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Hydrogels are commonly used in wound dressing, as they retain moisture, accelerate healing, and break down necrotic tissue. This process enhances patient comfort levels while simultaneously reducing pain caused by dead tissue. The purpose of this study was to investigate the in vivo toxicity of a dual hydrogel consisting of type I atelocollagen cross-linked with sodium hyaluronate hydrogel used for wound dressing. Porcine type I atelocollagen was cross-linked with sodium hyaluronate to form the hydrogel. For subcutaneous implantation, 0.5 ml of dual hydrogel was injected into two different sites of twenty rats per group. High density polyethylene rods were implanted subcutaneously to serve as a control material. Hematological assessment, blood biochemistry, histopathological, and histological evaluations were scored and graded after 4 weeks. A bioreactivity rating was used for evaluation of subacute toxicity. Differences observed in blood chemical analysis and hematological analysis between control and test groups were within normal variations and considered unrelated to the test article implantation. No significant implantation-related lesions were observed in any of the major organs of all test animals. The overall histopathological index of the test article implantation sites was evaluated as 0. The bioreactivity rating was evaluated as non-irritant after 4-week subcutaneous implantation. Overall, these results indicate that the dual hydrogel of type I atelocollagen and sodium hyaluronate is biologically and chemically safe for clinical application as a wound dressing.

Meniscus tears common injuries of the knee joint and a major osteoarthritis (OA) risk factor. Knowledge gaps that limit development of therapeutic approaches for meniscus injury and degeneration concern transcription factors that control the meniscus cell phenotype. Analysis of RNA-sequencing data from 37 human tissues in the Genotype-Tissue Expression (GTEx) database and RNA-sequencing data from meniscus and articular cartilage showed that transcription factor Mohawk (MKX) is highly enriched in meniscus. In human meniscus cells, MKX regulates expression of meniscus marker genes, OA-related genes and other transcription factors, including Scleraxis (SCX), SRY Box 5 (SOX5), and Runt domain-related transcription factor 2 (RUNX2). In mesenchymal stem cells, the combination of adenoviral MKX (Ad-MKX) and Transforming growth factor-β3 (TGF-β3) induced a meniscus cell phenotype. When Ad-MKX-transduced mesenchymal stem cells (MSCs) were seeded on TGF-β3-conjugated decellularized meniscus scaffold (DMS) and inserted into experimental tears in meniscus explants, they increased glycosaminoglycan content, extracellular matrix interconnectivity, cell infiltration into the DMS and improved biomechanical properties. Ad-MKX injection into mouse knee joints with experimental OA induced by surgical destabilization of the meniscus suppressed meniscus and cartilage damage, reducing OA severity. Ad-MKX injection into human OA meniscus tissue explants corrected pathogenic gene expression. These results identify MKX as a previously unidentified key transcription factor that regulates the meniscus cell phenotype. The combination of Ad-MKX with TGF-β3 is effective for differentiation of MSCs to a meniscus cell phenotype and useful for meniscus repair. MKX is a promising therapeutic target for meniscus tissue engineering, repair and prevention of OA.

The objective of this study was to examine FoxO expression and FoxO function in meniscus. In menisci from human knee joints with osteoarthritis (OA), FoxO1 and 3 expression were significantly reduced compared with normal menisci from young and old normal donors. The expression of FoxO1 and 3 was also significantly reduced in mouse menisci during aging and OA induced by surgical meniscus destabilization or mechanical overuse. Deletion of FoxO1 and combined FoxO1, 3, and 4 deletions induced abnormal postnatal meniscus development in mice and these mutant mice spontaneously displayed meniscus pathology at 6 mo. Mice with Col2Cre-mediated deletion of FoxO3 or FoxO4 had normal meniscus development but had more severe aging-related damage. In mature AcanCreERT2 mice, the deletion of FoxO1, 3, and 4 aggravated meniscus lesions in all experimental OA models. FoxO deletion suppressed autophagy and antioxidant defense genes and altered several meniscus-specific genes. Expression of these genes was modulated by adenoviral FoxO1 in cultured human meniscus cells. These results suggest that FoxO1 plays a key role in meniscus development and maturation, and both FoxO1 and 3 support homeostasis and protect against meniscus damage in response to mechanical overuse and during aging and OA.

