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During intervertebral disc (IVD) maturation, notochordal cells (NCs) are replaced by chondrocyte-like cells (CLCs) in the nucleus pulposus, suggesting that NCs play a role in maintaining tissue health. Affirmatively, NC-conditioned medium (NCCM) exerts regenerative effects on CLC proliferation and extracellular matrix (ECM) production. The aim of this study was to identify NC-secreted substances that stimulate IVD regeneration. By mass spectrometry of porcine, canine and human NCCM, 149, 170 and 217 proteins were identified, respectively, with 66 proteins in common. Mainly ECM-related proteins were identified, but also organelle-derived and membrane-bound vesicle proteins. To determine whether the effect of NCCM was mediated by soluble and/or pelletable factors, porcine and canine NCCM were separated into a soluble (NCCM-S; peptides and proteins) and pelletable (NCCM-P; protein aggregates and extracellular vesicles) fraction by ultracentrifugation, and tested on bovine and canine CLCs in vitro, respectively. In each model, NCCM-S exerted a more pronounced anabolic effect than NCCM-P. However, glycosaminoglycan (GAG) uptake from the medium into the carrier gel prevented more definite conclusions. While the effect of porcine NCCM-P on bovine CLCs was negligible, canine NCCM-P appeared to enhance GAG and collagen type II deposition by canine CLCs. In conclusion, porcine and canine NCCM exerted their anabolic effects mainly through soluble factors, but also the pelletable NCCM factors showed moderate regenerative potential. Although the regenerative potential of NCCM-P should not be overlooked, future studies should focus on unraveling the protein-based regenerative mechanism from NCCM produced from isolated NCs, e.g. by NCCM fractionation and pathway blocking studies.

Background: Mechanical and inflammatory processes add to osteoarthritis (OA). To what extent both processes contribute during the onset of OA after a cartilage trauma is unknown. This study evaluates whether local cartilage damage leads to focally confined or more generalized cartilage damage with synovial inflammation in the early development of joint tissue degeneration., Methods: In nine goats, cartilage damage was surgically induced on the weight bearing area of exclusively the medial femoral condyle of the right knee joint. The other tibio-femoral compartments, lateral femoral condyle and lateral medial tibial plateau, were left untouched. The contralateral left knee joint of each animal served as an intra-animal control. Twenty weeks post-surgery changes in cartilage matrix integrity in each of the four compartments, medial and lateral synovial tissue inflammation, and synovial fluid IL-1β and TNFα were evaluated., Results: In the experimental medial femoral plateau, significant macroscopic, histologic, and biochemical cartilage damage was observed versus the contralateral control compartments. Also the articulating cartilage of the experimental medial tibial plateau was significantly more damaged. Whereas, no differences were seen between the lateral compartments of experimental and contralateral control joints. Synovial tissue inflammation was mild and only macroscopically (not histologically) significantly increased in the experimental medial compartments. Synovial fluid IL-1β level was not different between experimental and contralateral control joints, and TNFα was overall beneath the detection limit., Conclusions: Local cartilage damage is a trigger for development of OA, which in early onset seems primarily mechanically driven. Early treatment of traumatic cartilage damage should take this mechanical component into consideration., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Intervertebral disc (IVD) degeneration is associated with most cases of cervical and lumbar spine pathologies, amongst which chronic low back pain has become the number one cause of loss of quality-adjusted life years. In search of alternatives to the current less than optimal and usually highly invasive treatments, regenerative strategies are being devised, none of which has reached clinical practice as yet. Strategies include the use of stem cells, gene therapy, growth factors and biomaterial carriers. Biomaterial carriers are an important component in musculoskeletal regenerative medicine techniques. Several biomaterials, both from natural and synthetic origin, have been used for regeneration of the IVD in vitro and in vivo. Aspects such as ease of use, mechanical properties, regenerative capacity, and their applicability as carriers for regenerative and anti-degenerative factors determine their suitability for IVD regeneration. The current review provides an overview of the biomaterials used with respect to these properties, including their drawbacks. In addition, as biomaterial application until now appears to have been based on a mix of mere availability and intuition, a more rational design is proposed for future use of biomaterials for IVD regeneration. Ideally, high-throughput screening is used to identify optimally effective materials, or alternatively medium content comparative studies should be carried out to determine an appropriate reference material for future studies on novel materials.

