time by age group., Introduction Microfracturing is a common primary cartilage repair technique for focal cartilage defects in the knee but sometimes fails. In this prospective study, second-line treatments of focal chondral or osteochondral defects in the knee after failed microfracturing by the use of a scaffold ACI are compared with first-line scaffold ACI treatments. Methods and Materials 28 patients (30.7 (17–51) years, defect size 4.5 (2.1–9.4) cm2) with failed microfracturing of focal cartilage defects in knee (ICRS III-IV°) were treated secondarily by scaffold ACI (MACI®). Their outcome was compared to primary treatments of equal defects of 28 matched patients (31.2 (14–49) years, defect size 4.4 (2.1–8.9) cm2) by scaffold ACI. The follow-ups were 27.1 (12–43) vs. 25.2 (12–39) months. Gender, defect origin, BMI and concomitant therapies were comparable. Results The Lysholm scores improved for second-line ACI (61.1 to 82.1) as well as for first-line ACI (60.9 to 86.4, both p⇠0.001). On a VAS pain decreased in second-line ACI (5.5 to 3.2) and in first-line ACI (4.8 to 2.6). Subjective knee function improved from 4.8 to 6.2 in second-line ACI and from 4.7 to 7.1 in first-line ACI (all p⇠0.001). No statistical differences between both groups were found. In revision cases 4/6 (second-line ACI) and 3/4 (first-line ACI) arthroscopic aspects of the repair tissue revealed normal or nearly normal results according to ICRS classification. MRI follow-ups showed an adequate filling of the defects, no prolonged effusion occurred. Conclusions Scaffold ACI is an efficient therapeutic option even for second-line cartilage repair after failed microfracturing for focal cartilage defects in the knee., Introduction Microfracture is an effective surgical treatment for isolated, full thickness cartilage defects. This study aimed to examine the clinical outcomes of patients who have undergone arthroscopic microfracture for chondral defects of the glenohumeral joint. Methods and Materials From March 2001 to August 2007, 18 patients who underwent arthroscopic microfracture of the humeral head and/or glenoid surface were retrospectively reviewed. All patients were examined by an independent, blinded examiner and completed surveys containing the Simple Shoulder Test (SST), American Shoulder and Elbow Score (ASES), and Visual Analog Scale (VAS). Preoperative scores were compared to postoperative outcomes at a minimum follow-up of one year. Results Three (16.67%) patients went onto shoulder arthroplasty and were considered failures, while two were lost to follow-up, for a total follow-up rate of 88.89%. Of the final thirteen patients, the mean age was 37.03 years (range, 18 to 55) with an average follow-up of 27.8 months (range, 12.1 to 89.2). Microfracture was performed on the humeral head in 8 cases, on the glenoid surface in 4 cases, and on both surfaces in 1 case. The average size of humeral and glenoid defects was 5.07 cm2 and 1.66 cm2, respectively. There was a statistically significant decrease (p⇠0.002) in the VAS following surgery as well as statistically significant improvements (p⇠0.002) in SST and ASES data. Twelve (92.3%) patients were satisfied with the surgery and would have the same procedure again. Conclusions Microfracture of the glenohumeral joint provides a significant improvement in pain relief and shoulder function in patients with isolated, full thickness chondral injuries., Introduction The purpose of this study was to determine the effectiveness of the microfracture technique according to function, pain control and activity level, in the treatment of knee cartilage lesions. Methods and Materials Thirty nine patients were treated with microfracture for ICRS grade III and IV cartilage lesions of the knee. Functional outcome was prospectively evaluated with a minimum 3-year follow-up (range, 36 to 79 months) by subjective rating, visual analogue scale and activity-based outcome scores, tested by paired t test and U Mann Whitney test. p⇠.05 was considered statistically significant. Results The mean age of our patients was 40.5 years (range, 19 to 74), 17 women and 22 men. Articular lesions involving medial or lateral compartment were 89.7% and 10.3% patellofemoral compartment. Partial meniscectomy were done in 24 patients (61.5%) and ACL reconstruction in 15 patients (39.5%). The mean postoperative Lysholm score was 80.9 (sd±17.7), there was a non statistically significant reduction in VAS score from 5.3 to 4.3 (p= 0.173), with a decrease in Tegner activity scale scores from 4.6 before injury to 3.6 postoperatively (p ⇠0.0001). We found no significant differences in Lysholm score between patients older or younger of 40 years old (p = 0.343) neither in patients with or without concomitant meniscectomy (p = 0.3). Conclusions According to our results, moderate functional results, non statistically significant reduction in pain relief and a decrease in activity level score, are obtained after three to six years follow up, with microfracture technique in the treatment of cartilage lesions., Introduction Pro-inflammatory cytokines play a pivotal role in bone tunnel widening after ACL reconstructive surgery. A new treatment option is to administer Autologous Conditioned Serum (ACS/Orthokine) containing endogenous cytokines including IL-1Ra and growth factors like IGF-1, PDGF and TGF-β produced from venous patients blood. The trial purpose was to establish whether the osteoclastic effect could be affected by ACS/ Orthokine, representing a better postoperative outcome. Methods and Materials In a prospective, randomized, double-blind, placebo-controlled trial with two parallel groups 62 patients were treated. Bone tunnel width was measured by CT-scans and efficacy was assessed by WOMAC and IKDC 2000 in patients receiving ACS (Group A) or Placebo (Group B) one year following ACL-reconstruction using autografts of m. semitendinosus and gracilis tendons (HS), as well as the patellar ligament (BTB). Results Tunnel widening during the first year was significantly lower in Group A than in Group B. The increase within the ACS group was significantly lower in the HS (ACS vs. Saline 6 months: p=0.032, 12 months: p=0.048) and in the BTB group (ACS vs. Saline 6 months: p=0.061 [n.s], 12 months: p=0.001). Clinical outcome (WOMAC, IKDC 2000) was better in patients treated with ACS at all data points and outcome parameters and significant in WOMAC stiffness subscale after 1 year. Conclusions The significant reduction of bone tunnel enlargement, the improvement of stiffness together with the consistently higher improvement of most other parameters demonstrates that ACS clearly induces a biological response different from saline treatment and warrant future investigations into the possible protective effects of ACS/ Orthokine., Introduction There is a lack of prospective, multicenter randomized clinical trials comparing cartilage repair products in development to surgical controls, making it difficult for surgeons to confidently develop treatment algorithms. BST-CarGel® is a new medical device being investigated for the repair of focal articular cartilage lesions for which a controlled clinical trial has been designed and implemented. Methods and Materials Eighty subjects (40 subjects/group) are being randomized to BST-CarGel® + microfracture or microfracture alone. The sample size was derived from large animal efficacy studies attempting to reach p⇠0.05 significance and 90% power. Eligible subjects from 18 to 55 years have single Grade 3 or 4 cartilage lesions up to 10 cm2 on the medial or lateral femoral condyles. Both groups follow identical 12 week post-operative physiotherapy programs and are non-weightbearing for 6 weeks. The primary endpoint is cartilage repair at 12 months assessed by blinded MRI for % lesion filling, and T2 MRI and dGEMRIC for repair tissue collagen characteristics and proteoglycan content, respectively. The secondary endpoint is improvement in pain and physical functioning at 12 months as assessed by the WOMAC questionnaire and tertiary endpoints are safety and quality-of-life by SF-36. Optional 13-month biopsies may provide additional quality information via microscopic methods Results A prospectively planned interim analysis was conducted at 6 months follow-up on 23 patients. Results indicated evidence of a positive effect of BST-CarGel® treatment on cartilage structure when compared to the microfracture control, and comparable safety to the control. Conclusions This study represents an important step toward developing evidence-based treatment algorithms for cartilage repair., Introduction Ideal treatment for osteochondral lesions of the knee (OLK) is still controversial. Although good results were obtained by Mosaicplasty and Autologous Chondrocytes Implantation, still well-known drawbacks are inevitable. The One step repair technique with bone marrow derived cells was previously presented in the ankle with satisfactory results. Aim of this study is to present the application of the “one-step” technique in the knee joint and evaluate the results. Methods and Materials From April 2006 to May 2007 13 patients with OLK underwent the One-step procedure. 7 cases affected the medial condyle, 2 cases the lateral condyle and 4 cases the patella. The condylar lesions underwent a completely arthroscopic procedure, while the patellar were treated by open access. Bone-marrow was harvested from the posterior iliac crest, and the cells were concentrated in the operating room and implanted at the lesion site on a hyaluronan-based scaffold. Platelet Rich Fibrin was added providing growth factors. Results The mean preoperative IKDC score was 34±13. The IKDC at 6 months follow-up was 68±15, at 12 months was 81±8 and at 24 months was 88±3 (8 patients). The control MRI at 12 and 24 months follow-up showed a good regeneration of the subchondral bone and the cartilagineous tissue. A biopsy of the regenerated tissue performed at 12 months showed a cartilagineous tissue in organization and a newly formed subchondral bone. Conclusions These results demonstrated that the one-step technique represents a good option for osteochondral lesion repair in the knee joint, overcoming the major drawbacks of previous techniques., Introduction Osteochondral Defect (OD) has a poor spontaneous regenerative capacity. To our knowledge there are no reports about therapy in humans using mesenchymal stem cells (MSCs) induced in vitro to chondrogenic differentiation over a 3D membrane. Aim: To describe the clinic, radiological, histological and molecular results at two-year follow-up. Methods and Materials Bone Marrow (BM) MSCs were obtained from a 32 years old patient carrying an OD ICRS4⇢10cm2 on the external femoral condyle of the left knee. MSCs were cultured and then repleate over a collagen type I membrane. 3 days before the surgical procedure, the cells were supplemented with TGF-b1 (10 ng/ml). After that, the membrane carrying cells was implanted over the top of the OD. Clinical and radiological follow up was performed every 3 months. 2 years after the surgical procedure a second-look arthroscopy was performed and biopsy specimens were obtained from the OD area. Results At two-years follow-up the patients have recovered articular functionality. The Lysholm score improved 80 points (15 to 95). In radiological studies was observed complete scaffold integration. The cartilage was normal at second-look arthroscopy. The biopsy histology (H-E and safranin-O) was normal. Western blot and real time PCR for SOX-9 and collagen II from biopsy were similar to normal cartilage. Conclusions We demonstrated successfully repair of OD ICRS4 ⇢10cm2 with autologous MSCs induced to chondrogenic differentiation over a 3D collagen type I membrane at two-year follow-up. We propose this technique as a new alternative in the treatment of OD ICRS⇢4., Introduction In degenerative arthritis, a full-thickness Outerbridge IV lesion is considered end-stage. However, an in-depth pathological study is not available. The Outerbridge IV lesion may have potential for repair in the proper environment. Cartilage regrowth has been reported in hips and in knees following high-tibial osteotomy. Our study purpose was to examine gross and microscopic characteristics and validate the potential for the cartilage aggregate as a source of repair. Methods and Materials Osteochondral specimens from the lesions were harvested from TKA patients. Multiple disc-shaped samples were prepared for tissue culture. Specimens were stained with Safranin-O (without fixation), which selectively stains the aggregates and adjacent intact cartilage. This technique quantitated the size of cartilage aggregates in live specimens and permitted monitoring of growth in culture and subsequent histology. Results Stained gross specimens showed cartilaginous aggregates on the surface and multiple small depressions. On microscopy, the cartilaginous aggregates were found to stain positive for glycosaminoglycans, type II collagen, and lubricin. Depressions were attributed to aggregate erosion, vascular rupture, and bone fragmentation. Cartilage aggregates grew over the surface of the exposed bone in culture. Histologic examination at 3 and 6 weeks revealed abundant cellular proliferation of spindle- and oval-shaped cells covering the surface. Conclusions The multiple small depressions could house various cell therapies. Cartilage aggregates proliferated in culture, confirming the hypothesis that they contribute to cartilage repair following reduction in joint pressure on the lesion. This experimental model is valuable to evaluate various therapeutics to enhance cartilage repair., Introduction ChonDuxTM is a biomaterial guided cartilage repair system consisting of (1) a photopolymerized hydrogel and (2) a biological adhesive that are used in conjunction with microfracture to enhance the environment for endogenous stem cells to repair cartilage. The purpose of this study was to evaluate the safety and feasibility of ChonDux in treating patients with symptomatic, focal chondral lesions. Methods and Materials Fifteen patients (age 27–57 yrs) without general osteoarthritis who had a single, symptomatic cartilage defect 2–4 cm2 in size on the medial femoral condyle in a stable knee were treated with ChonDux. Pain and knee function were assessed by the IKDC form at baseline, 3, 6, and 12 months. Repair tissue fill was determined by MRI. Data collected to date are presented. Results A significant improvement in knee function was observed. The average IKDC score increased from 38.4 at baseline to 69.0 at 6 months (n=14). The severity of pain dropped from 54% at baseline (n=15) to 25% at three months (n=15) and 24% at six months (n=14). The frequency of pain also progressively decreased from 76% (baseline) to 36% and 27% at 3 and 6 months, respectively. This coincided with significant tissue fill by MRI, averaging 83% at 3 months (n=9) and 85% at 6 months (n=12). Conclusions This study demonstrates Chondux is safe, easy to use, and provides clinical benefits (stable defect fill, reduction in pain, and improvement in knee function). Further studies will evaluate efficacy against microfracture controls. ChonDux may offer an effective, single surgery, off-the-shelf treatment option for cartilage repair., Introduction We need several approaches to improve the clinical results for cartilage defects. Methods and Materials 1. Tissue-engineered cartilage: We have been performing transplantation of tissue-engineered cartilage made ex vivo for the treatment of osteochondral defects of the joints (108 cases) as a second generation of chondrocyte transplantation since 1996. 2. Articulated Distraction Arthroplasty: Bone marrow stimulating procedure has two potential weak points to induce hyaline cartilage. One is compressive overload on the drilled or microfractured area at the early stage. In order to reduce the overload, we have developed external fixators which allow almost full ROM with joint distraction for clinical cases based on an animal study. 3. Future direction for cartilage repair with minimally invasive tissue-engineering technique: The most optimal procedure to repair cartilage defects is just injection of cytokines or growth factors and cells. Our completely novel approach is to use cell delivery system using an external magnetic field. Results 1. Sixty knees who had received transplantation of tissue-engineered cartilage for cartilage defects were followed up for at least 5 years. 2. This apparatus has been effective for clinical cases, although the number of the patients and the follow-up period were limited. 