The aim of this study was to examine the potential of platelet-derived growth factor (PDGF)-coated decellularized meniscus scaffold in mediating integrative healing of meniscus tears by inducing endogenous cell migration. Fresh bovine meniscus was chemically decellularized and covalently conjugated with heparin and PDGF-BB. In vitro PDGF release kinetics was measured. The scaffold was transplanted into experimental tears in avascular bovine meniscus explants and cultured for 2 and 4 weeks. The number migrating and proliferating cells at the borderline between the scaffold and injured explant and PDGF receptor-β (PDGFRβ) expressing cells were counted. The alignment of the newly produced ECM and collagen was analyzed by Safranin-O, picrosirius red staining, and differential interference contrast (DIC). Tensile testing of the explants was performed after culture for 2 and 4 weeks. Heparin conjugated scaffold showed immobilization of high levels of PDGF-BB, with sustained release over 2 weeks. Insertion of the PDGF-BB treated scaffold in defects in avascular meniscus led to increased PDGFRβ expression, cell migration and proliferation into the defect zone. Safranin-O, picrosirius red staining and DIC showed tissue integration between the scaffold and injured explants. Tensile properties of injured explants treated with PDGF-BB coated scaffold were significantly higher than in the scaffold without PDGF. In conclusion, PDGF-BB-coated scaffold increased PDGFRβ expression and promoted migration of endogenous meniscus cells to the defect area. New matrix was formed that bridged the space between the native meniscus and the scaffold and this was associated with improved biomechanical properties. The PDGF-BB-coated scaffold will be promising for clinical translation to healing of meniscus tears. Statement of Significance Meniscus tears are the most common injury of the knee joint. The most prevalent forms that occur in the inner third typically do not spontaneously heal and represent a major risk factor for the development of knee osteoarthritis. The goal of this project was to develop an approach that is readily applicable for clinical use. We selected a natural and readily available decellularized meniscus scaffold and conjugated it with PDGF, which we had previously found to have strong chemotactic activity for chondrocytes and progenitor cells. The present results show that insertion of the PDGF-conjugated scaffold in defects in avascular meniscus led to endogenous cell migration and proliferation into the defect zone with tissue integration between the scaffold and injured explants and improved tensile properties. This PDGF-conjugated scaffold will be promising for a translational approach to healing of meniscus tears.

Biologic augmentation for rotator cuff repair is a challenging treatment in patients with chronic large, massive, and irreparable rotator cuff injuries. Particularly, the use of an extracellular matrix (ECM) patch such as dermal tissue offered improved biomechanical properties in previous studies. Cytokines induce cell chemotaxis, proliferation, matrix synthesis, and cell differentiation. Moreover, osteoinductive growth factors such as bone morphogenetic protein-2 (BMP-2) affect the formation of new bone and fibrocartilage in lesions. However, the effects of using a dermal patch in combination with BMP-2 have not been evaluated to date, although many researchers have recognized the importance thereof. In this study, rhBMP-2-coated dermal patch (1 cm × 2 cm) isolated from human cadaveric donor was inserted in a rabbit model of chronic rotator cuff injury for in vivo evaluation. Bone mineral density and biomechanical strength were tested and histological and histomorphometric analyses were performed. The results showed that insertion of an rhBMP-2-coated acellular dermal patch not only significantly ameliorated new bone formation, it also improved biomechanical properties such as ultimate tensile strength. Thus, the use of this combination may improve the chronic rotator cuff injury-healing rate and clinical outcomes after rotator cuff repair. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1840-1846, 2017.

We report a compensation system for geometric errors of a linear axis to achieve the ultra-precision positioning. The system was miniaturized by integrating the drive and measuring components. A novel joint with three DOFs based on a flexure hinge was developed to obtain the ultra-precision positioning. The drive component, which was adapted to the parallel mechanism, had a high structural stiffness and rapid response characteristics. The rotational stiffness of the joint was calculated and the mode shapes were analyzed to verify the feasibility of three-DOF motion. The measurement component consisted of capacitive sensors and a reference mirror to measure the three geometric errors of the linear axis. In addition, the optimal position between the reference coordinate system and a sensor was determined to minimize the standard uncertainties of the measured errors. The compensation system was fabricated and evaluated experimentally. A compensation algorithm based on a recursive technique was developed to improve the positioning accuracy of the system. The geometric errors of the linear axis were measured and compensated for, and a marked improvement was observed compared to the case with no error compensation. The peak-to-valley error of the translational and rotational errors were reduced by 92% and 98%, respectively.