Objective: Hsa-miR-148a expression is decreased in Osteoarthritis (OA) cartilage, but its functional role in cartilage has never been studied. Therefore, our aim was to investigate the effects of overexpressing hsa-miR-148a on cartilage metabolism of OA chondrocytes., Design: OA chondrocytes were transfected with a miRNA precursor for hsa-miR-148a or a miRNA precursor negative control. After 3, 7, 14 and 21 days, real-time PCR was performed to examine gene expression levels of aggrecan (ACAN), type I, II, and X collagen (COL1A1, COL2A1, COl10A1), matrix metallopeptidase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and the serpin peptidase inhibitor, clade H (heat shock protein 47), member 1 (SERPINH1). After 3 weeks, DNA content and proteoglycan and collagen content and release were determined. Type II collagen was analyzed at the protein level by Western blot., Results: Overexpression of hsa-miR-148a had no effect on ACAN, COL1A1 and SERPINH1 gene expression, but increased COL2A1 and decreased COL10A1, MMP13 and ADAMTS5 gene expression. Luciferase reporter assay confirmed direct interaction of miR-148a and COL10A1, MMP13 and ADAMTS5. The matrix deposited by the miR-148a overexpressing cells contained more proteoglycans and collagen, in particular type II collagen. Proteoglycan and collagen release into the culture medium was inhibited, but total collagen production was increased., Conclusion: Overexpression of hsa-miR-148a inhibits hypertrophic differentiation and increases the production and deposition of type II collagen by OA chondrocytes, which is accompanied by an increased retention of proteoglycans. Hsa-miR-148a might be a potential disease-modifying compound in OA, as it promotes hyaline cartilage production., (Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Unlabelled: Histology is an important outcome variable in basic science and pre-clinical studies regarding intervertebral disc degeneration (IVD). Nevertheless, an adequately validated histological classification for IVD degeneration is still lacking and the existing classifications are difficult to use for inexperienced observers., Objective: Therefore the aim of this study was to develop and to validate a new histological classification for IVD degeneration. Moreover, the new classification was compared to the frequently used non-validated classification., Methods: The new classification was applied to human IVD sections. The sections were scored twice by two independent inexperienced observers, twice by two experienced IVD researchers and once by a pathologist. For comparison, the sections were also scored according to the classification described by Boos et al. by two experienced IVD researchers. Macroscopic grading according Thompson et al., glycosaminoglycan (GAG) content and age were used for validation., Results: The new classification had an excellent intra- and a good inter-observer reliability. Intraclass Correlation Coefficients (ICC) were 0.83 and 0.74, respectively. Intra- and inter-observer reliability were comparable for experienced and inexperienced observers. Statistically significant correlations were found between the new classification, macroscopic score, GAG content in the nucleus pulposus (NP) and age; Correlation coefficient (CC) 0.79, -0.62 and 0.68, respectively. The CCs of the Boos classification were all lower compared to the new classification., Conclusion: the new histological classification for IVD degeneration is a valid instrument for evaluating IVD degeneration in human IVD sections and is suitable for inexperienced and experienced researchers., (Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Purpose: To identify the mediator profile in healthy, pre-osteoarthritis (OA) and end-stage OA radiocarpal joints. We hypothesized that there would be an increase in soluble mediators in posttraumatic wrist OA., Methods: We obtained radiocarpal synovial fluid samples from 3 groups of patients: healthy control (n = 12) samples were collected during wrist ganglion resection; pre-osteoarthritic (n = 16) samples, during a 3-ligament tenodesis procedure for complete scapholunate dissociation; and end-stage OA (n = 20) samples in patients with proven radiological OA changes. Using a multiplex enzyme-linked immunosorbent assay, we measured 12 mediators: interleukin (IL)-1β, tumor necrosis factor-α, oncostatin-M, interferon-γ, IL-4, IL-6, IL-7, IL-8, IL-10, IL-13, IL-1RA, and osteoprotegerin. Statistical analysis was performed using analysis of variance and Bonferroni-corrected post hoc tests., Results: Mediators IL-6, IL-10, and interferon-γ were increased in OA wrists compared to healthy and pre-OA samples. Tumor necrosis factor-α, oncostatin-M, osteoprotegerin, IL-8, and IL-1RA were detected but not at increased levels in OA wrists. We found no differences between healthy and pre-OA joints in all 12 mediators. Mediators IL-4, IL-7, IL-13, and IL-1β were not detected in either healthy, pre-OA or end-stage OA samples., Conclusions: We identified no differences between healthy and pre-OA samples, suggesting no alteration in inflammatory status at the time of the 3-ligament tenodesis procedure. Consequently, mechanical disturbance seems to be the driving force toward OA and OA-associated inflammation in this stage of scapholunate dissociation. Increased levels of interferon-γ, IL-6, and IL-10 confirm inflammatory changes in the mechanically disturbed posttraumatic radiocarpal joint., (Copyright © 2013 American Society for Surgery of the Hand. Published by Elsevier Inc. All rights reserved.)