3. This magnetic system was demonstrated to be effective for animal studies. Conclusions Our new approaches are introduced., Introduction This paper assesses clinical and radiological outcome following cartilage repair in the knee using TruFit plugs, with a view to determining safe return to activity. Methods and Materials Prospective analysis of 31 patients undergoing articular cartilage repair using TruFit CB plugs (Smith & Nephew) for symptomatic chondral defects in the knee. Clinical and MRI analysis was performed post op at 6, 12, 18 and 24 months. 14 have minimum 12 months follow up and form the study group. Results 4 female and 10 male patients with mean age of 35.3 were included. 1 − 4 plugs were used in 6 Left and 8 Right knees, 3 for osteochondral lesions and 11 purely chondral. 4 procedures were performed mini-open and 10 arthroscopic. The trochlea was repaired in 3 and femoral condyle in 11 (medial 7, lateral 4). 6 had undergone previous microfracture and 1 previous OATs surgery. Mean IKDC improved from 42.6 pre op to 66.1 at 12 months and 78.9 at 24 months. The Tegner activity score improved from 3.2 preop to 4.6 at 12 months and 5.3 at 24 months. Similarly the Lysholm score also improved, from 53.2 preop to 72.7 and 81.8 (p⇠0.05). MRI evaluation showed that oedema like signal surrounding the plugs resolves around 6 months and the subchondral lamina is seen to reform. At 12 months T2 Mapping indicates that the neo-cartilage shows similar signal to native cartilage. Conclusions TruFit plugs appear to provide good results for repair of small lesions and MRI imaging including T2 mapping relates to clinical improvement., Introduction Recent knowledge regarding tissue biology highlights a complex regulation of growth factors in reaction to tissue damage. Platelet Rich Plasma (PRP), containing a natural pool of growth factors, can be obtained in a simple, low cost and minimally invasive way and be applied to the lesion site. In this prospective comparative study we evaluated if the treatment with PRP injections can reduce pain and increase function in cases of chronic refractory jumper's knee. Methods and Materials We treated 15 patients affected by chronic jumper's knee, who had failed previous nonsurgical or surgical treatments, with multiple PRP injections and physiotherapy, and we compared the clinical outcome with an homogeneous group of 16 patients primarily treated exclusively with the physiotherapic approach. Results No significant differences were obtained with the EQ VAS score and pain level evaluation at 6 months of follow up, as with time to recover and patient satisfaction, whereas a higher improvement of the sport activity level was achieved in the PRP group. Conclusions The clinical results are encouraging, indicating that PRP injections may have the potential to increase the tendon healing capacity in difficult cases with chronic refractory tendinosis., Introduction Pro-inflammatory cytokines play a pivotal role in osteoarthritis as well as after ACL reconstructive surgery. Therefore, controlling the harmful intra-articular influence of elevated interleukin (IL)-1β could be useful. A new treatment option is to administer Autologous Conditioned Serum (ACS/Orthokine) containing elevated amounts of Interleukin-1 receptorantagonist (IL-1Ra) produced from venous patients blood. The study objectives were to measure level and dynamics of IL-1β concentrations in two treatment groups in three post-operative periods. Methods and Materials In a prospective, randomized, double blind, saline-controlled group study we measured the IL-1β concentrations following ACL-reconstruction periodically in 3 synovial fluid and 4 serum samples in 20 patients receiving an injection series with either ACS (Group A) or Saline (Group B). Results The decrease of the IL-1b synovial fluid concentration was pronounced in the ACS Group and values were significant lower in the ACS Group at day 10 (p=0.017). In eight Group A patients serum IL-1β was detected on day 6. In four of them whose synovial fluid levels were over 10 pg/ml on day 6, serum I L-1β was detected on day 10. Correlation between serum and synovial fluid IL-1b appearance persists in patients after ACL surgery and ACS application. At the same time there was no such correlation in Group B. Conclusions The intraarticular application of ACS tends to result in a decrease of IL-1β and indicates a possible influence of ACS on the ACL healing process influencing the IL-1β levels on the basis of the serum IL-1β detection and warrant future investigations into the possible protective effects of ACS/Orthokine., Introduction Joint distraction as treatment of severe osteoarthritis has demonstrated progressive prolonged clinical benefit. The present study describes the first results on changes in tissue structure induced by joint distraction of end stage knee osteoarthritis. Methods and Materials Young patients (⇠60 yrs) with severe osteoarthritis of the tibio-femoral joint, considered for joint replacement, were treated with joint distraction. An external fixation frame bridging the knee joint was placed, distracting the joint 5 mm for a period of two months. In addition to clinical outcome, serum and urine samples were analyzed for biomarkers of cartilage turnover, radiographs were scored digitally for multiple OA related characteristics (KIDA), and MRIs were evaluated for area covered with cartilage, cartilage thickness and volume characteristics. Results The first thirteen patients (48±3yr) have reached a follow-up of 1 year. Clinical condition improved significantly and progressively. Radiographs demonstrated a significant increase in minimum joint space width. These results are corroborated by a gradual increase in cartilage synthesis markers (PIIANP and CS846) and decrease in breakdown markers (CTX-II, COMP) after an initial huge change in cartilage turnover during distraction. MRIs demonstrated a significant increase in cartilaginous tissue volume and covered bone area. Conclusions Absence of mechanical stresses on cartilage, in combination with maintained intermittent intra-articular fluid pressure, and significant increase in peri-articular bone turnover as induced by joint distraction, may be the underlying mechanisms for cartilage repair (demonstrated by different surrogate markers) being involved in the clinical benefits of this very promising treatment., Introduction Until now there are not well established therapies able to prevent the development of osteoarthrits. Methods and Materials We had in our clinical observations a group of young adults under 30 years old, who practiced different sports (athletics, running, ski, basketball, handball, football, gymnastics, tennis, bodybuilding, martial arts) during childhood and/or teenage, now presenting osteoarthritis-like symptomatology of the knee. Therapy with analgesics and/or nonsteroidal anti-inflammatory drugs were not efficient and physiotherapy was slightly helpful in some patients. We have no proof that some sports may trigger a premature osteoarthritis, except the therapeutical evidence that an early treatment with SYSADOA is beneficial by improving rapidly and significantly the osteoarthritis-like symptomatology mainly from the patient's point of view. Results Based on our hypothesis that there is a pre-arthritis state due to micro-lesions of the cartilage sustaining a repeated and intense effort during different sports, we recommended a combination of condroitin 1200 mg and glucosamine 1500 mg divided in 2 doses daily. The osteoarthritis-like symptomatology (crunches, stiffness, pain) was resolved within one month, but the therapy was continued for another month. We monitored the beneficial effect of SYSADOA for a period of 12 months. Conclusions An early therapy with SYSADOA might be a solution for the prophylaxis of knee osteoarthritis in young people after giving up sports. We intend to develop a long term study in order to monitor the effects of exercise on cartilage in the intact human joint in two situations: with physiologic loading conditions and with overloading during different sports., Introduction Although pharmacologic treatment remains the mainstay for treating rheumatoid arthritis, there is an increasing need for a method that biologically regenerates arthritic knee lesions as the patient's life span increases. Methods and Materials Case Presentation A 35-year-old Korean woman with RA was admitted for right knee joint pain. Plain radiographs revealed progression of arthritis with lateral joint space narrowing comparing with radiographs obtained four years previously. The cartilage fragment was sent to a commercial cell culturing facility (SW Cellontech, Seoul) for processing. Autologous chondrocyte implantation was performed six weeks after her initial surgery when 48′106 chondrocytes had been cultured. postoperatively, lateral joint space of the knee became wider and the patient could walk without pain. Results We treated rheumatoid arthritis of the right knee in a thirty-five-year-old Korean female patient by autologous chondrocyte implantation. Twelve months following surgery, the patient could walk without pain. Conclusions Therefore, we believe that autologous chondrocyte implantation appears to be effective for treating rheumatoid arthritis of the knee., Introduction The pathogenesis of osteoarthritis (OA) remains to be elucidated, partly due to limitations in investigative methods. We evaluated the microdialysis technique as a possible in vivo method to obtain dialysates from the intraarticular space and from the synovium of the knee. The technique enables local measurements of cytokines and biomarkers supposedly involved in the pathogenesis and understanding of OA. Methods and Materials Seven patients undergoing planned arthroscopy of the knee due to degenerative changes were included in the study. Following anaesthesia, 2 microdialysis catheters were positioned under ultrasonographic guidance, intraarticularly in the suprapatellar pouch and in the medial parapatellar part of the synovium. The microdialysis catheters were perfused with a solution of Ringer-acetate containing radioactively labelled glucose allowing for determination of relative recovery as an indicator of membrane permeability. The microdialysis catheters were left in situ to determine the actual position during arthroscopy and then removed peroperatively. Results From the perfusion 91% (SD 14%) of the volume, was recovered in the dialysate from the catheters placed in the synovium and 117% (SD 6%) from the intraarticular catheters. Relative recovery for intraarticular catheters was on average 55% (SD 17%) and for the catheters in the synovium 65% (SD 8%). Conclusions Microdialysis of the knee has previously been tried peroperatively2, but has not been methodologically evaluated. We have shown that it is possible to place microdialysis catheters in the synovium of the knee and intraarticularly by ultrasonographic guidance and furthermore to perform microdialysis with a relative recovery comparable to that of other tissues., Introduction There remain some debates on osteochondral autogenous transfer (OAT; mosaicplasty) for an early stage of osteoarthritis in knee joints (OA). The objectives of this study are to investigate effectiveness and validation of OAT for the early OA. Methods and Materials Consecutive series of eighteen cases (eighteen knees) were retrospectively reviewed. Six males, and 12 females, and average age at the surgery was 57.8±12.7 years old. Inclusion criteria was those patients who had isolated chondral lesion (ICRS grade-3 and −4) at medial femoral condyle, whose standing lateral femoro-tibial angle (FTA) was less than 180 degrees, and whose radiographic OA grading was less than 3 (Kellgren-Lawrence (K/L)). Several numbers of osteochondral plugs were transferred from lateral and medial edges of femoral groove. IKDC subjective score, FTA, and K/L grading were assessed pre-operatively and at the final follow-up (18.5±9.9 months in average). Results Post-operative IKDC score improved comparing to preoperative one. FTA significantly increased from 178±2.2 degrees pre-operatively to 179.5±3.0 degrees post-operatively (P=0.0030). In some cases where the pre-operative FTA was close to 180 degrees, the post-operative FTA increased being over 180 degrees. And there was moderate negative correlation between pre-operative IKDC score and the increase in FTA. Regarding K/L grading, nine cases did not change, while 9 cases deteriorated. Conclusions The present study showed favorable short-term clinical outcomes after OAT for the early knee OA where FTA was less than 180 degrees, although OAT cannot improve lower limb alignment. Further long-term clinical study is essential to evaluate the influence of increased FTA on the clinical outcomes., Introduction Mosaic arthroplasty is recognized as viable reconstructive option for cartilage lesions in the 2–4+ cm2 and range. Results have been comparable with more costly and complex cell based techniques, but donor site morbidity and the accuracy of graft harvesting and delivery remain problematic. Restoration of the joint surface relies on complex geometrical fitting of several cylindrical osteochondral plugs that can be re-arranged or rotated around their longitudinal axis to match the geometry of the recipient site. Complex, small radius or re-curved surfaces are difficult to reconstruct accurately resulting in gaps between grafts, protruding or recessed grafts; all of which are associated with a less favorable outcome. The objective of this work was to create a computerized planning and surgical guidance system that could improve outcomes in complex cases. Pilot studies are described here. Methods and Materials A patient-specific 3D virtual knee model is created by registration of CT and MRI imaging studies. Custom surgical planning software is used to define the diameter, location, surface geometry and depth of each plug, then these fitting parameters can be tested in the recipient site. A patient specific template incorporating multiple harvesting and insertion guides is formed in plastic using a rapid prototype printer and then sterilized. This surface template fits in a unique location on the articular surfaces, and provides accurate positioning of mosaic arthroplasty harvesting chisels, and drill guides as well as orientation of the grafts. Results This system has been tested in 8 sheep with impact-induced cartilage defects requiring 3–5 grafts and one patient with a medial condyle cartilage defect requiring 9 grafts. In all cases the immediate postoperative appearance was very good, operative time was similar to traditional surgeries and short term reduction in pain with improvement in mobility appear promising. Long term studies are ongoing. Conclusions Imaging and creation of patient-specific knee models allowed an unprecedented level of preoperative planning as well as production of plastic templates that allowed fast and precise transplantation of osteochondral grafts in areas of the knee with complex curved geometry., Introduction Autologous Osteochondral Transplantation (OCT) is an established method to treat articular cartilage defects in the knee. However, the potential for donor site morbidity remains a concern. Both the restoration of the original cartilage defect and the evolution of the donor site defects can be evaluated by bone scintigraphy. Thus, prospective bone scintigraphic evaluation was performed in patients who were treated with OCT. Methods and Materials In a group of 13 patients with a symptomatic articular cartilage defect bone scintigraphies were obtained pre-operatively, one year after osteochondral transplantation and finally at an average follow-up of 4 years (31–65 mnths). The evolution of bone scintigraphic activity was evaluated for both the recipient and the donor site. Parallel, clinical scoring was performed using the The Lysholm knee scoring, the Cincinnati knee rating system and the Tegner activity score. Results The bone scintigraphic uptake was elevated at the involved femoral condyle preoperatively and gradually decreased to normal levels in most cases after four years. The originally normal uptake at the trochlea, increased significantly one year after transplantation. Then, a gradual decrease in uptake occurred again at this donor site to remain significantly elevated at the final bone scintigraphy. A significant correlation was found between elevated bone scintigraphic activity and the presence of retro-patellar crepitus. Conclusions Elevated bone scintigraphic activity from an osteochondral lesion can be restored with OCT. However, increased scintigraphic activity is introduced at the donorsite, which reduces again with longer follow-up. Number and size of the harvested plugs appear to correlate with retropatellar crepitus and scintigraphic activity., Introduction Fresh osteochondral allograft transplant (OAT) to the distal femora is currently managed post-operatively with a limited weight-bearing (WB) protocol. This study prospectively assesses clinical and radiographic results of an immediate WB as tolerated