Background:Anterior cruciate ligament (ACL) reconstruction is the current standard of care for ACL tears. However, the results are not consistently successful; autografts or allografts have certain disadvantages; and synthetic grafts have had poor clinical results.Purpose:To determine if recellularization of decellularized tendons combined with mechanical stimulation in a bioreactor could replicate the mechanical properties of the native ACL and be successfully used for ACL reconstruction in vivo.Study Design:Controlled laboratory study.Methods:Porcine tibialis tendons were decellularized and then recellularized with human adult bone marrow–derived stem cells. Tendons were cultured in a tissue bioreactor that provided biaxial cyclic loading for up to 7 days. To reproduce mechanical stresses similar to those experienced by the ACL within the knee joint, the tendons were subjected to simultaneous tension and torsion in the bioreactor. Expression of tendon-specific genes and newly synthesized collagen and glycosaminoglycan were used to quantify the efficacy of recellularization and dynamic bioreactor culture. The ultimate tensile load to failure and stiffness of recellularized constructs were measured after dynamic stimulation. Finally, the tissue-engineered tendons were used to reconstruct the ACL in 24 pigs, and ultimate tensile load to failure and stiffness were assessed after 3 months.Results:Dynamic bioreactor culture significantly increased the expression of tendon-specific genes, the quantity of newly synthesized collagen and glycosaminoglycan, and the ultimate tensile load and stiffness of recellularized tendons. After in vivo reconstruction, the ultimate tensile load and stiffness of the tissue-engineered tendons increased significantly up to 3 months after surgery and were within 80% of the ultimate tensile load of the natural ACL.Conclusion:This translational study indicates that recellularization and dynamic mechanical stimuli can significantly enhance matrix synthesis and ultimate tensile load of decellularized porcine tibialis tendons. This approach to tissue engineering can be very useful for ACL reconstruction and may overcome some of the disadvantages of autografts and allografts.Clinical Relevance:Dynamic bioreactor cultivation of tissue-engineered tendons may overcome the limitations of autografts and allografts.

This study examines the hypothesis that injectable collagen gel can be an effective carrier for recombinant human bone morphogenetic protein-2 (rhBMP-2)'s localization to the healing tendon-bone interface. In 36 mature New Zealand White rabbits, the upper long digital extensor tendon was cut and inserted into the proximal tibial bone tunnel. Then a rhBMP-2-containing collagen gel was injected into the tendon-bone tunnel interface, using a syringe. Histological and biomechanical assessments of the tendon-bone interface were conducted at 3 and 6 weeks after implantation. In vitro testing showed that the semi-viscous collagen gel at room temperature was transformed into a firm gel state at 37°C. The rhBMP-2 release profile showed that rhBMP-2 was released from the collagen gel for more than 28 days. In vivo testing showed that fibrocartilage and new bone are formed at the interface at 6 weeks after injection of rhBMP-2. On radiography, spotty calcification appeared and enthesis-like tissue was produced successfully in the tendon at 6 weeks after injection of rhBMP-2. Use of the viscous collagen gel and rhBMP-2 mixture increased the fusion rate between the bone tunnel and tissue graft. This study demonstrates that viscous collagen gel can be an effective carrier for rhBMP-2 delivery into surgical sites, and that the injectable rhBMP-2-containing collagen gel may be applied for the enhancement of tendon-bone interface healing in the future. Copyright © 2015 John Wiley & Sons, Ltd.

In vitro experiment using degenerated human ligamentum flavum (LF) and herniated intervertebral disk (IVD).To investigate the role and effect of degenerated and herniated IVDs on LF hypertrophy and ossification.Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by aging and degenerative process. It is well known that degenerated IVDs spontaneously produce inflammatory cytokines. Therefore, we hypothesized that degenerated IVD may affect adjacent LF through secreted inflammatory cytokines.LF and herniated lumbar IVD tissues were obtained during surgical spinal procedures. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with disk supernatant from herniated IVDs. Secreted cytokines from IVD tissue culture were detected by enzyme-linked immunosorbent assay. After analysis of cytotoxicity, DNA synthesis was measured. Reverse transcription-polymerase chain reaction for mRNA expressions of types I, II, III, V, and XI collagen and osteocalcin, and histochemical stains were performed.Supernatant from tissue culture of herniated IVD showed increased production of interleukin-1α, interleukin-6, tumor necrosis factor-α, prostaglandin E2, and nitric oxide compared with disk tissue culture from traumatic condition. There was no cytotoxicity in LF cells treated with disk supernatant from herniated IVDs. There was significant increase in DNA synthesis, upregulation in mRNA expression of types III, XI collagen and osteocalcin, whereas variable expression pattern of type I and V, and strong positive stains for Von Kossa and alkaline phosphatase in LF cultures with disk supernatant.Degenerated and herniated IVDs provide an important pathomechanism in hypertrophy and ossification of the LF through inflammatory cytokines.