Objective: Soluble mediators in synovial fluid (SF) are acknowledged as key players in the pathophysiology of osteoarthritis (OA). However, a wide-spectrum screening of such mediators in SF is currently lacking. In this study, the levels of 47 mediators in the SF of control donors and osteoarthritic (OA) patients were compared., Materials & Methods: SF was collected from control donors (n = 16) and end-stage knee OA patients (n = 18) and analysed for 47 cytokines, chemokines and growth factors using several multiplex enzyme-linked immunosorbent assays (ELISAs). A Mann-Whitney U test was used to determine differences between OA and control controls. A principal component analysis (PCA) was performed to cluster the 47 mediators., Results: The majority of the mediators could be detected in both control and OA SF. Interleukin (IL)-6, interferon inducible protein (IP)-10, macrophage derived chemokine (MDC), platelet derived growth factor (PDGF)-AA and regulated on activation normal T cell expressed and secreted (RANTES) levels were found to be higher in OA compared to control SF (P < 0.001). Leptin, IL-13, macrophage inflammatory protein (MIP)-1β, soluble CD40 (sCD40L) levels were higher and eotaxin and granulocyte colony-stimulating factor (G-CSF) levels were lower in OA SF than in control SF, albeit borderline significant (P < 0.05). The PCA enabled identification of six clusters of mediators, which explained 76% of the variance., Conclusions: The current study provides the first extensive profile of cytokines, chemokines and growth factors present in control and OA SF. Increased levels of mediators such as MDC and IL-6 imply involvement of inflammatory processes and might be associated with the influx of inflammatory cells in OA synovial tissue. Moreover, the performed cluster analysis indicated multiple clusters, which could indicate different pathophysiological pathways in the joint., (Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: The combination of chondrocytes and mononuclear fraction (MNF) cells might solve the expansion induced dedifferentiation problem of reimplanted cells in autologous chondrocytes implantation as sufficient cells would be available for direct, one-stage, implantation. Earlier in vitro work already showed a positive stimulation of cartilage specific matrix production when chondrocytes and MNF cells were combined. Therefore, this study aimed to evaluate cartilage regeneration using a one-stage procedure combining MNF cells and primary chondrocytes for the treatment of focal cartilage lesions in goats compared to microfracture treatment., Design: Freshly created focal cartilage defects were treated with either a combination of chondrocytes and MNF cells embedded in fibrin glue or microfracture treatment. After 6 months follow-up local regeneration as well as the general joint cartilage health were evaluated using validated scores and biochemical assays., Results: Macroscopic (P = 0.015) scores for the cartilage surface at the treated defect were, after 6 months, significantly higher for the chondrocyteMNF treatment compared to microfracture-treated defects, but microscopic scores were not (P = 0.067). The articulating cartilage showed more (P = 0.005) degeneration following microfracture treatment compared to chondrocyteMNF treatment. Biochemical glycosaminoglycans (GAG) evaluation did not reveal differences between the treatments. Both treatments had resulted in a slight to moderate cartilage degeneration at other locations in the joint., Conclusion: In conclusion, treatment of focal articular cartilage lesions in goats using a combination of MNF cells from bone marrow and unexpanded chondrocytes leads to better macroscopic regeneration compared to microfracture, however needs further fine-tuning to decrease the negative influence on other joint compartments., (Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: Delayed gadolinium enhanced magnetic resonance imaging (MRI) of cartilage (dGEMRIC) facilitates non-invasive evaluation of the glycosaminoglycan content in articular cartilage. The primary aim of this study was to show that the dGEMRIC technique is able to monitor cartilage repair following regenerative cartilage treatment., Design: Thirty-one patients with a focal cartilage lesion underwent a dGEMRIC scan prior to cartilage repair surgery and at 3 and 12 months follow-up. At similar time points clinical improvement was monitored using the Knee injury and Osteoarthritis Outcome Score (KOOS) and Lysholm questionnaires. Per MRI scan several regions-of-interest (ROIs) were defined for different locations in the joint. The dGEMRIC index (T1gd) was calculated for each ROI. Repeated-measures analysis of variance (RMANOVA) analysis was used to evaluate improvement in clinical scores and MRI T1gd over time. Also regression analysis was performed to show the influence of local repair on cartilage quality at distant locations in the knee., Results: Clinical scores and the dGEMRIC T1gd per ROI showed a