protocol following fresh OAT with single or multiple cylindrical grafts to the femoral condyle. Methods and Materials Patients with grade IV ICRS defects treated with OAT were allowed immediate full WB. Validated outcome evaluations were collected pre- and post-operatively. CT scans were assessed for degree of osseous incorporation within modified Cahill/Berg zones. Results Six month data from 32 patients (age= 35.2, 15 to 66) with femoral lesions (5.8 cm2, range 0.8 to 19.6 cm2) secondary to focal arthritis (10), OCD (19) and AVN (2) is reported. Statistically significant, p⇠0.05, improvement was observed in all measures versus baseline: KOOS (Pain 57 to 77, Symptoms 58 to 70, ADL 68 to 89, Sports 41 to 65, QOL 23 to 46), IKDC (45 to 56) and SF-36 (56 to 72). CTs indicate grafts implanted to direct WB regions had ⇢75% incorporation (21 of 27 grafts) compared to ⇠50% incorporation in the indirect WB regions (9 of 16 grafts). Greater improvement was seen with single grafts (SG) n=19, compared to multiple grafts (MG) n=13. Indirect WB region inclusive grafts were larger (10.3cm2 vs. 4.2cm2) requiring MG. Incorporation of ⇠50% was associated with fragmentation. Conclusions Fresh OAT with full WB shows osseous incorporation and improved short term clinical outcomes. SG implantation is associated with stable incorporation. MG treatment, which includes indirect WB regions, can lead to fragmentation., Introduction Osteochondral autograft transfer (OAT) procedures involve taking small cylindrical grafts from areas of decreased weight bearing in the knee and transferring them to accurately prepared recipient defect sites. The morbidity of the donor sites, which are selected at the periphery of the articular surfaces, from areas of relatively low load per unit area, has been poorly studied. Methods and Materials We report on a competitive athlete who underwent an autologous osteochondral transplantation for a full-thickness osteochondral defect of the right medial femoral condyle. The patient developed significant morbidity in the donor site. During arthroscopic revision surgery, biopsy of the donor site showed a foreign-body giant cell reaction. Results A revision arthroscopic autologous osteochondral transplantation was performed with good postoperative outcome. The symptoms resolved unremarkably. Physical examination revealed negative patello-femoral signs. The patient has a negative step up-step down test, a negative patellofemoral grinding test, and a negative patellofemoral compression test. The patient returned to sports activity, but not at the previous competitive level. When last reviewed 2 years post-operatively, he was fully active and walked normally with a Lysholm score of 99. Conclusions OATS certainly has a role in managing OCD lesions of the knee, the potential donor-site morbidity and its potential impact on the functional status of the patient have to be taken into consideration when counseling patients about this procedure. Further investigations are necessary to clarify the donor-site morbidity that can occur after OAT., Introduction Large osteochondral defects in the weight-bearing zones of femoral condyles in young and active patients were treated by autologous transfer of the posterior femoral condyle (MegaOATS). This salvage procedure aims at painfree mobility of patients. Methods and Materials 18 subsequent patients were included betweeten July 1999 and December 2000. 16 patients (4 females, 12 males) were evaluated using the Lysholm score and x-rays. A random test of 8 individuals underwent MRI analysis. The average age at the date of surgery was 37,4 (15–59) years, the mean followup 55,2 (46–62) months. The mean defect size was 5,4 cmÂ2 (3,1–7,1). Trauma or osteochondrosis dissecans were pathogenetic in 81%. Results The Lysholm score showed significant increase from preoperatively median 65.0 to postoperative median 86.0 points (p=0.001). 15 patients returned to sport activities. X-rays showed a rounding of the osteotomy edge in 12 out of 14 patients and a partial remodelling of the posterior femoral condyle in 11 patients. Preoperative osteoarthritis in 9 patients was related to a significant lower increase of the Lysholm score (p=0,038). All MRI examinations showed vital and congruent grafts. Conclusions Patients significantly improved in the Lysholm score, in daily life activity levels and often returned to recreational sports. 15 patients were comfortable with the results and would undergo the procedure again. Thus, MegaOATS is recommended as a salvage procedure for young individuals with large osteochondral defects in the weight-bearing zone of the femoral condyle., Introduction Osteochondral autografts (OCA) brings hyaline cartilage when treating full thickness patellar chondral lesions, offering a good alternative for its management. The objective of the present study is to evaluate clinical and functional results of full thickness patellar chondral lesions treated with OCA. Methods and Materials Consecutive series of ten cases were treated in a seven year period evaluated with Lysholm and International Knee Documentation Committee (IKDC) scores. Average follow up 37,3 (12 to 89) months. All patients were diagnosed with patellar instability, six with associated patellofemoral malalignement (PFM). Results Eight cases treated with mosaicplasty (six PFM, two traumatic injuries), two treated with Osteochondral Autograt Transfer System (OATS) (one PFM, one traumatic injury). Average associated procedures for each patient 1,4 (1 to 4). Eight OCA performed in the medial facet, one in the lateral facet, one in the central patellar area. Average chondral lesion area 1,2 (0,9 to 2) cm2. Autograft harvested from superior lateral trochlea in eight cases and from superior medial trochlea in two. Average of 1,9 (1 to 4) grafts used in each patient. Average graft diameter 6,95 (4,5 to 8) mm, average graft length 10,8 (10 to 12) mm. Average post op scores: Lysholm 95 (90 to 100) points, 60% excellent (6 cases) and 40% good (4 cases). IKDC 93,6 (92 to 96) points. No postoperative complications were registered until conclusion of this review. Conclusions Patellar OCA is a good surgical alternative for the treatment of full thickness patellar chondral lesions, offering good and excellent clinical results in the midterm follow up., Introduction The purpose of this study were to evaluate the time course change of glycosaminoglycan concentrations after high tibial osteotomy using delayed gadolinium enhanced MR imaging of cartilage. Methods and Materials A fifty year old male patient who had HTO due to grade IV secondary osteoarthritis was examined using 1.5T MRI. dGEMRIC was performed before and 1,3,6,9 months after the operation. dGEMRIC index which represent GAG concentrations were calculated and color images of articular cartilage layers were described. ICRS grading of the articular cartilage was performed arthroscopically at the time of opening wedge HTO using TomoFix system. Results The articular cartilage of lateral femoral condyle was divided into two layers; low GAG content superficial layer and high GAG content deep layer. Arthroscopic findings revealed medial compartment to be grade IV and lateral compartment to be grade II and III, which were observed in MRI in the same way. Before the operation, dGEMRIC index was 431m, and decreased to 385ms after 3 months, then recovered to 427ms after 6 month, and maintained to 421ms after 9 months. Conclusions dGEMRIC had the potential to evaluate the extracellular matrix of articular cartilage after high tibial osteotomy non-invasively. Temporary decrease and recovery of GAG content was observed after surgery, which indicates extracellular matrix may have self remodeling process after biomechanical changes due to high tibial osteotomy., Introduction The aim of this study was to determine to what extent combination treatment with high tibial osteotomy (HTO) plus chondroabrasion and microfractures could improve clinical symptoms in patients with medial compartmental osteoarthritis in genu varum. Methods and Materials The study population was 40 patients, 20 of which underwent HTO alone (group A) and 20 others received combination treatment with HTO plus chondroabrasion and microfractures (group B). Final assessment was conducted at 5 years after the operation. Assessment included clinical response as measured by the International Knee Documentation Committee (IKDC) and Lysholm scores and patient satisfaction as evaluated on a satisfaction scale. Results A statistically significant improvement in Lysholm scores (P⇠0.05) was noted in both groups but no statistically significant difference in pre- and postoperative IKDC scores was found between the two groups: 80% of group A and 85% of group B patients scored A at postoperative IKDC. Regarding the Satisfaction Score, there was no significative difference between the two groups in preoperative self assessment, while postoperative subjective satisfaction was significantly higher in patients belonging to group A (p=0,004). Conclusions In patients with medial chondropathy, correction of the mechanical axis by osteotomy remains the treatment of choice in those wishing to continue accustomed levels of sports activities. Because it is a biological choice, osteotomy performed together with chrondoabrasion and microfractures allows a fibrocartilagenous layer to regenerate with characteristics very similar to cartilage in areas where the cartilage is still undamaged., Introduction Combined High Tibial Osteotomy (HTO) with Matrix Induced Autologous Chondrocyte Implantation (MACI) has not yet been described in the literature. We have conducted a prospective study of combined procedures in patients with end stage knee osteoarthritis and have investigated the cartilage in-fill on the weight bearing surface of the medial femoral condyle in the hostile environment of the arthritic knee. Methods and Materials There were 18 combined surgical procedures in 15 patients aged between 27 and 58. Functional evaluation using the 5 KOOS-domains independently was compared with preoperative values. MRIs after 3 months 1, 3 and 5 years were scored according to Marlovits. Results There was significant improvement in all KOOS-domains when 1-year results were compared with preoperative values (p⇠0.05). These improvements were maintained at 3 and 5 years. Two patients have been offered salvage Total Knee Arthroplasty after graft failure at 5 years. Histological investigation after the death of one patient (unrelated cause) 18 months after surgery demonstrated full-thickness hyaline-like articular cartilage. MRI improvements were modest from a mean Marlovits-score of 2.2 to 2.4 with subtotal or complete in-fill in only 6 of 18 knees. Conclusions One third of the patients had good cartilage in-fill indicating the possibility of combining HTO with the MACI-technique. However, there was no convincing MRI evidence of substantial in-fill in 12 of the 18 knees. Based on our findings it is difficult to justify the addition of MACI to HTO for young patients with unicompartmental osteoarthritis. The improvements in clinical outcome could be readily attributed to the osteotomy alone., Introduction Our aim was to use a minimally invasive method for management of the young active patient with medial compartment osteoarthritis, without the risk of serious complications. Methods and Materials Prospective data of 28 consecutive knees who underwent Medial opening wedge high tibial osteotomy for medial compartment osteoarthritis using monolateral external fixator, combined with microfracturing was analysed. Mean age was 47 years, follow up was 23 months (range 7 to 61). Results Mean Lysholm scores improved from 63.6 (42 to 85) to 81.6 at latest follow-up (46.5–100) (p⇠0.001). Similarly, the mean Tegner activity scale improved from 1.7 to 3.3 (p⇠0.001). Average IKDC score at last follow-up was 68 (35.6 − 100). Mean pain score improved from 5.5 to 3.5. (p⇠0.001). All but 4 patients reported improved pain scores. The mean mechanical axis angle was improved from 6.07 degrees of varus, to 3.39 degree valgus. The average joint space improved from 2.4 mm to 2.61 mm after surgery. The mean increase was 2.1 mm. Superficial pin site infection occurred in 6 patients (21.4%) and settled with oral antiobiotics in all cases. One patient had persistent patellofemoral (PF) pain. Conclusions The use of hemicallotasis for high tibial osteotomy (HTO) in association with extensive microfracturing of the medial compartment provides a viable, minimally invasive method for management of the young active patient with medial compartment osteoarthritis, without the risk of serious complications. In the short term even with advanced full thickness cartilage damage, this provides effective pain relief, helps put off more major arthroplasty alternatives and improves activity levels., Introduction Aim of this study was to introduce an improved 3D-MOCART score using the possibilities of isotropic 3D-MRI in the post-operative evaluation of patients after matrix-associated autologous chondrocyte transplantation (MACT) as well as to compare the results to the conventional 2D-MOCART score using standard MR sequences. Methods and Materials One hundred consecutive MR scans in sixty patients at standard follow-up intervals of 1,3,6,12,24, and 60 months after MACT of the knee joint were prospectively included. The mean follow-up interval of this cross-sectional evaluation was 21.4±20.6 months; the mean age of the patients was 35.8±9.4 years. MRI was performed at a 3.0 Tesla unit. All variables of the standard 2D-MOCART score where part of the new 3D-MOCART score. Furthermore additional variables and options were included with the aims to utilize the capabilities of isotropic MRI, to include the results of recent studies, and to adapt to the needs of patients and physician in a clinical routine examination. Standard MR sequences were performed to assess the 2D-MOCART score; an isotropic 3D-TrueFISP sequence was prepared to evaluate the new 3D-MOCART score. Results The correlation between the standard 2D MOCART score and the new 3D MOCART was for the eight variables defect fill, cartilage interface, surface, adhesions, structure, signal intensity, subchondral lamina, and effusion; highly significant correlation with a Pearson coefficient between 0.566 and 0.932. The variable bone marrow edema correlated significantly with a Pearson coefficient of 0.257. Conclusions In the clinical routine follow-up after cartilage repair, the 3D MOCART score, assessed by only one high-resolution isotropic MR sequence, provides comparable information than the standard 2D MOCART score, with the possible advantages of isotropic 3D MRI., Introduction There is a clear need for a patient-based questionnaire to measure the outcome of cartilage therapy. The KOOS has been designed for active patients and validated for several stages of osteoarthritis. The aim of this study was to evaluate the clinimetric properties of the KOOS for regenerative cartilage therapy. Methods and Materials A total of 40 patients treated with regenerative cartilage therapy were used in a test-retest setup, with an intermediate of two days. The patients were asked to complete the Dutch KOOS and complementary questionnaires (SF-36, Lysholm, EQ-5D) to evaluate the clinimetric properties of the Dutch KOOS in terms of internal consistency (Crohnbach's alpha), reliability (IntraClassCorrelation (ICC) and Bland Altman plots), construct validity (Spearman's Rank correlation), and floor and ceiling effects. Results For the KOOS subscales and total score the Crohnbach's alpha ranged from 0.74–0.96. The overall ICC of the KOOS was 0.97 while the subscales ranged from 0.87–0.95 and the Bland Altman plots showed a small individual variance between the two assessments in time for each subscale of the KOOS. The Spearman's Rank correlation between the subscales of the KOOS and representative subscales of the SF-36, Lysholm and EQ-5D ranged from 0.59–0.70 with all p-values lower than p⇠0.001. We observed no floor effects while the largest ceiling effect was 10.3%. Conclusions This is the first study evaluating the clinimetric properties of the KOOS for cartilage therapy. We conclude that the validity and reliability of the KOOS is suitable to measure the clinical condition of patients after regenerative cartilage therapy., Introduction There are many different techniques in use to measure the success of cartilage repair. These range from patient self-assessment scores (such as WOMAC) to semi-quantitative imaging scores (such as MOCART). By standardizing image acquisition techniques and using phantoms with known values, it is possible to obtain consistent quantitative measurements using MRI. Without performing a biopsy, it is difficult to assess the composition of the repair tissue. MR imaging biomarkers (T1 and T2 decay times) can serve as proxies for tissue composition. Methods and Materials To obtain structural measurements in cartilage two gradient echo sequences can be used; one which yields good delineation between bone and cartilage (T1-weighted SPGR TR=39ms, TE=7ms, FA=20°, Spacing=1.5mm), and a second that provides good delineation between cartilage, fluid and soft tissue (T2*-weighted GRE TR=29ms, TE=15ms, FA=40°, Spacing=1.5mm). dGEMRIC techniques can be utilized to obtain T1 maps of the repair tissue and surrounding cartilage. Multi-echo sequences are used to obtain T2 maps of the repair tissue and the surrounding cartilage. Results Structural measurements such as defect volume, repair tissue volume and thickness and surrounding cartilage thickness can be obtained from standardized MR imaging sequences. Tissue composition or quality measurements can be obtained utilizing dGEMRIC or T2 imaging sequences. Conclusions Structural measurements determine how much repair tissue has grown, but not the composition of the tissue. The use of tissue-composition biomarkers (T1 and T2) provide further insight into the quality of the repair tissue. Choosing the right biomarkers depends upon several factors including the trial phase, size, cost and anticipated outcome., Introduction Autologous chondrocyte transplantation (ACT) is an increasingly used procedure cartilage defects in the knee. In this cell based technique, it is felt that increasing time results in maturation of the tissue and improved mechanical, histological, and clinical properties. A prospective study of 118 patients was performed to assess the improvement in clinical outcome (IKDC) related to the follow-up interval. Methods and Materials 118 patients (80m, 38f) underwent matrix assisted ACT (NOVOCART™3D, B. Braun - Aesculap, Germany) from 11/03 −12/06 and were prospectively evaluated using IKDC scoring. The total sample was divided into two subgroups: ⇠24 month followup (n= 63, mean 14,7 month, range 3–24); and ⇢24 months(n=55, mean 36 month, range 25–48). Results The subgroup I (⇠24 month, mean 14,7) shows an improvement in IKDC score of 13,7 pts (31,7%). Within the total sample (avg. follow-up: 24,6 months) the IKDC increased 19 pts (47,5%). Subgroup II (⇢24 months, mean 36,1) improved 25,4 pts (68,3%) compared to the preoperative status. A relationship between increasing followup and increasing improvement was identified. Conclusions Our data suggests that the clinical improvement following matrix-ACT increases depending on the follow-up period. Matrix-ACT remains a technique for cell transplantation, and the initial graft does not demonstrate the mechanical and histological properties of articular cartilage. However, as the tissue matures it gains in strengths and durability within the years after transplantation. This process seems to continue over at least 3 years. Our clinical observation correlates with histological data, showing an improvement of tissue quality between 1 and 3 years after ACT [Roberts S Arthritis Res Ther. 2003]., Introduction Osteoarthritis (OA) is a chronic disease, characterized by gradual loss of articular cartilage and functional limitations. A few studies have compared a muscular strength of subjects with OA degrees I and II. The aim of this study was verify the effectiveness of exercise therapy in these patients. Methods and Materials Were selected 10 men (52,9 ± 6,52 years) who performed a test on the isokinetic dynamometer (Biodex System 3) to assess the maximal isometric torque (MIT), the maximal concentric (MCIT) and eccentric isokinetic torque (MEIT) of the quadriceps femoral and the MCIT of the hamstrings muscles. The individuals have undergone a rehabilitation program for 11 weeks and reassessed at the 5th and 11th week. To determine the effect of treatment were analysed used Friedman test (p≤0,05). Results The result showed statistically significant difference in MCIT of the hamstrings, at 90°/s before and after the 11 weeks (p=0.06) and between the 5th and 11weeks (p=0.0005); at 180°/s, for the same torque, was found differences for the same periods (p=0.005 and p=0.02). For the MIT of the quadriceps, MCIT and MEIT wasn't found statistical differences, however, we can observe a qualitative increase of 20% in the MIT of quadriceps (178.8 to 216.7Nm) before and after 11 weeks and 13%, over the same period, for the MEIT at 90°/s (217,55 to 246,51Nm) showing a progress report. Conclusions The rehabilitation program was effective in improving concentric and eccentric strength especially after the sensory-motor training. It is suggested that the rehabilitation training for OA should emphasize strengthening and training sensory-motor., Introduction For a successful outcome after cell based cartilage repair in the knee an optimal patient selection and accompanying physiotherapy planning seems fundamental. In contrast to athletes, patients encompass difficulties to achieve preset goal settings in rehabilitation. Methods and Materials This qualitative study was performed to obtain improved insights on the rehabilitation processes after cartilage repair in the knee. Physiotherapists, specific experienced in guiding patients after local cartilage repair in the knee, attended 4 panel discussions on physiotherapy planning over a 2 year period. Results Experience based suggestions are: 1/Patients have better adherence if there is specific communication on rehab goals and methods every 3 months following surgery. 2/Physiotherapists can set realistic goals if they are well informed on exact location, size and contours of the repair, concomitant procedures and previous pathologic knee conditions. 3/Since patient and therapist often over-estimate the local load ability of the repair zone, rehab protocols with fixed timelines for progression are not recommended. 4/Patient tailored rehab approach is needed to gain knee function progressive and safe. Moreover it allows the physiotherapist to focus systematically on the quality of movements of the operated knee. Conclusions Experienced based recommendations are: The progress in exercise demands should be paced by the biological process that is on-going in the knee joint. Exercises that improve proprioception and neuromuscular control need to be accentuated in low and moderate load conditions. These two movement quality aspects are believed to be most important. Strengthening exercises, which can overload the repair zone, are specifically planned in later phases of rehabilitation., Introduction The purpose of this study was to analyze the morphological characteristics and incorporation of the TruFit CB scaffold plugs used for OATS donor lesion backfill with cartilage-sensitive MRI and T2 mapping. Methods and Materials Twenty-six patients (mean age 28.72 yrs) underwent OATS for chondral defects of the knee or talus with Trufit plug backfill of donor site lesions. 43 cartilage-sensitive MRI's and 25 T2-mapping studies were performed at various postoperative intervals (range: 1–39 months, mean 16.4 months). The donor sites were assessed for plug morphology, displacement, hypertrophy, subchondral edema, bony overgrowth, percentage fill, degree of incorporation, ICRS score of the adjacent and opposing articular cartilage, and T2 mapping of the repair tissue. Results Longitudinal analysis revealed favorable plug appearance at early follow-up (≤6months) with deterioration at intermediate follow-up (ff12months). Plug appearance substantially improved, however, with longer follow-up (≥16 months). Hyperintense signal was associated with a short postoperative interval (p=0.02) or multiple plug configuration (p=0.01). Abnormal morphology was associated with a short postoperative interval (p=0.02) or large plug size (p=0.003). Incomplete defect fill and poor plug incorporation were both associated with intermediate postoperative duration (p=0.02 and p=0.006 respectively). T2 mapping scores significantly improved with increasing postoperative duration (p⇠0.004). Interface T2 scores were better with single compared to multiple plug configurations (p=0.03). Conclusions The MRI appearance of Trufit CB plugs used for filling an osteochondral defect of the knee demonstrates a predictable pattern of postoperative maturation that reflects biological incorporation. Plug appearance significantly improves with increasing postoperative duration with T2-mapping scores that approach native articular cartilage., Introduction The orthopedic community has not found a satisfactory treatment for articular cartilage defects of the knee. The purpose of this retrospective clinical study was to evaluate the short-term results of the treatment of knee osteochondral defects with a synthetic resorbable biphasic implant (TruFit Plug; Smith & Nephew, San Antonio, TX). Methods and Materials Fifteen skeletally mature patients with symptomatic, full-thickness cartilage lesions of the femoral condyles, between 1 and 2.5 cm in diameter, were treated with implantation of bioabsorbable implant. Implants were press-fit into holes drilled into the defect. All patients were evaluated both preoperatively and postoperatively with the Lysholm knee score, International Knee Documentation Committee (IKDC) Standard Evaluation Form, and magnetic resonance imaging of the joint. Results Fifteen patients of a mean age of 39 years were followed-up for a mean time of 9.1 months. The mean Lysholm score improved from 43.6 preoperatively to 87.5 postoperatively. Excellent or good outcome was accomplished in 12 patients. Using the IKDC assessment, 11 patients reported their knee as being normal or nearly normal. Congruency of the articular surface was restored in 13 patients on magnetic resonance imaging. Abnormal marrow signal in the subchondral bone beneath the region of implant was present in all patients. Conclusions The TruFit implant is an effective and safe method of treating symptomatic full-thickness chondral defects of the femoral condyles in appropriately selected cases. However, further studies with long-term follow-up are needed to determine if the implanted area will maintain structural and functional integrity over time., Introduction We have been developing a new tissue engineering technique for cartilage repair which involves a scaffold-free tissue engineered construct (TEC) bio-synthesized from synovium-derived mesenchymal stem cells (MSCs). The objective of the present study was to perform an atomic force microscopic analysis and observation on the surface structure and stiffness of mature and immature porcine cartilage-like tissues repaired with the TEC. Methods and Materials Synovium-derived MSCs from the immature porcine knee joints were cultured though 4 to 7 passages. After an addition of ascorbic acid 2-phosphate, the cells were allowed to undergo active contraction for 8 hours to develop the TEC. A cylindrically shaped, cartilaginous defect created on the medial condyle of immature and mature pigs. The TEC was allografted to the defect. Six months after surgery, a cylindrically shaped specimen of repaired tissue was extracted and subjected to a surface observation and micro-indentation test using an atomic force microscope. Results The surface of the TEC-repaired immature and mature cartilage exhibited tubercles of approximately 2–4 mm in height, which was significantly smaller than those observed in the non TEC-repaired cartilage. The stiffness of the immature and mature normal cartilage were 23.8 and 15.0 (x 10–3 N/m), respectively, with a significant difference between two groups. The stiffness of the TEC-repaired cartilage was significantly lower than those of the normal cartilage. Conclusions The present study suggested that the TEC did not enhance the healing of the superficial layer of cartilage-like tissues in both immature and mature porcine femoral cartilage. (Supported, in part, by NEDO (06001904-0) & MEXT (BERC)), Introduction The present study was performed to determine the static and dynamic compressive properties as well as permeability of a cartilage-like tissue repaired with a tissue engineered construct (TEC) (Ando. Biomat. 2007). Methods and Materials Synovium-derived cells from porcine knee joints were cultured though 4 to 7 passages. After an addition of ascorbic acid the matrices were allowed to undergo active contraction to develop TEC. The TEC was allografted to a round-shaped, cartilaginous defect in the porcine femur. Six months after surgery, the static and dynamic compression tests as well as permeability test were performed for a cylindrically shaped repaired tissue specimen extracted from the femur. Results Histological observation indicated that the defect was filled with TEC-repaired cartilage-like tissues. The modulus of the specimens was similar (820 kPa) to that of normal cartilage (620 kPa), and was significantly higher than that of TEC-untreated tissues. However, the accumulated strain was significantly increased (11%) as compared with normal cartilage (8%) at 40s. The permeability of the surface layer of the TEC-repaired tissue was significantly larger (19×10–15 m4/Ns) than that of normal cartilage. Conclusions It is suggested that the surface layer restricts the interstitial fluid outflow in normal cartilage, which contributes adequate compressive properties to the tissue. Although the TEC-repaired tissues exhibited better compressive properties than TEC-untreated tissues, the dynamic compressive property was slightly deteriorated. This may be attributable to a 5-fold higher permeability measured in the surface layer in the TEC-repaired tissues (Supported, in part, by NEDO (06001904-0) & MEXT(BERC))., Introduction The purpose of the present study is to evaluate the efficacy of synthetic resorbable scaffolds transplantation of the talar dome with MRI and clinical short-term follow-up. We therefore present surgical technique steps and early results at one year obtained with preformed in shape and size bone graft substitutes in repair of III and IV degree full thickness osteochondral defects of the talus. Methods and Materials The utilized implant is a cylinder composed of poly(D,L-lactide-co-glycolide) to which calcium sulphate and surfactant are added to enhance bone in-growth and make implant's surface more hydrophilic. The three-dimensional porous cylindrical implant with interconnected pores is press fit into the site for close apposition and encourage migration of repair tissue as blood and marrow into the scaffold. The two layer construct of the implantmimics the mechanics of the surrounding tissues, bone and cartilage, in order to facilitate from the beginning the nature of the repair tissue that will be formed. The plugs are available in different sizes (5,7,9 mm) are preformed in order to match the talus dome surfaces. The first 15 patients were included in the study (7 women, 8 men). Every patient has underwent arthroscopic ankle asse ssment to evaluate size, location and degree of defects and has underwent implantation of TrufitTM cylindrical resorbable scaffold. Majority of synthetic bone substitutes implanted were 7 mm in diameter. Results All surgical procedures have been completed uneventfully. Patient have been controlled clinically and by serial ankle MRI's and showed statistically significant improvement of AOFAS scores associated to healing of defect and integration of bone plugs in absence of adverse reactions. Conclusions Preliminary results enable us to conclude that porous, resorbable scaffolds can be used in treatment of cartilage defects offering a secure support to secondary bone in-growth with the advantage of being applied in one single step procedure, enabling patients to quickly move back to previous daily and sport activities, Introduction A cohort of young patients has recently been described with end-stage glenohumeral arthritis following shoulder stabilization surgery. This study investigates the demographics of patients with post-arthroscopic glenohumeral arthritis and elucidates the factors associated with the development of their condition. Methods and Materials Between 2003 and 2008, 20 patients (mean age 21.8 years) were referred for management of glenohumeral arthritis developing after stabilization surgery. Records were reviewed for initial injury, surgical treatment, and symptom development. Standardized shoulder surveys were provided at referral and surgical follow-up. Results Post-surgical pain pump usage was a contributing factor for degenerative glenohumeral changes in 16 patients, with the remainder related to failed anchor placement (2) and radiofrequency device usage (2). All patients had a recurrence of pain at 5.6 months post-operatively, which was accompanied by a decreased range of motion in 15 cases. Grade 4 chondral damage encompassed over 50% of the humeral head, while glenoid involvement was variable (0% to 100%). Of the 20 patients, 18 have undergone a post-referral