In this study, a measurement method of double ball-bar is proposed to measure the geometric errors of an ultra-precision roll mold machine tool. A volumetric error model of the machine tool is established to investigate the effects of the geometric errors to a radius error and a cylindricity of the roll mold. A measurement path is suggested for the geometric errors, and a ball-bar equation is derived to represent the relation between the geometric errors and a measured data of the double ball-bar. Set-up errors, which are inevitable at the double ball-bar installation, also are analyzed and are removed mathematically for the measurement accuracy. In addition, standard uncertainty of the measured geometric errors is analyzed to determine the experimental condition. Finally, the proposed method is tested and verified through simulation.

Lee K-I, Lee J-S, Jung H-H, Lee H-Y, Moon S-H, Kang K-T, Shim Y-B, Jang J-W. Inactivation of enveloped and non-enveloped viruses in the process of chemical treatment and gamma irradiation of bovine-derived grafting materials. Xenotransplantation 2012; 19: 365–369. © 2012 John Wiley & Sons A/S. Abstract Background: Xenografts, unlike other grafting products, cannot be commercialized unless they conform to stringent safety regulations. Particularly with bovine-derived materials, it is essential to remove viruses and inactivate infectious factors because of the possibility that raw materials are imbrued with infectious viruses. The removal of the characteristics of infectious viruses from the bovine bone grafting materials need to be proved and inactivation process should satisfy the management provision of the Food and Drug Administration (FDA). To date, while most virus inactivation studies were performed in human allograft tissues, there have been almost no studies on bovine bone. Methods: To evaluate the efficacy of virus inactivation after treatment of bovine bone with 70% ethanol, 4% sodium hydroxide, and gamma irradiation, we selected a variety of experimental model viruses that are known to be associated with bone pathogenesis, including bovine parvovirus (BPV), bovine herpes virus (BHV), bovine viral diarrhea virus (BVDV), and bovine parainfluenza-3 virus (BPIV-3). The cumulative virus log clearance factor or cumulative virus log reduction factor for the manufacturing process was obtained by calculating the sum of the individual virus log clearance factors or log reduction factors determined for individual process steps with different physicochemical methods. Results: The cumulative log clearance factors achieved by three different virus inactivation processes were as follows: BPV ≥17.73, BHV ≥20.53, BVDV ≥19.00, and BPIV-3 ≥16.27. On the other hand, the cumulative log reduction factors achieved were as follows: BPV ≥16.95, BHV ≥20.22, BVDV ≥19.27, and BPIV-3 ≥15.58. Conclusions: Treatment with 70% ethanol, 4% sodium hydroxide, or gamma irradiation was found to be very effective in virus inactivation, since all viruses were at undetectable levels during each process. We have no doubt that application of this established process to bovine bone graft manufacture will be effective and essential.

Study design. A prospective case control study. Objectives: To investigate the risk of a fall using functional mobility tests in patients with lumbar spinal stenosis (LSS) via a comparison with patients with knee osteoarthritis (KOA). Summary of Background Data: LSS is a degenerative arthritic disease in the spine that results in decreasing function, impaired balance and gait deficit with increased levels of leg and back pain. This physical impairment might result in an increased risk of fall later in the disease process as shown in knee osteoarthritis. However, there has been no study regarding the association between the risk of a fall and LSS. Methods: The study was an age- and weight-matched case control study consisting of two groups, one group consisting of 40 patients with LSS who were scheduled to undergo spine surgery (LSS group), the other group consisting of 40 patients with advanced OA in both knees, scheduled to undergo TKA on both knees (KOA group). For both groups, four functional mobility tests such as a six meter walk test (SMT), sit-to-stand test (STS), alternative step test (AST), and timed up and go test (TUGT) were performed. Results: There was no difference in demographic data between both groups except for BMI. For the SMT and STS, the patients in the LSS spent significantly more time performing these tests than the patients in the KOA. For the AST, however, patients in the KOA group presented a statistically worse performance in functional mobility, compared to the LSS group. The mean TUGT time was not statistically different between the two groups. Conclusions: The current study highlights that patients with symptomatic LSS have a comparable risk of a fall to patients who had degenerative KOA based on the results of functional mobility tests (SMT, STS, AST, and TUGT). Language: en