statistically significant improvement (P < 0.01), from baseline, at 12 months follow-up. Also, improvement from baseline in T1gd of the ROI defining the treated cartilage defect showed a direct relationship (P < 0.007) to the improvement of the T1gd of ROI at other locations in the joint., Conclusions: The dGEMRIC MRI protocol is a useful method to evaluate cartilage repair. In addition, local cartilage repair influenced the cartilage quality at other location in the joint. These findings validate the use of dGEMRIC for non-invasive evaluation of the effects of cartilage regeneration., (Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Background: The intervertebral disc (IVD) is dependent on nutrient provision through a cartilage layer with underlying subchondral bone, analogous to joint cartilage. In the joint, subchondral bone remodeling has been associated with osteoarthritis (OA) progression due to compromised nutrient and gas diffusion and reduced structural support of the overlaying cartilage. However, subchondral bone changes in IVD degeneration have never been quantified before., Objective: The aim of this study is to determine the subchondral bone changes at different stages of IVD degeneration by micro-CT., Methods: Twenty-seven IVDs including the adjacent vertebral endplates were obtained at autopsy. Midsagittal slices, graded according the Thompson score, were scanned. Per scan 12 standardized cylindrical volumes of interest (VOI) were selected. Six VOIs contained the bony endplate and trabeculae (endplate VOIs) and six accompanying VOIs only contained trabecular bone (vertebral VOIs). Bone volume as percentage of the total volume (BV/TV) of the VOI, trabecular thickness (TrTh) and connectivity density (CD) were determined., Results: An increase in BV/TV and TrTh was found in endplate VOIs of IVDs with higher Thompson score whereas these values remained stable or decreased in the vertebral VOIs., Conclusion: The increase in bone volume combined with the increase in TrTh in endplate VOIs strongly suggest that the subchondral endplate condenses to a more dense structure in degenerated IVDs. This may negatively influence the diffusion and nutrition of the IVD. The endplate differences between intact and mild degenerative IVDs (grade II) indicate an early association of subchondral endplate changes with IVD degeneration., (Copyright © 2010 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Background: In degenerative intervertebral discs (IVDs) collagen type X expression and calcifications have been demonstrated, resembling advanced osteoarthritis (OA), which is associated with hypertrophic differentiation, characterized by the production of collagen type X, Runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), alkaline phosphatase (ALP) and calcifications., Objective: The aim of this study was to determine if hypertrophic differentiation occurs during IVD degeneration., Methods: IVDs from all Thompson degeneration grades were prepared for histology, extraction of nucleus pulposus (NP) and annulus fibrosis (AF) tissue (N=50) and micro-CT (N=27). The presence of collagen type X, OPG and Runx2 was determined by immunohistochemistry, with OPG levels also determined by Enzyme-linked immunosorbent assay (ELISA). The presence of calcification was determined by micro-CT, von Kossa and Alizarin Red staining., Results: Immunohistochemical staining for collagen type X, OPG, Runx2 appeared more intense in the NP of degenerative compared to healthy IVD samples. OPG levels correlated significantly with degeneration grade (NP: P<0.000; AF: P=0.002) and the number of microscopic calcifications (NP: P=0.002; AF: P=0.008). The extent of calcifications on micro-CT also correlated with degeneration grade (NP: P<0.001, AF: P=0.001) as did von Kossa staining (NP: P=0.015, AF: P=0.016). ALP staining was only incidentally seen in the transition zone of grades IV and V degenerated IVDs., Conclusion: This study for the first time demonstrates that hypertrophic differentiation occurs during IVD degeneration, as shown by an increase in OPG levels, the presence of ALP activity, increased immunopositivity of Runx2 and collagen type X., (Copyright © 2010 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

The progression of cartilage matrix damage to generalized degeneration is associated with specific pathophysiological and clinical aspects. Reliable detection of stage-related characteristics of cartilage disease serves both a therapeutic and prognostic goal. Over the past years, several (pre)clinical diagnostic modalities for cartilage pathologies have been advocated. Each modality focuses on different aspects of the disease. Early diagnosis, before irreversible damage has occurred, opens up the possibility for better treatment and improves the patients' prognosis. This article gives an overview of the diagnostic modalities available for monitoring cartilage pathology and focuses on reliability, clinical value, current status, and possible applications.