surgery, including 13 biologic resurfacing procedures. Nine patients with glenohumeral resurfacing have completed a shoulder function survey at their most recent follow-up (mean 2.5 years). A statistically significant improvement was achieved on both the SST and ASES scales. Conclusions Severe glenohumeral arthritis in young adults is a devastating complication of stabilization surgery. Although not a universal finding, the use of postoperative glenohumeral pain pumps is concerning. The use of biologic resurfacing is promising for this cohort, but further long-term follow-up data is required to determine on the efficacy., Introduction This study tested the hypothesis that MAP kinase inhibitors enhance chondrogenesis and suppress hypertrophic changes during chondrogenesis from MSCs. The effect of PD98059,(an ERK1/2 inhibitor), and SB203580,(a p38 inhibitor), were tested on bone marrow-derived mesenchymal stem cells (BMMSCs) and adipose-tissue-derived mesenchymal stem cells (ATMSCs). Methods and Materials In vitro pellet cultures were carried out using 2.5 × 105 MSCs in chondrogenic medium containing 5 ng/ml of TGF-p2 for BMMSCs, and 5 ng/ml of TGF-p2 and 100 ng/ml of BMP-7 for ATMSCs. From the 14th day of culture, subsets of the pellets were additionally treated with PD98059 [0, 1mM, 10mM] or SB203580 [0, 1mM, 10mM]. After two more weeks of in-vitro culture, pellets were harvested for analysis. Results Treatment of PD98059 increased DNA contents and GAG amounts in both BMMSCs and ATMSCs. Real time PCR analysis showed COL1A1 mRNA decresed to almost a quarter in PD98059 treated BMMSCs, but did not chang in ATMSCs. The mRNA levels of SOX-9 and COL2A1 increased several fold in BMMSCs and ATMSCs after PD98059 treatment. The gene expression of Runx-2, and, to a lesser degree, COL10A1, decreased after PD98059 treatment in both BMMSCs and ATMSCs, whereas, SB203580 elevated their expressions in both cell types. Type I collagen expression was significantly declined by PD98059 in both BMMSCs and ATMSCs, but was not significantly changed by SB203580 in both cells type. Safranin-O and type II collagen expression were increased in both BMMSCs and ATMSCs by PD98059. Whereas SB203580 had moderate increase in Safranin-O and type II collagen expression in BMMSC and no observable effect in ATMSCs. On the other hand type X collagen and Runx-2 protein expressions were reduced by PD98059 in both BMMSCs and ATMSCs, but were slightly increased by SB203580 in both cells type. Conclusions Our study demonstrates the usefulness of the ERK 1/2 inhibitor, PD98059 for the promotion of chondrogenesis and the suppression of hypertrophic changes. This finding could be helpful for cartilage tissue engineering from MSCs.

Probiotic microorganisms have been shown to provide specific health benefits when consumed as food supplements or as food components. The main problem of such products is the poor survival of the probiotic bacteria in the low pH of gastric fluid. However the use of synthetic excipients for enteric coating to prevent the exposure of microorganisms to gastric fluid is limited in food supplementary industry. Therefore the aim of this study was to develop an enteric coating formulation containing shellac as a natural polymer. Shellac possesses good resistance to gastric juice; the major disadvantage of this polymer is its low solubility in the intestinal fluid [1, 2]. Thus films containing different ratios of shellac and water-soluble polymers (sodium alginate, hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidon (PVP)) or plasticizers (glycerol and glyceryl triacetate (GTA)) were prepared in order to analyse the films’ melting temperatures (Tm), the changes in enthalpy (ΔH), their capability of taking up water, and their solubility in different media. The release characteristics of the films were studied by loading pellets with Enterococcus faecium M74 and coating them with formulations containing different amounts of shellac and polymer or plasticized shellac. Using dissolution tests, performed according to USP XXXI paddle method, the resistance of the coatings to simulated gastric fluid (SGF, pH 1.2) and the release of cells in simulated intestinal fluid (SIF, pH 6.8) was investigated. The trials showed that an increasing amount of plasticizer results in a decrease of Tm and ΔH of the films whereat glycerol had a superior plasticization effect to GTA. The compatibility of films made of water-soluble polymers and shellac was also concentration dependent. HPMC and PVP showed superior compatibility with shellac compared to sodium alginate, since films containing shellac and more than 10% [w/w] sodium alginate tended to separate into two phases. In the end five formulations containing shellac and either 5% [w/w] glycerol, 10% [w/w] PVP, 20% [w/w] PVP, 10% [w/w] HPMC, or 5% [w/w] sodium alginate emerged as feasible for enteric coating purposes., It was the purpose of this study to develop an oral oligonucleotide delivery system based on thiolated polymer/ reduced glutathione (GSH) system providing protective effect towards nucleases, permeation enhancement and controlled drug release. Polycarbophil-cysteine conjugate (PCP-Cys) was synthesized by the covalent attachment of cysteine to polycarbophil via amide bond formation. Incubation of 30-mer phosphorothioate oligonucleotide with DNase I and freshly collected intestinal fluid were performed in order to evaluate the protective effect of the polymer. Based on PCP-Cys conjugate together with GSH, permeation studies were performed on Caco-2 monolayer cell culture and on freshly excised intestinal rat mucosa in Ussing chambers. Additional drug release studies of tablets containing PCP–Cys, reduced GSH, and the oligonucleotide were performed in 100 mM phosphate buffer pH 6.8. PCP-Cys displayed 223±13.8 μmol thiol groups per gram polymer. After 4 h the unprotected ODNs, which were incubated with DNase I and intestinal fluid, were significantly degraded by 61 % and 80 %, respectively. In contrast, less than 41 % (in DNase I) and 60 % (in intestinal fluid) of the ODNs were degraded in the presence of 0.02 % (m/v) of PCP-Cys. Permeation studies demonstrated due to the addition of PCP-Cys/GSH an 8-fold and 10-fold increase in the apparent permeability coefficient (Papp) on Caco-2 monolayer and on intestinal rat mucosa in comparison to the buffer only, respectively. Tablets containing PCP– Cys, GSH, and oligonucleotide showed a sustained drug release over 2 h. According to these results the PCP-Cys/GSH system might be a promising tool for the oral administration of oligonucleotide., Mupirocin-loaded microparticles (MP) were designed to control drug release at the skin surface assuring that drug remains localized at the application site and does not unnecessarily enter into the systemic circulation [1]. These reservoirs release active ingredient over an extended period of time maintaining effective drug concentration on the skin, at the same time reducing undesired side effects. The goal of this research was to design controlled release MP with acrylic polymer using spray-drying technique and assess influence of feed composition (in terms of native drug/polymer physical form and solvent used) and preselected drug loadings (1:5 and 2:1 (w/w) drug:polymer proportion) on MP performance under the same processing conditions. Physicochemical properties of MP were evaluated using thermal (MDSC, TGA), spectroscopic (FT-IR) and X-ray analyses and correlated with encapsulation efficacy and in vitro drug release achieved. Morphology and particle size were determined using low angle laser light scattering (LALLS) and scanning electron microscopy (SEM). Spray-drying of feed dispersion has formed partially coated crystalline MP with reduced encapsulation efficacy, irregular morphology and poor ability to control drug release irrespective of drug loading. Conversely, solid dispersions prepared from spray-drying feed solution have shown that drug/polymer miscibility, morphology and in vitro drug release were dependent on drug loading and solvent used [2]. The superior control of drug release from MP was achieved for the higher drug loading (2:1 (w/w) drug:polymer proportion) using solvents in the following order: methanol ≈ methanol+ethanol(50:50) > isopropyl alcohol+acetone (40:60). MP were amorphous, with smooth and spherical morphology. The higher polymer loading (1:5 (w/w) drug:polymer proportion) yielded less control over drug release regardless of solvents used, with MP exhibiting significantly different morphologies. Acrylic-based solid dispersions were confirmed as suitable microcarriers for controlled drug release using simple and scaleable spray-drying technique., Monoglycerides, Phytantriol and a few other lipophilic molecules self-assemble in bulk in presence of water to form well defined liquid crystalline phases. Their structure can be tuned by temperature variation and/or by addition of oils. This leads to gel-like or fluid systems with a large internal interface between water and oil domains with different bulk viscosities. These nanostructured phases can be dispersed in the excess water phase by addition of an external stabilizer and energy input leading to internally self-assembled particles, so-called ISAsomes [1–4]. These ISAsomes are potential carrier systems for hydrophilic, amphiphilic and lipophilic functional molecules. The hierarchical structure can be extended to a next level by gellifying the continuous aqueous phase by the addition of polymers like κ–Carrageenan or Methylcellulose. This leads to a new type of hydrogel, loaded with ISAsomes [5, 6]. Differently to the original oil-continuous bulk phase, the viscosity of this, now water-continuous, system can be varied in a wide range by composition. These gels can even be dried into foils and re-dispersed on demand. Finally, we can use the oil-continuous nanostructured bulk phase to create concentrated, stable water in oil emulsions having a paste-like consistency and water content from 50% up to 90% by volume. No additional stabilizer is needed to create these systems. They have a great potential as delivery systems for functional molecules in very different fields like pharmaceutical and cosmetic applications, as well as in food science and agro-chemistry., Background: Lactose intolerance is the inability to metabolize lactose because of the absence of the enzyme lactase. It is estimated that 75–90% of birth lactase levels are lost by most people after weaning. The prevalence of lactase deficiency ranges widely with the ethnic background from 2–15% among Northern Europeans to 95–100% among Asians [1]. Nowadays, lactose intolerance is usually controlled by strict adherence to a loactose-free or lactose-reduced diet. Moreover, as an alternative, capsules or tablets containing microbial-derived ß-galactosidase are available. However, this treatment is inconvenient for patients since these formulations have to be administered immediately before or together with lactose-containing diet because of their short-acting effect. Aim Therefore, the present work is aimed to develop an innovative long-acting peroral formulation for the treatment of lactose intolerance. Methods: Biodegradable and biocompatibale polymeric microcarriers (2.78±1.05μm in diameter) were manufactured from poly(D,L-lactide-coglycolide) (PLGA) using spray-drying. They were functionalized with β-galactosidase from Kluyveromyces lactis and targeted with wheat germ agglutinin (WGA), which might prolong the residence time of particles in the small intestine. The particle-bound enzyme activity, the mucoadhesive as well as the cytoadhesive properties were assessed. Results: The highest particle-bound enzyme activity (1470 U ß-galactosidase per gram PLGA) was obtained with hexamethylene diamine as a spacer using carbodiimide method representing a 6-fold increase as compared to particles without spacer. Surface immobilisation of WGA enhanced considerably the particle binding to porcine mucin layer (mucoadhesion) and Caco-2 cell monolayers (cytoadhesion). Conclusions: PLGA-microparticles, surface-modified with active ß-galactosidase as enzyme substitute and WGA as a targeter, are able to bind to enterocytes and thereby to prolong the intestinal residence time. It is a promising approach towards a promising approach towards a more convenient therapy of lactose deficiency and intolerance., Skin is main target for UV-oxidative stress and their antioxidant defenses can be quickly overcome. The consequences are reflected at the molecular and cellular level. Here, most important organelle included in survival pathway are mitochondria. Recently significant activation of mitochondria-mediated signaling cascades by resveratrol (RSV) has been discovered [1]. RSV is a naturally occurring polyphenolic phytoalexin, which has many beneficial biological effects but on the other hand possess some limitations. Few of these are: poor water solubility, high metabolic rate and frequent dosage–dependent effect in the cellular environment [2]. One of recent progresses that can satisfy all these obstacles of RSV is design of nanosized drug delivery system [3]. The protective effects of RSV in solution or loaded into SLN (SLN-RSV) on radiated keratinocytes were studied comparing with the effects on non-radiated cells. Cosequences of damaged effect of UV-radiation was slightly changed cell morphology, as some fragments of actin fibers appeared and mitochondria activity was weakened and they were dispersed over whole cytoplasm. However, only SLN with incorporated RSV at 100 μM preserved normal cell morphology and improved mitochondria activity, while the other samples (RSV at 10 or 100 μM and also SLN-RSV at 10 μM) showed any significant difference regarding radiated control cells. These effects are ascribed to the transfer of RSV by nanoparticles intracellularly – integrating among mitochondria, where RSV can best realize its “anti-stress” potential. To conclude, the results clearly revealed the hypothesis that loading of RSV into SLN significantly improve bioavailability and diminish UV-related damage of keratinocytes, and thus providing better photoprotection of cells compared to the application of RSV in solution., Cationic SLN formulations were developed and optimized in terms of cationic lipid/surfactant ratio and production parameters. 