Study design In vitro and in vivo experiment using degenerated human ligamentum flavum (LF) and Type 5 adenovirus construct with bone morphogenetic protein-2 (BMP-2) cDNA. Objectives To demonstrate in vitro and in vivo osteogenic effect of BMP-2 gene transfer to human LF and to propose genetically modified LF as a substitute for autogenous bone graft in spinal fusion. Summary of background data Spinal fusion is still considered to be an important option for treating various spinal disorders. To induce solid spinal fusion, osteoinductive and/or osteoconductive agents have been widely adopted. Autogenous LF, however, has never been seriously considered as a carrier for ex vivo osteoinductive gene therapy for spinal fusion. Methods In vitro experiment: Degenerated human LF was harvested and cultured. Type 5 adenovirus lacZ (Ad/lacZ) and BMP-2 construct (Ad/BMP-2) were produced. LF cell cultures were then exposed to Ad/BMP-2. Expressions of osteocalcin and BMP-2 mRNA were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Western blot analysis was performed to detect osteocalcin protein. Alkaline phosphatase and von Kossa stains were used to detect osteogenic markers and bone nodule formation, respectively. In vivo experiment: Human LF tissues treated with Ad/lacZ, Ad/BMP-2, and saline were implanted into the subcutaneous tissue of nude mice. After 4 weeks, nude mice were radiographed and killed. Implanted LF tissues were harvested and histologically stained. Results LF cell cultures with Ad/BMP-2 revealed strong expression of BMP-2 and osteocalcin mRNA in RT-PCR and osteocalcin protein in western blot analysis. LF cell culture with saline showed baseline expression of BMP-2, osteocalcin mRNA, and osteocalcin protein, respectively. Furthermore, LF cell culture with Ad/BMP-2 demonstrated the expression of alkaline phosphatase and bone nodule formation in the aforementioned histochemical stain. LF tissues with Ad/BMP-2 revealed de novo osteogenesis in nude mice, whereas LF with Ad/lacZ or saline showed only remaining LF tissue without sign of bone formation. Conclusion Human LF cells transduced with Ad/BMP-2 exhibited the expression of osteogenic phenotype and bone nodule formation. Additionally, genetically modified human LF with BMP-2 cDNA clearly demonstrated de novo osteogenesis, which supports the concept that biologically modified LF can be a substitute for autogenous bone graft in spinal fusion surgery.

OBJECTIVE: Most patients with muscular dystrophy (MD) develop progressive scoliosis after losing ambulatory status, but some cases develop severe scoliosis at a skeletally immature age before losing ambulatory status. Only a few studies have been conducted in skeletally immature patients with severe scoliosis. The purpose of this study was to assess the functional and cosmetic outcome in skeletally immature patients with severe scoliosis. METHODS: Preoperative, immediate postoperative, and final follow-up radiographs were analyzed in 10 consecutive skeletally immature patients with respect to the Cobb angle degree and the pelvic obliquity angle correction, how long the correction was maintained, and the development of the crankshaft phenomenon. In the functional assessment, the ability to sit balanced, according to the Mulcahy method, and the ability to use hands, according to the Rhyu method, were evaluated. Furthermore, the degree of subjective satisfaction was evaluated in these patients. RESULTS: The average age of the patients was 10.4 years, and the average follow-up period was 33 months with minimum 2 years' follow-up. All 10 patients survived and were available at the follow-up. The mean Cobb and pelvic obliquity angles were 80 degrees and 17 degrees at the time of the surgery, 31 degrees and 3.7 degrees immediately after the surgery, and 35 degrees and 4.7 degrees at the time of the final follow-up, respectively. The initial mean Cobb angle correction averaged 61%, with 78% of pelvic obliquity corrected. These corrections were maintained over time in most cases. At the time of the surgery, the mean volume of blood loss was 1111 mL, with an average operation time of 411 minutes. There were no major complications. At the time of the last follow-up, no patient showed development of the crankshaft phenomenon. The average score for the ability to sit balanced improved from 4.4 to 6.6 according to the Mulcahy evaluation method. The scores for hand use were 2.2-2.7. However, the forced vital capacity of the lungs decreased from a preoperative 48% to 46.1%. CONCLUSIONS: These results indicate that even in very young MD patients with severe scoliosis, acceptable curve correction can be achieved and maintained with surgery. The improved pelvic obliquity and scoliosis angle stabilized the spine, freeing the upper extremities and allowing productive activities characteristic of childhood.