Objective: Regardless of recent progress in the elucidation of intervertebral disc (IVD) degeneration, the basic molecular characteristics that define a healthy human IVD are largely unknown. Although work in different animal species revealed distinct molecules that might be used as characteristic markers for IVD or specifically nucleus pulposus (NP) cells, the validity of these markers for characterization of human IVD cells remains unknown., Design: Eleven potential marker molecules were characterized with respect to their occurrence in human IVD cells. Gene expression levels of NP were compared with annulus fibrosus (AF) and articular cartilage (AC) cells, and potential correlations with aging were assessed., Results: Higher mRNA levels of cytokeratin-19 (KRT19) and of neural cell adhesion molecule-1 were noted in NP compared to AF and AC cells. Compared to NP cytokeratin-18 expression was lower in AC, and alpha-2-macroglobulin and desmocollin-2 lower in AF. Cartilage oligomeric matrix protein (COMP) and glypican-3 expression was higher in AF, while COMP, matrix gla protein (MGP) and pleiotrophin expression was higher in AC cells. Furthermore, an age-related decrease in KRT19 and increase in MGP expression were observed in NP cells. The age-dependent expression pattern of KRT19 was confirmed by immunohistochemistry, showing the most prominent KRT19 immunoreaction in the notochordal-like cells in juvenile NP, whereas MGP immunoreactivity was not restricted to NP cells and was found in all age groups., Conclusions: The gene expression of KRT19 has the potential to characterize human NP cells, whereas MGP cannot serve as a characteristic marker. KRT19 protein expression was only detected in NP cells of donors younger than 54 years., (Copyright 2009 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: The purpose of the current study was to investigate the feasibility of applying defect-size femoral implants for the treatment of localized cartilage defects in a 1-year follow-up model., Methods: In 13 goats, a medial femoral condyle defect was created in both knees. Defects were randomly treated by immediate placement of an oxidized zirconium (OxZr) (n=9) or cobalt-chromium (CoCr) implant (n=9) or left untreated (n=8). Six un-operated knee joints served as a control. Animals were sacrificed at 52 weeks. Joints were evaluated macroscopically. Cartilage quality was analyzed macroscopically and microscopically and cartilage repair of untreated defects was scored microscopically. Glycosaminoglycan (GAG) content, release and synthesis were measured in tissue and medium. Implant osseointegration was measured by automated histomorphometry., Results: Cartilage repair score of the defects was 13.3+/-3.0 out of 24 points (0=no repair, 24=maximal repair). Articular evaluation scores decreased (indicative of degeneration) in untreated defects and in defects treated with either implant (P<0.05). Macroscopical, microscopical and biochemical analysis showed that the presence of untreated defects and the implants caused considerable degeneration of medial tibial plateau, and to a lesser extent of the lateral compartment. Mean bone-implant contact was extensive and not different between materials (39.5+/-28.1% for OxZr and 42.3+/-31.5% for CoCr) (P=0.873)., Conclusions: Considerable cartilage degeneration was induced in the articulating cartilage of the medial tibial plateau 1 year after creating an osteochondral defect in the medial femoral condyle. Treating this defect with a small metal implant, made of either OxZr or CoCr, could not prevent this degeneration. Further optimization of defect-size implants and their placement is required to make this the therapy of choice for the treatment of local cartilage defects., (Copyright 2009 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: Newly developed regenerative cartilage interventions based on the application of 3D-scaffolds require a further evaluation of the surgical techniques involved. The present study compared four different scaffold fixation techniques [fibrin glue (FG), transosseous (TS) fixation, biodegradable pin (BP) fixation and continuous cartilage sutures (CS)] to implant a custom-printed porous PEOT/PBT1000/70/30 scaffold in a human cadaver knee model., Methods: After implantation, the knees were subjected to a vertically oriented loaded continuous passive motion (CPM) protocol. The fixation techniques were evaluated after 60 and a subsequent 150 motion cycles, focusing on area coverage, outline attachment and scaffold integrity. After the total of 210 cycles, also an endpoint fixation test was performed., Results: The fixation techniques revealed marginal differences for area coverage and outline attachment after 60 and 150 cycles. The FG scored higher on scaffold integrity compared to TS (P<0.05) and CS (P=0.01). Endpoint fixation was highest for the CS, whereas FG showed a weak final fixation strength (P=0.01)., Conclusions: This study showed that optimal fixation cannot be combined always with high scaffold integrity. Special attention devoted to scaffold properties in relation to the fixation technique may result in an improvement of scaffold fixation, and thus clinical cartilage regenerative approaches involving these scaffolds., (Copyright 2009 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: Regeneration of hyaline cartilage has been the focus of an increasing number of research groups around the world. One of the most important outcome measures in evaluation of its success is the histological quality of cartilaginous tissue. Currently, a variety of histological scoring systems is used to describe the quality of osteoarthritic, in vivo repaired or in vitro engineered tissue. This review aims to provide an overview of past and currently used histological scoring systems, in an effort to aid cartilage researchers in choosing adequate and validated cartilage histological scoring systems., Methods: Histological scoring systems for analysis of osteoarthritic, tissue engineered and in vivo repaired cartilage were reviewed. The chronological development as well as the validity and practical applicability of the scoring systems is evaluated., Results: The Histological-Histochemical Grading System (HHGS) or a HHGS-related score is most often used for evaluation of osteoarthritic cartilage, however the Osteoarthritis Research Society International (OARSI) Osteoarthritis Cartilage Histopathology Assessment System seems a valid alternative. The O'Driscoll score and the International Cartilage Repair Society (ICRS) II score may be used for in vivo repaired cartilage. The 'Bern score' seems most adequate for evaluation of in vitro engineered cartilage., Conclusion: A great variety of histological scoring systems exists for analysis of osteoarthritic or normal, in vivo repaired or tissue-engineered cartilage, but only few have been validated. Use of these validated scores may considerably improve exchange of information necessary for advances in the field of cartilage regeneration., (Copyright 2009 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: For many years, the Histologic/Histochemical Grading System (HHGS) for osteoarthritis monitoring has been used as a histological scoring system for the quality of cartilage. There are, however, some limitations using this grading system. The goal of the investigation presented in this paper was to examine the hypothesized advantage of the recently introduced Osteoarthritis Research Society International (OARSI) Cartilage Histopathology Assessment System (OOCHAS) as compared to the most frequently used HHGS by means of reliability, reproducibility, and variability evaluation as well as the correlation analysis between the two systems in goat knee articular cartilage., Methods: Nine hundred and thirty-six sections of Dutch Milk goat articular knee cartilage were scored using light microscopy. Three observers applied the HHGS for all sections and subsequently, the OOCHAS. The same scoring procedure was repeated after a minimum interval of 1 week. For each system the reliability, reproducibility and variability as well as the correlation between both systems were determined., Results: The reliability of the OOCHAS was higher as compared to the HHGS. Both the HHGS as well the OOCHAS have an excellent intra- and inter-observer reproducibility and variability and a good positive correlation between the scores., Conclusions: Although the HHGS has proven to be an excellent tool for histological scoring of cartilage quality, we recommend the OOCHAS as the premium choice while stressing the importance of further research investigating the correlation of the histological results to macroscopic and biochemical parameters.