5wt% of Compritol 888 ATO (COM, Gattefosse) or Imwitor 900(IMW,BASF) were used, 0.4–1.0wt% of cetyltrimethylammonium bromide (CTAB, Sigma) and 0.1–1.0 wt% of Lutrol F68 (BASF) were tested. Formulations were produced in triplicate. SLN were prepared by modified microemulsion method [1], which was further optimized in terms of velocity and time of high shear homogenization step. Freeze-drying was performed on DuraDry MP at −45°C/184 mT during 6 days. Samples were freeze-dryed with 5.0% or 10.0% of Glucose (Sigma) or Sucrose (BDH Chemicals) or without cryoprotectants. Reconsitution was performed by rehydratation, vortexing during 3min and applying ultrasound during 30s. A Zetasizer NanoSeries (Malvern) was used to determine average hydrodynamic diameter (z-ave), polydispersity (PdI) and zeta potential (ZP) Optimized formulation consisted of 5.0% solid lipid, 0.5% CTAB and 0.25% Lutrol F68, yielding particles with z-ave=159nm, PdI=0.34 and ZP=54.8 mV (IMW) and z-ave=180nm, PdI=0.272 and ZP=56.2 mV (COM). These parameters remained unchanged during 8 days of storage at 8°C. Samples dried without cryoprotectants yielded light powdery product which could be redispersed easily; freeze-dried samples with saccharides tested yielded solid products. Cationic SLN formulations intended for gene delivery were designed and optimized. Particles with z-ave below 200nm and low PdI were produced. These SLN showed good stability of 15 days .Suitability of cationic SLN for freeze-drying without cryoprotectants was confirmed [2]., Pharmaceutical secondary manufacturing has long stood in stark contrast to the drug discovery end of the business, as well as to other sectors, when it comes to innovation such as continuous manufacturing. Is that about to change? The traditional business model is breaking down with consequent pressures on all parts of the pharma value chain. Manufacturing’s contribution to improving yield, reducing time, cost and waste is increasingly critical. Regulation which previously had insisted on batch testing is now moving to be much more supportive of real time product release and process analysis, heralding a future where the validation and establishment of continuous manufacturing will be easier. Integration of each process stage is crucial for continuous manufacturing. However, pharma companies tend to work with ‘islands of automation’ where every unit of operation is more or less an independent from an integration point of view. Another key barrier is that these changes require companies to work in a more multi-disciplinary way, crossing system worlds. For the first time, if you are in analytics you have to speak to people in process control and so on. Multi-disciplinary teams are an absolute must but not everyone is ready for that. The relatively slow uptake of process analytical technology (PAT), crucial for continuous manufacturing, following the FDA’s 2004 PAT initiative, highlights the challenge facing companies. Mindset changes and internal culture changes will be key to the successful introduction of PAT as a first step towards continuous manufacturing. The companies that are first to overcome the barriers will not just reap the reward of increased competitiveness. Because of the stage of its introduction, they have the opportunity to implement the technology to a higher level than in industries where it is already established. From being a laggard, secondary manufacturing has the potential to become a trendsetter., Granular flows are of paramount importance in industry and nature. To understand, e.g., the effect of various process parameters on the final product performance is critical. In industry, the flow in agitated mixing equipment basically consists of granular flow over a blade. While previous experimental and numerical work [1, 2] could reveal some features of this flow situation, information like single-blade mixing efficiency or the principal parameters that influence this flow are still scarce. We performed excessive high-speed video imaging of dry and wet granular flow under a controlled atmosphere in a 3D mixer as well as in a novel 2D flow setup. We use particle image velocimetry (PIV) to measure the velocity of the granular bed from the top (3D system) as well as in a cross section of the blade. Discrete-Element-Method (DEM) simulations are used to help interpreting our experimental results. The ultimate goal was to quantify the mixing in bladed mixers (e. g., high-shear granulators). We show results for the instantaneous, time-averaged as well as fluctuating velocity fields for the flow over a single blade. Our results indicate that the bed height in front of the blade has a significant effect on the flow pattern. Also, the flow situation changes when multiple blades are used and the blade-to-blade distance is changed. Finally, we quantify the mixing efficiency of a single-blade passage using image analysis., Porous particles are present in various technical applications as well as in the field of medical treatment. The latter involves amongst others the production of highly porous and respirable particles to serve as solid inhalants and substitute intestinal medication. One promising way of manufacturing such biotechnological therapeutic agents is the lyophilisation of single drops or sprays – a complex technological approach that still requires research and process layout. Besides experiments, numerical flow simulations are capable of providing insight into the physics that influence the transport and mass and heat transfer related to porous particles. Predicting the corresponding multiphase flows requires knowledge of the behaviour of porous particles in comparison to solid ones. By performing fully-resolved direct numerical simulations (DNS) of single complex particles correlations between particle properties and their fluid dynamic behaviour are to be established. In this work, the Lattice-Boltzmann method (LBM) is used to simulate the flow around virtual lyophilisates and to calculate drag, lift and torque coefficients [1]. A future aim is to perform simulations of the sublimation process of frozen spray droplets which are numerically realized in form of heterogeneous particles. In a first approach, the highly-porous lyophilisates are modelled by creation of simplified aggregates consisting of spherical primary particles (Fig. 1, left). This base structure is covered by a mutable hull-shaped matrix to represent the second phase of the frozen solution of the medical agent which is shrinking due to sublimation (Fig. 1)., Numerical methods such as Finite Element Analysis (FEA) or Computational Fluid Dynamics (CFD) are considered to be well established technologies in industrial design and engineering. While the methodology of numerical simulation is fully recognized in conventional industry (e.g. automotive, aviation, construction, machinery) even (especially) for critical application, pharma-industry and biotechnology have just started to carefully consider numerical simulation methods as valueable tool to create a sound understanding of their processes. The high level of regulation in this specific industry as well as the strong drive for evidence based validation (verfication) may have contributed to the fact that numerical simulation methods are hardly applied in pharma industry and biotech. The paper in hand shall demonstrate on typical applications how the CFD approach can be applied to design and optimize processes, to create additional knowledge about specific process interactions, to provide better understanding about correlations between process parameters and product quality, to supply a sound data base for process validation and thus to increase process flexibility. In four different references various aspects of the CFD methodology are discussed. The operational performance of a fermenter is presented with specific focus on gas distribution (oxygen) depending on mixer and vessel design and on shear stress exposure of the cell culture. Also, a stirred and heated storage vessel for thermal sensitive emulsions is investigated. Especially the temperature distribution within the products related to the temperature of the heating jacket and stirrer parameters shall be analysed. A third application considers a very specific freeze technology for protein-buffer-solutions. The freeze and thaw process is analysed by CFD under specific consideration of temperature induced concentration shifts in the protein-buffer. Finally a standard vessel-agitator set-up is discussed and strategies for process optimizations are presented based on CFD. Based on this specific problem it shall be demonstrated how CFD can support process-upscaling from lab to industrial size., This contribution describes the dynamic optimization of a pharmaceuticals batch dryer. The optimal control problem is based on the control vector discretization or single shooting method, thus a non-linear programming (NLP) problem is obtained. The target of the open loop optimization problem is the batch time minimization and the control variables are the On/Off intervals of the mixer and the mixer rotation speed. In order to implement a relevant scenario from practical point of view the following constraints are applied: the on and off intervals have same duration, the stirrer speed is constant during the entire batch drying process and the final moisture specification must be met. Finally, an upper limit on the fines fraction in the particle size distribution is set. The dryer model [1] consists of a system of partial differential equations coupled to a population balance model which describes the breakage process. The optimization problem presents several local minima and low gradient regions, thus a gradient based method fails to converge to the minimum. Therefore, a genetic algorithm-pattern search (simplex) method is used to ensure convergence of the optimization problem. The relevance of the work is highlighted by recent contributions which deal with the operation optimization of this unit operation [2–5]., In the pharmaceutical industry spray coating is a frequently used method to apply a film layer on the surface of tablets or pellets. The function of coating ranges from the controlled release of active pharmaceutical ingredients (APIs) to taste masking and coloring. A widely used technique is drum coating, where tablets are placed in a rotating drum and the coating liquid is sprayed onto the moving tablet bed surface. One of the most central quality attributes is the uniformity of coating, both inter-tablet and intra-tablet. Besides experimental work [1], numerical simulations have become an extremely important tool in process design and development. Coater performance depends on a range of different parameters, which could be essential for the quality of the final product. In order to quantify the impact of different CPPs (critical process parameters) on the related CQAs (critical quality attributes), the combination of advanced numerical methods on different scales is necessary. In this work we present an innovative approach to analyze, understand and optimize important steps of the pharmaceutical tablet coating process by means of numerical simulations. The final outcome is the application of the developed methods to industry-scale processes. Concerning our numerical multi-scale approach, the Discrete Element Method (DEM) is used for the simulation of the tablet bed mixing. Furthermore, a detailed numerical analysis of the interaction between spray droplets and tablet surface is performed with multiphase Computational Fluid Dynamics (CFD) methods [2]. On a larger scale, the CFD approach is also adapted to investigate the coater internal flow in terms of drying air and liquid spray efficiency. Summarizing, the presented combination of simulation methods helps to gain a deeper understanding of the spray coating process, providing a further step away from trial-and-error towards Quality-by-Design approach., The impact of liquid drops on wetted and dry surfaces is a complex two-phase flow phenomenon with high technical relevance especially in surface coating. It is also of increasing interest in the development of novel drug dosage forms in pharmaceutical engineering. The complex physics after the impact, which is still not understood in full detail, has been investigated in numerous experimental and analytical studies. Based on dimensional analysis and/or simplifying assumptions various semi-empirical correlations and analytical solutions have been derived to describe the individual stages of the motion of the liquid (see, e. g. [1, 2]). The present work numerically investigates the whole process of the liquid spreading after the impact using the CFD software FLUENT. Impact velocities and surface wettabilities were varied in the considered cases. It is shown that the Volume of Fluid based approach, which has become a standard numerical method for two-phase flow simulations, generally captures the typical flow features, like splashing (see Fig. 1), qualitatively and quantitatively very well. As such the computational results agree very well with the experimental data and analytical asymptotic solutions obtained for the individual regimes in literature. Notable discrepancies were however observed at the late stages of the liquid spreading on dry surfaces, where the outer rim of the liquid lamella eventually starts to recede. This deficiency points at the great numerical challenge to capture accurately the dynamics at the contact line between the liquid, gas, and the solid surface. The strong sensitivity of the incipience of liquid receding to the contact physics becomes very evident here., Magnetoliposomes (MLs) are phospholipid vesicles encapsulating magnetic nanoparticles utilised as contrast agents for targeted molecular Magnetic Resonance Imaging (MRI). There are different methods of preparation and characterization of liposomes containing ultra small paramagnetic iron oxide ions (USPIOs) i. e. lipid film hydration method [1] or reverse phase evaporation [2]. In this work as magnetic core we have used commercially available dextran coated iron particles (Molday-ION, purchased by Bio-Pal, USA), with a colloidal size of 30 nm. Liposomes were prepared by thin lipid film hydration method: a predetermined mixture of lipids made of phosphatidylcholine, with or without cholesterol and small amounts of PEG-ylated lipids (which sterically stabilize the liposome reducing their usual rapid uptake from the immune system) was dissolved in organic solvent and evaporated to dryness. Then the dry lipid film was hydrated with a buffered aqueous solution of different concentration of dextran magnetite. The resulting MLs, which were heterogeneous in their size, were centrifuged suddenly after the hydration step to remove excess dextran magnetite; some MLs were sonicated before centrifugation with an Ultra Sonicator (50 min, 130 Watt) to obtain a homogeneous particle size. Physical characterization was performed by dynamic light scattering (DLS) to determine the mean particles diameter and the size distribution. These data were compared to morphological images obtained by transmission electron microscopy (TEM). Chemically, the absolute amount of Fe in MLs was determined by a colometric test using potassium thiocyanate (KSCN) as reagent. MRI measurements were performed in vitro in agarose gel phantoms to evaluate the intensity of the contrast agent on T1 and T2 relaxation time. In vivo, the biodistribution and the cleareance of the particles over time were studied in mice. MLs have shown a high negative and positive enhancement in MRI and due to this strong contrast effect they will be modified for non invasive diagnosis of atherosclerotic plaques by targeted molecular imaging., Ultrasound active liposomes currently have gained a lot of interest as therapeutic agents for targeted drug delivery. A well known problem is their fast clearance from the circulation and thus the short time frame for drug release. The use of ultrasound as external trigger for drug release is attractive because it can be easily focused and only few adverse effects on the healthy tissue can be observed. To prolong the blood circulation kinetics the liposome surface can be modified with poly(ethylene glycol) (PEG) derivatives to prepare so called ‘Stealth’ liposomes[1]. Lin and Thomas described that the PEG content in the liposome membrane influences the drug release during ultrasound application [2]. A new ultrasound contrast agent composed of dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine and varying concentrations of polyethyleneglycol-40stearate was prepared using the thin-film hydration method. It was demonstrated that concentrations of 1 mol% and 10mol% polyethyleneglycol-40stearate show very good ultrasound activity. This result was unexpected due to the small size of only 100–200nm. The aim of this study was to determine the physico-chemical properties and the structural behavior of these contrast agents. Therefore 31P-NMR measurements, AFM investigations and TEM images were correlated. A combination of these methods should enable us to distinguish between organisations such as micelles, SLN and liposomes., Specially engineered drug particles can solve solubility and formulation problems, which are major challenges for the pharmaceutical industry. Particle size reduction of poorly water soluble drugs results in increased dissolution rate and higher bioavailability or better processability as demonstrated in previous studies [1, 2]. High-pressure homogenization can be used to decrease particle size, since it induces cavitation forces and causes highly localized increase in temperature and pressure within the fluid, resulting in decrease of particle size and inhibition of agglomeration. The current study is focused on properties inherent to particle engineering as well as methods for production and characterization of micro- and nanocrystals. The meloxicam, a low molecular weight analgetic for oral administration, exhibits a slow dissolution, therefore, it was formulated as micro- or nanosized particles by high-pressure homogenization. The samples were freeze-dried and characterized regarding particle size, morphology, structural analyses (DSC, XRPD) and in vitro dissolution rate. The final formulations were free of organic solvent and contained micro- (4–8 μm) or nano-sized (400–1000 nm) meloxicam crystals. Meloxicam in vitro dissolution rate was significantly improved and its particle size was found to be dependant on stabilizers used. To conclude, the new meloxicam formulations can potentially expand its application also to alternative administration routes (e. g. pulmonary and intranasal)., The availability and use of appropriate Process Analytical Technology (PAT) is fundamental to achieving the transformed style of pharmaceutical manufacture that is envisaged by the regulators. Implementing the knowledge-driven, risk-based approach to development and manufacture described in ICH Q8, Q9 and Q10 Guides, and adopting more efficient, modern operating practices, depends on being able to reliably identify, analyze and control those parameters that dictate pharmaceutical performance. This paper examines the potential contribution of laser diffraction particle sizing technology within this context, focusing on the use of industrially proven on-line solutions for real-time