Decellularized tissues have been successfully used in tissue engineering and regenerative medicine for the purpose of removing antigens present in the cellular components. However, this decellularization technique uses ionic solutions or chemical treatments such as enzyme treatments that might damage the biophysical properties or reduce the physical strength of tissue. This study aimed to improve the strength of decellularized tissues. We designed a tissue bioreactor that can repeatedly deliver physical stimulation, such as tensile and torsional deformation, to the upper and lower parts of a tissue. To decellularized porcine Tibialis tendons, we used an enzymatic solution to remove the primary cells, and then applied ultrasonic cleansing using a combination of ionic solution and distilled water to destroy residual cells by differing from the osmotic pressure between the inside and outside of the cell membrane. The total DNA content of decellularized tissue was decreased by 77% compared with that of the original tissue and the ultimate tensile strength of the decellularized tissue was 20% lower than that of the normal tissue. Decellularized tissues were then cultivated in the tissue bioreactor with repeated physical stimulation of 110% tension, 90° torsion, and frequency of once per a second, and the ultimate tensile strength was found to be greater than that of the normal ligament at 7 day culture. This study showed that decellularization using enzyme and mechanical treatment is safe and use of a tissue bioreactor can increase the physical strength of tendons, making this a potential mechanism to reconstruct human ligaments.

Study Design: In vitro experiment using degenerated human ligamentum flavum (LF) and various inflammatory cytokines. Objectives: To examine the effect of inflammatory cytokines on LF cells and to identify their roles in the pathogenesis of LF hypertrophy and ossification. Summary of Background Data: Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by the degenerative processes (ie, increased collagen synthesis and chondroid metaplasia) of ligament fibroblasts. Degenerated intervertebral disk spontaneously produces inflammatory cytokines, which might affect the adjacent LF through local milieu of the spinal canal. Methods: The interlaminar portion of the LF was collected during surgical spinal procedures in 15 patients (age range, 49–78 y) with lumbar spinal stenosis. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with various inflammatory cytokines: interleukin (IL)-1α, IL-6, tumor necrosis factor-α (TNF-α), prostaglandin E2 (PGE2), and nitric oxide (NO). Cytotoxicity was analyzed by MTT assays. DNA synthesis was measured with 3H-thymidine incorporation, and mRNA expression of types I, III, V, and XI collagen and osteocalcin were performed by reverse transcription-polymerase chain reaction. Histochemical stains such as Von Kossa were also performed to detect bone nodule formation. Results: There was no cytotoxicity in the LF cells treated with each cytokine. There were significant increases in DNA synthesis and upregulated mRNA expression of types I, V, XI collagen and osteocalcin in LF cultures treated with various cytokines. LF cultures treated with IL-6, TNF-α, PGE2, and NO showed positive Von Kossa staining, indicating bone nodule formation from LF cells. Conclusions: Inflammatory cytokines (IL-6, TNF-α, PGE2, and NO) seem to play a crucial role in hypertrophy and ossification of LF. Degenerated, herniated intervertebral disks, and facet arthrosis may influence LF through inflammatory cytokines and cause hypertrophy and ossification of LF.