Objective: Currently, the surgical treatment of localized cartilage defects has limitations. Alternatively, localized cartilage defects may be treated with small biocompatible metal cartilage tacks. Our purpose was to investigate the applicability of defect-size femoral implants. Different bearing materials, cobalt-chromium (CoCr) and oxidized zirconium (OxZr), were tested to evaluate the effect on opposing cartilage quality and osseointegration at different insertion depths., Methods: In 18 adult female New Zealand White rabbits, a medial femoral condyle defect was filled with either an OxZr or a CoCr implant (Ø articulating surface 3.5 mm; fixating pin of 9.1 mm length), placed flush, 1mm deep or 1mm protruding with respect to the level of the surrounding cartilage. Animals were sacrificed after 4 weeks. Tibial cartilage quality was scored microscopically and osseointegration measured by automated histomorphometry., Results: Considerable articulating cartilage erosion was found in all conditions. Tibial cartilage quality was least compromised when both implants were placed flush compared to deep (P=0.01) or protruding position (P=0.004) and was better for OxZr compared to CoCr (P=0.011) when left protruding, while no differences were found when placed deep of flush. Most bone formation around the fixating pin was observed in a protruding position (P=0.01). In deep position, more bone-implant contact was observed with CoCr compared to OxZr (P=0.02)., Conclusions: OxZr and CoCr implants showed good osseointegration when used as a localized cartilage defect treatment in the rabbit knee; however, opposite cartilage damage was observed in all cases. Placement flush to the surrounding cartilage seems essential and when left protruding OxZr may be less erosive. In conclusion, caution is warranted using small metal implants for the treatment of localized cartilage in the human patient.

Various in vivo and in vitro studies suggest that joint homeostasis may have a crucial effect on the quality of regeneration tissue resulting from cartilage tissue engineering techniques. The goal of the current study was to evaluate the effect of synovial fluid (SF) from injured knee joints on in vitro chondrogenesis. Chondrocytes were isolated from a healthy human femoral condyle (post-mortem) and expanded in monolayer for 2 passages. Subsequently, the chondrocytes were redifferentiated for 14 days on collagencoated filters, cultured either in the presence or absence of 10% SF. SF was obtained from 12 injured human knee joints. After 14 days of culture, SF supplementation resulted in a significant downregulation of final proteoglycan (PG) content (7.3 +/- 1.8 mg versus 15.6 +/- 1.3 mg; p = 0.0001), PG content normalized to DNA (0.7 +/- 0.5 mg/microg versus 3.0 +/- 0.6 mg/microg; p < 0.05), relative collagen type II mRNA levels normalized to GAPDH mRNA levels (0.2 +/- 0.3 versus 7.0 +/- 5.6; p < 0.001), and differentiation index (collagen type II/I mRNA ratio; 0.1 +/- 0.2 versus 6.0 +/- 2.9; p < 0.001) as compared to control culture conditions. Additionally, SF-supplemented media resulted in significantly increased cellularity, as reflected by DNA content, compared with control media (1,369 +/- 683 microg versus 514 +/- 72 microg; p < 0.0001). Morphology, and collagen type I, X, and aggrecan mRNA levels were not significantly affected. In conclusion, this study demonstrates that SF from injured human knee joints significantly affects in vitro chondrogenesis and therefore may provide a viable target for future improvement of ACT by refinement of culture techniques, patient selection, or pretreatment of affected joints to restore joint homeostasis.