measurement and automated process control. Particle size has a controlling effect on the behaviour of many pharmaceutical products, often influencing critical to quality parameters such as dissolution rate, bioavailability and product stability. Where this is the case, the robust and proven technique of particle sizing by laser diffraction has for many become the method of choice. Non-destructive, with measurement rates sufficiently rapid for real-time monitoring, laser diffraction transitions easily, from the laboratory through to on-line measurement within the pilot plant, or in a manufacturing environment. With on-line analysis in place, the design, optimization and control of unit operations such as granulation, spray drying and, perhaps most importantly, milling becomes simpler and much more effective. Case studies illustrate this, the most compelling describing the use of on-line laser diffraction technology to fully automate control of a mill such that the particle size of the exiting active is maintained within specification despite variations in feed quality., The aim of Process Analytical Technology (PAT) is to gain deeper insight in pharmaceutical manufacturing processes, replacing empirical approaches by knowledge-based procedures. A detailed understanding of the key parameters of a process and their impact on the product quality allows companies to “build in quality” instead of “testing it into the final product”. From this point of view, PAT has to be tailored for a direct implementation to manufacturing lines to measure in real-time the key parameters of the process. Here, we present results on real-time quantitative in-line monitoring of powder mixing processes by near-infrared (NIR) spectroscopy [1, 2]. Spectral data collection and data analysis was performed in real-time based on partial least squares models developed via dynamic calibration. Different strategies are followed: I) Application of a single reflection probe to determine the mixing endpoint. II) A novel multiple reflectance probe measurement system for a spatially resolved monitoring of the powder flow kinetics within a blender. The parameters from the mixing process are linked to keyparameters of powders, obtained from rheometric investigations based on experimental design. Beside the batch processes monitored here with NIR spectroscopy, we also introduce hyper spectral imaging for monitoring fast continuous processes and non-invasive product quality analysis of solid dosage forms., Near-infrared spectroscopy (NIRS) is a rapid and nondestructive analytical method. The use of near-infrared spectroscopy in the pharmaceutical industry has been rapidly increasing over the past decade, with particular interest in the analysis of solid dosage forms, such as tablets and capsules [1]. This article describes use of NIR spectroscopy to determine of content the active pharmaceutical ingredient (metformin hydrochloride) in tablets, as well as strength tablets. Our goal is to create a necessary basis of spectral data and develop an optimal chemometric models that will later be transferred and used for monitoring the process. The calibration set was made up of 100 laboratory and 180 production samples in which the content of the active ingredient varied from 90 % to 110% of the declared value. The set of calibration samples, used for determination of the tablet hardness, included 70 laboratory and 70 production samples, which were compressed using four levels of compression force. The API content is determined by the validated HPLC method, while Erweka hardness tester was used to determine tablet hardness. NIR spectra analysis by using method of partial least squares [2]. The best result for drug content determination was obtained for the raw spectrum, where the coefficient of correlation for calibration model was 0.99945, RMSEC 0.552, and RMSEP 0.570, with seven factors used. The model which gave the best results for the tablet hardness determination was obtained by second-derivative spectra (coefficient of correlation: 0.99616, RMSEC: 0.506 and RMSEP: 0.841). According to the obtained results, it was concluded that both models ensured a high coefficient of correlation as well as low level of error of the calibration model and prediction. It can be concluded that NIR spectroscopy, a rapid and nondestructive technique, is a powerful tool that can be easily implemented, with appropriate technical solutions, in the production process, thus ensuring its on-line or in-line monitoring., Background: The use of nano-sized materials (NMs) offers exciting new options in technical and medical applications. Adverse effects on cells and organs, however, have also been reported. It is known that NMs may interfere with conventional assays. It may be suspected that inter-cell line differences in the sensitivity to NMs also exist. NMs may also interact physicochemically with plasma membranes. Cells in suspension, where a relatively large area is exposed, may therefore react different from adherent cells. As NMs by interaction with the replicative machinery may inhibit proliferation, also the proliferation rate (length of the doubling time) may be an important parameter. Methods: To assess the effect of the medium 20–1000 nm large carboxyl polystyrene particles (CPS) were tested in the presence of different amounts of fetal bovine serum. Potential interference was evaluated by testing in the cytotoxicity screening assays WST-1, MTT, MTS, Neutral Red, Sulforhodamin B, leucine incorporation and ATP content. IC50 concentrations were compared between 20 different cell lines. Results: The size of small (20–60 nm) CPS increased markedly from medium with 0% to 10% FBS; for larger CPS only minimal differences were seen. 20 nm CPS in medium with 0% FBS acted cytotoxic in all cell lines. 20 nm CPS in 5% and 10% FBS and larger CPS in all media were only minimally cytotoxic to nonphagocytic cells. In phagocytic cells also CPS of ≥ 500 nm acted cytotoxic. Half maximal inhibition concentrations (IC50) in a given cell line did not differ markedly between the screening assays but varied more than 10 times between the cell lines. IC50 values were significantly lower in suspension cells. Large cells and human cell lines with long doubling times were more resistant to the cytotoxic action of 20 nm CPS. Conclusions: In addition to medium composition, growth characteristics, proliferation and cell size of the cell line used for testing may influence the IC50 values of NMs. Cells in suspension are especially sensitive to the cytotoxic action of small carboxyl polystyrene nanoparticles., Praziquantel (PZQ) is a drug active against all species of Schistosoma and it is the drug of choice for the treatment of schistosomiasis [1]. Due to its low water solubility and risk of parasite resistance or tolerance to PZQ, it would be useful to develop a novel pharmaceutical product that could increase its therapeutic efficacy and improve the bioavailability. Solid lipid nanoparticles (SLN) combine the advantages of different colloidal carriers and also avoid some of their disadvantages in relation to the stability and possibility of large scale production. Thus, the aim of this work was to develop SLN containing PZQ (SLN-PZQ) and evaluate the cytotoxicity in the HepG2 cell line. The SLN were produced by a modified of the oil-in-water microemulsion method [2] using stearic acid as lipid core and poloxamer 188 as surfactant. The PZQ was incorporated in lipid core for the production of SLN-PZQ. Particle size was measured by dymamic light scattering (DLS) and the electrophoretic mobility was measured by laser Doppler anemometry. The cytotoxicity of PZQ (dissolved in ethanol) and SLN-PZQ was examinated in the HepG2 cell line using AlamarBlue assay [3]. The prepared SLN-PZQ had a mean particle size of 480.4nm with a zeta potential of −36.5mV. In HepG2 cell cultures, the tested SLN-PZQ suggested a decreased toxicity of the drug when delivered by SLN, in comparison to a conventional PZQ solution of similar concentration. The degree of toxicity was shown to be dose-dependent., Many current dermatological preparations, sunscreens and other cosmetics, contain nanosized particles (NPs), which were observed to be rapidly internalized by keratinocytes [1]. Besides their ability to enter into cells, NPs can be responsible for higher toxicity compared to bigger particles, mainly due to their larger surface area and enhanced chemical reactivity. Application of NPs increase the risk of cell damage and consequently cause alterations in mitochondria, actin filaments, cell membranes, etc., usually resulting in diminished cell functions or even cell death [2]. Despite the fact that ZnO and/or TiO2 NPs are frequently used, only few long-term toxicological studies are available. Our research was focused on long-term investigations, where the effects of repeated application of ZnO and TiO2 NPs on keratinocytes were evaluated. The preliminary short-term experiments showed no adverse effects of ZnO and TiO2 NPs on viability and morphology of keratinocytes in concentrations up to 15 μg/ml. Furthermore, TiO2 NPs did not significantly affect cell viability as well as cell growth in concentrations up to 100 μg/ml. Oppositely, ZnO NPs decreased cell viability in concentrations above 20 μg/ml sharply, and caused detachment of keratinocytes. Thus, the keratinocytes can survive short-term exposure to low concentrations of ZnO and TiO2 NPs, however, question regarding cell ability to maintain essential functions during their life span arises. Therefore, the concentration that did not show any cytotoxicity in short-term experiments was used for long-term investigations.The results after 3 months continuous treatment with 10 μg/ml NPs showed no significant changes in generation of reactive oxygen species (ROS) compared to untreated cells. Microscopical observation of cells continuously exposed to TiO2 indicated unchanged cell surface and presence of endosomes filled with TiO2 NPs, which were enlarged compared to endosomes of untreated control cells. Contrary, exposure to ZnO caused significant decrease in cell number as well as appearance of dense nuclei and disappearance of visible actin filaments, elevated ROS levels and decreased mitochondrial activity, indicating oxidative stress as a main cause for cytotoxicity of ZnO NPs. To sum up, the results indicate that the right selection of NPs is crucial for formulation of safe dermal products., Introduction: The oral cavity acts as a complex barrier and displays the first main hindrance against uncontrolled uptake of a variety of substances. The oral mucosa represents 60% of the total surface area within the oral cavity, offers a good opportunity for drugs to be absorbed and shows a 4 to 4.000 times greater permeability than the skin.The barrier function of this tissue is mainly guaranteed by i) the saliva, ii) the mucus layer, iii) the cell junctions in the epithelium and iv) the membrane coating granules. However, as the permeability of drugs through the oral mucosa is limited, new delivery carriers have to be developed. Nanostructured materials (NMs) are small enough to overcome this tissue. Within the development of such nano-carriers, the efficacy as well as the safety are important factors that cannot be neglected. Currently, no standardized physiological in vitro models, evaluating the permeability, transport route and toxic effects of NMs are available. Methods: NMs (polystyrene particles (PS), silver particles Ag)) were primarily characterized in terms of size and surface charge in physiological media. The penetration of the particles was investigated through excised buccal mucosa from pig. The in vitro permeability studies were carried out with Franz diffusion cells. It is a fact that the diffusion barriers in the buccal mucosa are only functional within living cell layers. Therefore, the viability (MTT) and the structural integrity of the membrane (methylene blue/PBS with and without EDTA) were investigated. The transport studies were carried out with Transwells® and the Trans-Epithelial Electric Resistance (TEER) was measured. The particle uptake into the cells was recorded with fluorescence/electron microscopy and the cell damage was evaluated. Results: The results demonstrate that the permeability of the particles depends on the size, surface charge, hydrophobicity and particle concentration. Particles in sizes of 20 nm (PS) and 35 nm (Ag) permeated the mucus layer and penetrated in the stratum superficiale of the top third region of the epithelium. They did not affect the tight junctions and were taken up by the cells. The cellular uptake correlated with the cell damage. Particles in sizes of 150 nm (Ag) and 200 nm (PS) aggregated in the saliva, were entrapped in the mucus layer and could not enter the top third region of the epithelium. Additionally, they also did not affect the tight junctions and no cellular uptake could be recorded. Conclusion: Independent from the material small (20–35 nm) NMs showed a much higher penetration than larger (150–200 nm) NMs., Studies on nasal epithelial models are important to develop vehicles for systemic nasal drug delivery, and also for targeting drugs to brain via the nasal route. RPMI 2650 human nasal septum carcinoma cell line was used in our experiments as an in vitro cell culture model for toxicity and permeability assays. For toxicity tests cells were cultured in 96-well plates, and MTT dye conversion and lactate dehydrogenase release were determined after treatments. For permeability tests RPMI 2650 cells were cultured on collagen-coated Millipore CM inserts (hydrophilic PTFE membranes, pore size: 0.4 μm, surface 4.2 cm2) in 6-well plates. Fluorescein was selected as a marker of paracellular permeability, and TEER and Papp were measured. RPMI 2650 cells passaged at high cell density grew as confluent multilayers on inserts. To induce barrier properties several treatments and culture conditions were tested. The effects of serum, hydrocortisone, cAMP and air-liquid interface on RPMI 2650 cell layers were examined. Hydrocortisone and cAMP increased the tightness of the nasal epithelial barrier, and induced the expression and junctional localization of claudin-1, claudin-4, ZO-1, and β-cathenin visualized by immunohistochemistry and confocal microscopy. The changes in cell and junctional morphology were confirmed by electron microscopy. The resistance of the monolayers reached 240 ± 13 Ωcm2, and Papp of 2.7 ± 0.2 10−6 for fluorescein indicating a barrier typical for nasal epithelium. The model was used to test the non-toxic nasal doses of absorption enhancers Tween 80, Cremophor RH40, Transcutol P, and water soluble sucrose esters and to determine their effects on paracellular permeability. Tween 80 and Cremophor RH40 decreased TEER by 50 % and significantly increased Papp values of RPMI 2650 layers. We have successfully established a human in vitro nasal epithelial model that can be used to study the effects of absorption enhancers and their mode of action., Introduction: Nanotechnologies and Nanomedicine are promising fields of scientific research. Experts expect a huge economic and social impact from Nanotechnologies and Nanomedicine in the next decades. New properties associated with size smaller than 100 nm opens a new world of applications. At the same time, possible toxicological aspects associated with nanotechnologies are being discussed in the scientific community. Nanotoxicity, possibly resulting from altered chemical and physical behaviour of nanoparticles compared to bulk material, is a major concern nowadays. These phenomena are believed to be related to the extreme high specific surface area and altered electrochemical properties of nanoparticles. The investigation of nanomaterials in terms of their toxicological behaviour is an extremely multidisciplinary challenge. Toxicology of non-nano chemicals is a well established procedure. Unfortunately, Nanotoxicology is quite more complicated and standard protocols in Toxicology can not be used. Nanomaterials have to be investigated in detail for different parameters like size, size distribution, shape, charge, etc. Furthermore, interaction with different matrices like blood, interstitial fluid, buffers, etc. influences significantly nanomaterials like, agglomeration, size distribution, charge, etc. In consequence, nanotoxicology needs new standardised protocols which are able to deal with the extreme complexity of nanomaterials and their toxicological behaviour! Focus: To build a national contact point for nanotoxicology, the BioNanoNet as a network company, has initiated the build up of the European Center of Nanotoxicology (www.euro-nanotox.at). This virtual center unites all Austrian Experts in the field of Nanotoxicology and helps to develop standardized methods. EURO-NanoTox initiates research projects