STUDY DESIGN In vitro experiment using rabbit nucleus pulposus (NP) cells seeded in atelocollagen scaffolds under the stimulation of growth factors. OBJECTIVE To demonstrate the effect of anabolic growth factors in rabbit NP cells cultured in atelocollagen type I and type II. SUMMARY OF BACKGROUND DATA Atelocollagen provides intervertebral disc (IVD) cells for a biocompatible environment to produce extracellular matrix. IVD cells with exogenous transforming growth factor-beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) also render an increase in matrix synthesis. However, the effect of anabolic growth factors in NP cells cultured in atelocollagens was not elucidated before. METHODS Rabbit NP cell was harvested, enzymatically digested, and cultured. The NP cells were seeded to atelocollagen type I and type II scaffolds, and then cultures were exposed to TGF-β1 (10 ng/mL) and/or BMP-2 (100 ng/mL). DNA synthesis was measured using [4H]-thymidine incorporation. Newly synthesized proteoglycan was measured using [35S]-sulfate incorporation. Reverse transcription-polymerase chain reactions (RT-PCRs) for mRNA expression of aggrecan, collagen type I, collagen type II, and osteocalcin were performed. RESULTS Rabbit NP cells cultured in atelocollagen type I scaffold showed an increase (1.7 to 2.4-fold) in DNA synthesis in response to TGF-β1 and/or BMP-2 (P < 0.05), whereas NP cultures in atelocollagen type II demonstrated a 30% increase in DNA synthesis only with combination of both growth factors compared with control (P < 0.05). Rabbit NP cells in atelocollagen type II scaffold with TGF-β1 and combination of both growth factors exhibited robust 5.3- and 5.4-fold increases in proteoglycan synthesis (P < 0.05), whereas any cultures in atelocollagen type I failed to show any significant increase compared with control. Rabbit NP cells in atelocollagen type I and type II scaffolds with TGF-β1 and/or BMP-2 demonstrated the upregulation of aggrecan, collagen type I, and collagen type II mRNA expression compared with saline control (P < 0.05). The response in transcriptional level was more robust in atelocollagen type II than in type I. In any event, there is no recognizable expression of osteocalcin (P < 0.05). CONCLUSION NP cells in atelocollagens under the stimulation of TGF-β1 and BMP-2 exhibited anabolic responses in transcriptional and translational levels. Hence, such an approach can provide a suitable engineered tissue for IVD regeneration with potential for robust refurbishment of matrix.

Objective The aim of the current study was to determine whether a hydroxyapatite (HA)/beta-tricalcium phosphate (β-TCP) ratio of 20/80 impregnated with recombinant human bone morphogenetic protein (rhBMP-2) enhances new bone formation and to evaluate the dose-dependent response of rhBMP-2. Study Design Critical-sized calvarial defects were made in rats, and biphasic calcium phosphate (BCP) with different rhBMP-2 doses was loaded into rat calvarial defects. The animals were allowed to heal for either 2 or 8 weeks. Results The percentages of new bone after 2 and 8 weeks of healing were significantly greater in the rhBMP-2–treated groups (at all doses) than in the control groups. The percentage of remaining BCP was significantly lower at 8 weeks than at 2 weeks in all groups that included BCP. Conclusions rhBMP-2 administered using a BCP carrier significantly induces new bone formation. A dose-dependent response was not shown in the present study.

Study Design: An in vitro experiment. Objectives: To evaluate the mRNA expressions of matrix components, and analyze the cellular proliferation and proteoglycan synthesis of human intervertebral disc cells in response to dexamethasone and TGF-β1 Summary of Literature Review: Corticosteroids are responsible for the regulation of a diverse range of biological processes through modulation of the expression of target genes. The direct injection of methylprednisolone to the intervertebral disc (IVD) has been shown to cause degeneration and calcification of the disc in rabbits. Systemic administration of hydrocortisone induced degeneration of notochordal cells, which accelerated the aging process of the disc in mice. Transforming growth factor beta-1 (TGF-β1) is known as a potent agent for the proliferation, differentiation and matrix synthesis of IVD. Materials and Methods : IVD cells were isolated from ten patients, and subsequently cultured. Various doses of dexamethasone (DEX) and/or TGF-β1 were administered to the IVD cultures. DNA and proteoglycan syntheses were measured by the incorporation of [3H]-thymidine and [35S]-sulfate, respectively. RT-PCRs were performed for the expressions of aggrecan, collagen types I and II, and osteocalcin mRNA. R e s u l t s: Cultures with DEX showed increased cellular proliferation and decreased proteoglycan synthesis (p