Cartilage regeneration based on isolated and culture-expanded chondrocytes is studied in a variety of in vitro models, but with varying morphological quality of tissue synthesized. The goal of the present study was to investigate the extent of the influence of expansion and redifferentiation conditions on final tissue morphology by comparing 2 expansion and redifferentiation methods. Chondrocytes from 9 human donors were expanded in medium without growth factor supplementation (basic expansion condition [BEC]) or in medium with basic fibroblast growth factor (bFGF) supplementation (growth factor supplemented expansion condition [GFSEC]). After expansion, cells were either redifferentiated in pellet culture or seeded on collagen type II-coated filters. Post-expansion mRNA levels of collagen type I and II and Sox-5, -6, and 9, measured by semiquantitative real-time polymerase chain reaction (PCR), suggested that expansion in GFSEC results in increased dedifferentiation compared to BEC. However, after 28 days of redifferentiation culture, morphology of tissue synthesized by GFSEC-expanded chondrocytes scored significantly higher on the Bern scale compared to BEC (6.4 +/- 0.3 points vs. 4.5 +/- 0.3 points in pellet culture and 6.0 +/- 0.4 points vs. 4.5 +/- 0.3 points on collagen-coated filters; p < 0.05). Expansion in GFSEC compared to BEC increased proteoglycan (PG) synthesis rate at day 9 (4.0-fold in pellet culture and 1.9-fold on collagen-coated filters; p < 0.01), PG release (6.7-fold in pellet culture and 3.2-fold on collagen-coated filters; p < 0.001), and final PG content at day 28 (1.6-fold in pellet culture and 1.5-fold on collagen-coated filters; p < 0.05). Redifferentiation on collagen-coated filters compared to pellet culture increased PG synthesis rate at day 9 (5.2-fold in BEC-expanded chondrocytes and 2.6-fold in GFSEC-expanded chondrocytes; p < 0.01), PG release (4.2-fold in BEC-expanded chondrocytes and 3.1-fold in GFSECexpanded chondrocytes; p < 0.01), and final PG content (1.3-fold in BEC-expanded chondrocytes and 1.9- fold in GFSEC-expanded chondrocytes; p < 0.01). Moreover, as visualized via electron microscopy, chondrocytes and organization of extracellular matrix cultured on filters was more similar to those found for hyaline cartilage. In conclusion, chondrocyte expansion in GFSEC and redifferentiation on collagen-coated filters resulted in most optimal chondrogenesis.

Objective: In vitro models of chondrogenesis often depart from chondrocytes harvested from less-affected areas of osteoarthritic joints. However, there are indications that these chondrocytes are phenotypically different from chondrocytes from healthy joints and thus might differ in their capacity to generate hyaline cartilage. The goal of this study was to compare the chondrogenic capacity of chondrocytes from healthy and OA joints., Design: Chondrocytes isolated from nine healthy and nine OA knee joints were expanded in monolayer for two passages. Chondrocytes from passages 1 and 2 were analyzed for expression of (de)differentiation and hypertrophy markers and were seeded at passage 2 on collagen-coated filters for redifferentiation culture to study cartilage matrix formation., Results: The collagen II/I mRNA ratio, reflecting differentiation, decreased from passage 1 to 2 in both chondrocytes from OA joints and chondrocytes from healthy joints (P<0.05), without a significant difference between the two donor types. At passage 1, levels of the cartilage transcription factors Sox-5, Sox-6 and Sox-9 appeared to be higher in chondrocytes from OA joints (n.s.), but this was not seen at passage 2. However, a clear difference was observed in collagen type X expression, which was high in chondrocytes from OA joints at both passages, while undetectable in chondrocytes from healthy joints (P<0.01). Tissue generated by chondrocytes from healthy joints redifferentiated for 28 days, showed a significantly better morphology, as assessed by histological scoring (P<0.01) and higher proteoglycan content (P<0.05), compared to chondrocytes from OA joints. Matrix turnover parameters, i.e., proteoglycan synthesis and degradation rate, were not significantly affected by donor tissue origin., Conclusions: These results suggest that clear differences between chondrocytes from healthy and OA joints exist and that these are not completely abolished during the process of de- and redifferentiation. Therefore, in vitro cartilage regeneration models, which use chondrocytes from OA joints, should be interpreted with care.

The culture of spermatogonial cells under well-defined conditions would be an important method for elucidating the mechanisms involved in spermatogenesis and in establishing tissue regeneration in vivo. In this study, a serum-free culture system was established, with type A spermatogonia isolated from adult vitamin A-deficient mice. At days 1, 3 and 7 of culture, the viability and proliferation of cells were monitored. The viability of the cells decreased by day 7 to 10% of the cells present. Proliferation occurred mainly during day 1, when 1% of the germ cells was proliferating. Co-labelling for a germ cell marker (heat shock protein-90alpha, Hsp90alpha), and a marker used to detect dividing cells (bromodeoxyuridine, BrdU), showed that this proliferation was restricted to germ cells. In an attempt to improve these parameters, medium containing fetal calf serum (FCS) was used. Viability was not influenced by serum, but proliferation was markedly enhanced. However, after day 7 of incubation with FCS, co-immunolocalization for Hsp90alpha and BrdU showed a preferential proliferation of somatic cells. Comparison of cultures of adult cells with cultures of prepubertal germ cells, commonly used in studies of spermatogenesis, showed that prepubertal germ cells are twice as viable. In addition, a different proliferation profile was observed, with a peak at day 3. Here, a distinct proliferation of somatic cells was also noted. The results from the present study indicate that the origin of isolated germ cells partly determines culture outcome and that cultures of prepubertal germ cells may not be representative for adult spermatogenesis. Moreover, adding FCS to the culture medium invokes the risk of profound and undesirable effects on cell composition, also underlining the need for identification of germ cells during culture.