together with its members and is links Austrian activities in the field of nanotoxicology to European activities. Within this contribution the European Center for Nanotoxicology will be presented. The general strategy of a stepwise approach to investigate nanomaterials in respect of their potential toxic properties will be shown. Furthermore, some details for the investigation of skin penetration of quantum dots will be addressed., Powder mixing is crucial for many processing stages within the pharmaceutical, catalysis, food, cement, and mineral industries, to name a few. A significant problem hindering process design is the paucity of information about the effects of changing process parameters on mixing efficiency, especially in the case of continuous mixing. The main target of this talk is to highlight continuous mixing and to examin the effects of different process and design parameters. Interestingly, continuous processing has been utilized extensively by petrochemical, food, and chemical manufacturing but has yet to reach the pharmaceutical industry to a meaningful extent. Recent research efforts indicate that a well-controlled continuous mixing process illustrates the capability of scale-up and ability to integrate on-line control ultimately enhancing productivity. In this talk we will decribe the use of engineering methodologies to design continuous pharmaceutical manufacturing systems for solid dose products. We will examine the performance of gravimetric feeders and continuous mixers, and their integrated dynamics, and develop guidelies for the optimal design and operation of the integrated system. Variance components will be characterized, and the monitoring of performance using PAT tools will be discussed., Introduction: Xanthan (XAN) is a well known negatively charged biopolymer that adopts different conformations in media, which are still poorly understood. XAN tablets in contact with water hydrate, forming a gel layer that regulates the drug release rate [1]. The hypothesis was that XAN molecular structure influences the drug release from matrix tablets. Thus, detailed studies on molecular as well as on macro-scale level were performed. Experimental: To determine XAN molecular conformations, its solution in water at pH 7.0 or 1.2 was deposited on freshly cleaved mica, dried and images were taken in air using tapping mode AFM. In order to determine Young’s modulus and investigate properties of single XAN molecule, nanoindentation as well as nanofishing were performed. Ingress of media into XAN tablet, gel structures and drug release were studied by NMR and MRI, oscillatory rheometer and dissolution USP Apparatus II. Results and discussion: Our results reveal that XAN adopts single chain conformation in water at pH 7.0, whereas at 1.2 double stranded structures are formed. This was confirmed by AFM images as well as by calculated parameters: persistence length of XAN in water was 210.37 compared to 116.83 in pH 1.2. Young modulus of XAN film in water was lower (1.52 GPa) compared to pH 1.2 (1.94 GPa). Higher rigidity of XAN films at pH 1.2 was further confirmed by formation of XAN gels, where elastic G’ and loss G” moduli were higher in the whole deformation range. Double stranded molecules form more cohesive gels, while single stranded are more flexible, due to the higher hydration of negatively charged polymer. MRI results proved much higher hydration and consequently the swelling of XAN tablets at pH 7.0 than at 1.2 and the thicker gel layer as well. Surprisingly, drug release in pH 1.2 media was faster regardless to firmer gel structure. Conclusion: High rigidity and low hydration of XAN matrix in pH 1.2 are the consequence of XAN molecular structure regulating drug diffusion in tablets., Dry powder coating is a relatively new technique to coat substrates without the use of any organic solvent or water. The film forming polymer is applied as a powder to the cores to be coated leading to reduced process times [1]. Additionally, excipients like plasticizers, preferably in liquid form, are often needed to soften the polymer in order to get a functional film. A curing step is sometimes necessary to achieve film formation depending on the characteristics of the polymer used. One critical parameter in dry powder coating is the coating efficiency which describes how much of the applied coating material actually adheres to the cores. Therefore, another main task of the excipients used is to enhance the adhesion of polymer powder. The aim of this work was to evaluate excipients, like plasticizers and polymers, regarding their suitability for dry powder coating. Dry powder coating was performed in a rotary fluid bed (GPCG1.1, Glatt, Binzen, Germany). Polymer powder and liquid were fed simultaneously via a three-way nozzle to the fluid bed. Coating efficiency was calculated by dividing the mass of adherent coating material by the mass of applied coating material. Theophylline pellets were used as cores and ethylcellulose, hydroxypropylmethylcellulose acetate succinate (HPMCAS) or Eudragit RS® as coating polymer. Various liquids were characterized with respect to several properties, like viscosity, spreading behavior and droplet size. Coated pellets were investigated via dissolution testing and scanning electron microscopy before and after curing steps to evaluate whether film formation was achieved. The spreading behavior of the liquids seems to be one key factor affecting the coating efficiency. It can be investigated by measuring contact angles of liquids on the polymer or predicted by calculating surface energies. However, there are still other parameters impacting the coating efficiency of the process like viscosity of the liquid, which influences on the one hand the kinetic of spreading as well as the droplet size generated by the spraying nozzle. The film forming properties of the polymers used were very different. Using ethylcellulose a curing step was required to obtain dense films whereas Eudragit RS® formed films without any curing., Coated pellets offer several advantages related to safety and effectiveness of the medicinal product such as reproducibility of gastric emptying and of absorption, and predictable plasma levels with lower probability of dose dumping due to modified release [1,2]. Layered pelletizing involves a process in which drug in solution, dispersion or powdered form is loaded onto inert starting cores. Starter inert cores have several benefits, such as they serve as nuclei with standardized shape, therefore the product shows more exactly defined surface for functional coating [3]. The objective of this study was to investigate the effects of formulation and manufacturing parameters on the pellet quality after the layering and coating process applying 3 types of inert core materials (sugar, microcrystalline cellulose, and isomalt). An additional objective was to investigate and compare the effect of the core material on the in vitro drug release of water soluble and poorly water-soluble drugs when they were coated with a permeable polymer membrane. The drug layering and coating process were followed by non-destructive tests such as image analysis and NIR spectroscopy. Flowability, hardness, friability, wettability and morphology of starting cores as well as of finished products were investigated. According to the non-destructive analysis and physical tests, all pellets manufactured from different core materials demonstrated satisfactory quality attributes. Since the type of inert core material may significantly influence the dissolution profile, the solubility characteristics of both the drug and core material should be considered., Hot Melt Extrusion, proven state-of-the-art technology in the field of plastics, is becoming increasingly important in the pharmaceutical industry. In the Hot Melt Extrusion process, the polymer is processed above the glass transition temperature to mix the active component with the thermoplastic binder and / or the polymer on a molecular level. The most significant benefits of HME processing are the absence of water and solvents, which leads to fewer processing steps and lower manufacturing costs. In addition the short residence time is beneficial for many heat- and shear sensitive drugs and the free volume in the twin screw gives the processor the opportunity to devolatilise the compound in a controlled and reproducible environment. Because of their excellent mixing behaviour and the degassing possibilities, co-rotating twin-screws are particularly suitable for Hot Melt Extrusion. The modular concept of the Twin Screw Extruder enables the individual adaptation of the processing section to the different requirements. Individual steps such as dispersive mixing, degassing and pressure build-up take place very effectively in this way. The closely intermeshing screws with their tight, self-wiping profiles eliminate dead zones over the whole length of the process section. The effect: consistently higher degree of process reliability and optimal self-cleaning. Coperion has references in the pharmaceutical field for twin screw extruders in sizes from 18 to 70 mm screw diameter. Typical applications for Coperion’s twin screw extruders ZSK: – Hot Melt Extrusion– Production of Transdermale Adhesives– Extrusion of Micro-Pellets– Production of Drugs with Delayed Ingredient Release– Low-Temperature Melt Extrusion– Potent-Extrusion– Co-Extrusion, The control of particulate processes and their understanding can be improved by modern measuring techniques. The paper describes the optical probe system Parsum IPP 70 as an example of the modern measuring techniques which is based on the spatial filtering technique (SFT). Measuring principles are the fibre-optical spatial filtering velocimetry and the fibre-optical spot scanning in order to determine simultaneously the size and the velocity of particles. Therefore the measured particle size distributions are chord length distributions which can be recalculated. A calibration is not necessary. The Parsum IPP 70 is a compact and highly robust measuring system for application as an in-line particle size analyzer for processes involving larger particulate sizes up to 6000 μm. It allows a data rate up to 20,000 particles per second and the system can track continuously a large variety of process parameters. Different options of the measuring system are available: tube length up to 4 m, air supply system for dispersion and cleaning, comprehensive software support, Ex-Zones and pharma solutions. The application is given for grinding/dosing, agglomeration, fluidized bed processes, mixing and coating, sieving, wet and dry granulation, spray drying. The Parsum IPP 70 is a powerful tool for process control of fluid bed granulation in pharmaceutical industry. The advantages are: increase of process transparency, short response time in the event of process disturbances, continuous control of product quality, full feedback control for automated solutions, no need of samples and laboratory analysis. Results are given by Petrak et al. [1]. Examples show also the ability to prove the model goodness of a fluid bed process by using IPP 70 in-line-SFT [2–4]., To establish a micro fluidic system that covers the whole production process of drug-loaded solid lipid nanoparticles especially high pressure emulsification and dispersion processes must be taken into account. The advantages of micro fluidic systems in a continuous production imply a high surface-to-volume ratio (material and energy transfer) and a narrow residence time distribution (homogeneously defined stresses). Detailed investigation is needed for process understanding and to improve micro channel geometries. The micro fluidic geometries were designed to selectively expose product flows to different main break-up mechanisms (shear stress, elongational stress, turbulent stress, particle-particle and particle-wall impacts). Within these groups structural modifications were carried out to evaluate their influence on emulsification and dispersion efficiency. Two formulations (to simulate the dispersion and emulsification process, respectively) were exposed to the specified micro channel geometries (including channel height variation) at different pressures, optionally using multiple homogenisation cycles in addition. The emulsification process in general shows a decrease in particle size by elevated pressure and higher number of cycles, as expected. Regarding the pressure drop, straight or bent channel geometries show a much lower breakup efficiency compared to either orifice-like or Y-shaped geometries. The latter two types over all show comparable efficiencies. Applying minor structural modifications to several geometries results in significant differences in particle size. As an example, a cascading quadruple orifice geometry shows significantly smaller particle sizes when the flow first interacts with the smallest orifice in comparison to passing the smallest orifice at the end. The dispersion of solid particles (hydrophilic alumina Alu C) shows similar results although the drop in particle size is less pronounced in these experiments. This is due to the high binding forces between primary particles., A pharmacokinetic (PK) model describes the absorption, distribution, metabolism and excretion of a pharmaceutical, thus providing, once properly validated, quantitative predictions of the consequences of various dosing regimens. Pharmacological activity might be linked to properties of a time course for a particular compartment (such as half-life or area-under-curve, AUC), or directly modelled by coupling the PK model with a pharmacodynamic (PD) model of the physiological effect of the agent. Due to the high replication and mutation rates of the human immunodeficiency virus (HIV), which ultimately results in strains resistant to any single agent, a multiple-agent strategy has to be adopted. Highly active anti-retroviral therapy (HAART) uses two or more agents that target different components of the HIV replication cycle, but these agents typically produce adverse side effects in patients. A number of authors have applied techniques from nonlinear control to this problem, but in general the explicit link between the drug dose and the physiological effect, via the PK, is neglected so that the controls derived are continuous in time. In [1] the control problem is addressed via feedback linearization applied to a PD model of HIV dynamics. A single-compartment PK model is then used to estimate a dosing regime that gives rise to the required continuous control law. In [2] a fixed-point approach was taken to determine the doses required to obtain a particular AUC or target profile. Although a constructive method is shown for determining the required dosing scheme, full solution trajectories and their integrals are required. An iterative method for constructing the dosing regime is proposed here that provides the fixed-point solution from [2] but is computationally easier to implement, being based on the underlying system equations. After each iteration the current dosing scheme is updated based on a linear operator applied to the difference between achieved and desired target vectors. The iterative method is applied to the coupled PK-PD model from [1] in order to directly construct a dosing regime, rather than indirectly via a continuous efficacy profile., The pharmaceutical industry uses million of animals for in-vivo studies to discover or to develop novel pharmaceutics. Time and costs involved and the extrapolation to human physiology of the data obtained are the main difficulties of these studies. The alternative to laboratory animals is the development of invitro and in-silico models (which are physical and mathematical models respectively). In literature, different in-vitro models are proposed to reproduce the gastrointestinal tract taking in account both the mixing and the biochemical features. However, these models are not able to reproduce closely the real physiology. To describe the drug release in the human body, different kinds of mathematical models (in-silico models) are proposed. Among these, physiologically-based pharmacokinetic models are more complex and complete: the body is divided into compartiments, corresponding to real structures of the body, each with a specific function. Despite their reliability, these models require the fitting of a lot of parameters. The aims of this work are to realize an in-vitro model reproducing the mechanical and biochemical features of the gastrointestinal tract and to develope (and validate) a novel physiologically-based model incorporating the pharmacokinetic and physiological parameters. The proposed in-silico model is simple and is characterized by a limited number of parameters. Haematic drug levels after different kind of administrations have been successfully simulated.