To date, there have been no prospective, objective studies comparing the accuracy of the MRI, myelo-CT and myelography. The purpose of this study is to compare the diagnostic and predictive values of MRIs, myelo-CTs, and myelographies. Myelographies with dynamic motion views, myelo-CTs, MRIs and exercise treadmill tests were performed in 35 cases. The narrowest AP diameter of the dural sac was measured by myelography. At the pathologic level, dural cross-sectional area (D-CSA) was calculated in the MRI and Myelo-CT. The time to the first symptoms (TAF) and the total ambulation time (TAT) were measured during the exercise treadmill test and used as the standard in the comparison of correlation between radiographic parameters and walking capacity. The mean D-CSA by CT was 58.3 mm(2) and 47.6 mm(2) by MRI. All radiographic parameters such as AP diameters and D-CSA have no correlation to TAF or TAT (p > 0.05). Our data showed no statistically significant differences in the correlation of the patients' walking capacity to the severity of stenosis as assessed by myelography, myelo-CT and MRI.

Study design: An in vitro experiment. Objectives: To assess the effect of pulsed sinusoidal EMF on human intervertebral disc (IVD) cells. Literature Review Summary: Electromagnetic field (EMF) is known to modify some relevant physiological parameters of cells cultured in vitro, such as proliferation, synthesis, secretion of growth factors and transcription. EMF induces bone formation in delayed, non union and spinal fusion models. Also, the exposure of EMF has been shown to protect against the hazardous effect of smoking in the rabbit IVD. Materials and Methods: Human IVD cells were three-dimensionally cultured in alginate beads and exposed to a 650Ω, 1.8millitesla magnetic flux density, 60Hz sinusoidal wave of EMF. The cultures were divided into the control and EMF groups, with various exposure times. The cytotoxicity, and DNA and proteoglycan syntheses were measured by the MTT assay, and [3H]thymidine and [35S]-sulfate incorporation, respectively. RT-PCRs were performed for aggrecan, and collagen types I and II mRNA expressions. Results: There was no recognizable cytotoxicity in the EMF group, but cellular proliferation was stimulated (p

Study Design: In-vitro experiments using human mesenchymal stem cells (MSCs), intervertebral disc (IVD) cells and type 5 adenovirus/transforming growth factor-β1 construct (Ad/TGF-β1). Objectives: To determine the effect of MSC-based gene therapy for matrix regeneration of IVD cells. Summary of Literature Review: MSCs are known to be multipotent in tissue regeneration. In degeneration of IVD, cellular replacement with genetic modification other than that of IVD cells may prove an enhanced mechanism for the regeneration of IVD cells. Materials and Methods: MSCs and IVD cells were cultured and an adenovirus construct containing TGF-β1 cDNA (Ad/TGF-β 1) was also produced. In the first step, the MSCs were transduced with Ad/TGF-β1, then mixed with IVD cells in various proportions and three dimensionally cultured. [methyl-H]Thymidine and [ S]Sulfur incorporation for DNA and proteoglycan synthesis, respectively, were measured. RT-PCR was performed to assess the aggrecan and collagen types I and II mRNA expressions Results: Mixed cultures of MSC and IVD cells showed relatively similar amounts of newly synthesized proteoglycan compared with cultures of IVD cells only. In mixed cultures transduced with Ad/TGF-β1, there were significant decreases in newly synthesized proteoglycan with increasing the proportions of MSCs, which was also found with the aggrecan and collagen type II mRNA expressions. However, the collagen type I mRNA expression increased with increased proportions of MSCs transduced with Ad/TGF-β1. Conclusion: Cell therapy with MSCs and IVD cells provided a mechanism for cellular augmentation. However, MSC-based gene therapy coupled with IVD cells did not maintain a chondrogenic phenotype.

The rate constants of the derivatives of N-(2,4-dinitrophenyl)-benzimidoyl chloride were determined at various pH and a rate equation which can be applied over wide pH range was obtained. The reaction mechanism of hydrolysis of N-(2,4-dinitrophenyl)-benzimidoyl chloride which has not been studied carefully earlier in acidic and basic solution can be fullly explained by the rate equation obtained. The rate equation reveals that, beow pH 7.00, the hydrolysis of benzimidoyl chloride proceeds through reaction to form a carbonium ion intermediate.Above pH 8.5, however, the hydrolysis proceeds through the type reaction which depends on hydroxide ion and imidoyl chloride concentration. At pH 7.0∼8.5, two reactions occur competitively