The aim of this study was to isolate and purify bovine type A spermatogonia. Testes from 5-7-month-old calves were used to isolate germ cells using a two-step enzymatic digestion. During the isolation and purification steps, the viability of cells was determined using live/dead staining. The identity of type A spermatogonia during isolation and purification was determined under a light microscope equipped with a Nomarski lens. Isolated cells were characterized further by using specific markers for type A spermatogonia, including Dolichos biflorus agglutinin (DBA) and c-kit. The cell suspension was transplanted into immunodeficient recipient mouse testes and the colonization was assessed 1-3 months after transplantation, to assess the stem cell population among the isolated cells. After isolation, a cell suspension was obtained containing about 25% type A spermatogonia, which was enriched further by differential plating and separation on a discontinuous Percoll gradient. Finally, fractions containing 65-87% pure type A spermatogonia were obtained. Large and small type A spermatogonia with different numbers and sizes of nucleoli were found. DBA stained both large and small type A spermatogonia and its application in fluorescence-activated cell sorting (FACS) resulted in comparable percentages of type A spermatogonia to those determined by morphological examination under a light microscope equipped with a Nomarski lens. Nearly all of the large type A spermatogonia showed strong c-kit immunoreactivity, indicating that these cells had undergone at least an initial differentiation step. In contrast, approximately half of the small type A spermatogonia were negative for c-kit, indicating the presence of the spermatogonial stem cells in this population. At 3 months after transplantation, groups of bovine type A spermatogonia were found in most tubule cross-sections of the recipient mouse testes, showing the presence of spermatogonial stem cells among the isolated cells.

The contribution of vacuolar H+-ATPases (V-ATPases) to collagen degradation was investigated in soft connective tissue explants (periosteum). Immunolocalisation showed faint to intense staining of cells throughout the periosteum. The V-ATPase inhibitors, bafilomycin A1 and folimycin, decreased overall collagen degradation by 40 and 50% after 24 and 48 h, respectively. The participation of V-ATPases in intracellular degradation of collagen was demonstrated by the decrease of the amount of phagocytosed collagen in fibroblasts upon inhibition of pump activity. The inhibition of degradation was not due to a reduction in activity of gelatinase A, an enzyme previously found to mediate collagen degradation, as assessed by zymographic analysis of tissue and conditioned medium. Bafilomycin A1 even induced an increase of gelatinase A and B levels in both fractions. In conclusion, acidification by V-ATPases may represent an important mechanism in extracellular and intracellular collagen degradation in soft connective tissue., (Copyright 1998 Academic Press.)

The degradation of soft connective tissue collagen is considered to depend on the activity of various proteolytic enzymes, particularly those belonging to the group of matrix metalloproteinases and cysteine proteinases. In the present study, we investigated the contribution of these enzymes to this process. Using a general inhibitor of MMPs (SC44463), collagen degradation was strongly inhibited, by about 40% after 24 h and up to 80% after 72 h of culturing. Blockage of cysteine proteinase activity (with leupeptin or E-64) reduced breakdown at these time intervals by 50% and 20%, respectively. Given the abundant presence of gelatinases--in particular gelatinase A (MMP-2)--in the tissue, the effect of an inhibitor selective for gelatinases (CT1166) was studied. Gelatinase inhibition resulted in a dose-dependent decrease of collagen breakdown up to 90% after 48 h. The ability of gelatinase A to degrade collagens was demonstrated by the induction of breakdown in devitalized explants by addition of activated gelatinase A, or by activation of endogenous enzyme with 4-aminophenylmercuric acetate. This latter effect was not found with plasmin, an activator of MMPs other than gelatinase A. Finally, the relevance of gelatinase A to the in vivo degradation of soft connective tissue collagen was implicated by the significant correlation found between its activity and the collagen turnover rates of four soft connective tissues (tooth pulp, periodontal ligament, molar gingiva and skin). We conclude that collagen degradation in soft connective tissue is mediated by MMPs and to a lesser extent by cysteine proteinases. Our data are the first to attach a key role to gelatinase A in this process.