Regulating Fibrocartilage Stem Cells via TNF-α/Nf-κB in TMJ Osteoarthritis.

In this study, we investigate harnessing fibrocartilage stem cell (FCSC) capacities by regulating tumor necrosis factor α (TNF-α) signaling for cartilage repair in temporomandibular joint osteoarthritis (TMJOA). Stem cell specifics for FCSCs were characterized in the presence of TNF-α. Etanercept as a TNF-α inhibitor and BAY 11-7082 as an Nf-κB inhibitor were used to study TNF-α regulation of FCSCs. Lineage tracing was performed in Gli1-CreERT+;Tmfl/fl mice when etanercept (1 mg/kg, every 3 d) or isometric vehicle was subcutaneously injected to trace specific changes in FCSCs. Surgically induced TMJOA Sprague-Dawley rats were generated with BAY 11-7082 (5 mg/kg, every 3 d) or vehicle subcutaneous injection to investigate the functional role of TNF-α/Nf-κB in TMJOA. Anterior disc displacement (ADD) rabbits were used to analyze the therapeutic effect of etanercept as a TMJOA intra-articular treatment with etanercept (0.02 mg in 100 µL, every 2 wk) or isometric vehicle. In vitro, TNF-α inhibited proliferation of FCSCs and increased FCSC apoptosis. TNF-α activation interfered with osteogenic and chondrogenic differentiation of FCSCs, while etanercept could partially recover FCSC specificity from TNF-α. FCSC lineage tracing in Gli1-CreERT+;Tmfl/fl mice showed that the chondrogenic capacity of Gli1+ cell lineage was markedly suppressed in osteoarthritis cartilage, the phenotype of which could be significantly rescued by etanercept. Specifically blocking the Nf-κB pathway could significantly weaken the regulatory effect of TNF-α on FCSC specificity in vitro and in TMJOA rats in vivo. Finally, intra-articular etanercept treatment efficiently rescued TMJ cartilage degeneration and growth retardation in ADD rabbits. Inhibition of TNF-α signaling reduced Nf-κB transcripts and recovered FCSC specificities. In vivo, etanercept treatment effectively rescued the osteoarthritis phenotype in TMJOA mice and ADD rabbits. These data suggest a novel therapeutic mechanism whereby TNF-α/Nf-κB inhibition promotes FCSC chondrogenic capacity for cartilage transformation in TMJOA.

Engineering self-assembled neomenisci through combination of matrix augmentation and directional remodeling.

Knee meniscus injury is frequent, resulting in over 1 million surgeries annually in the United States and Europe. Because of the near-avascularity of this fibrocartilaginous tissue and its intrinsic lack of healing, tissue engineering has been proposed as a solution for meniscus repair and replacement. This study describes an approach employing bioactive stimuli to enhance both extracellular matrix content and organization of neomenisci toward augmenting their mechanical properties. Self-assembled fibrocartilages were treated with TGF-ß1, chondroitinase ABC, and lysyl oxidase-like 2 (collectively termed TCL) in addition to lysophosphatidic acid (LPA). TCL + LPA treatment synergistically improved circumferential tensile stiffness and strength, significantly enhanced collagen and pyridinoline crosslink content per dry weight, and achieved tensile anisotropy (circumferential/radial) values of neomenisci close to 4. This study utilizes a combination of bioactive stimuli for use in tissue engineering studies, providing a promising path toward deploying these neomenisci as functional repair and replacement tissues. STATEMENT OF SIGNIFICANCE: This study utilizes a scaffold-free approach, which strays from the tissue engineering paradigm of using scaffolds with cells and bioactive factors to engineer neotissue. While self-assembled neomenisci have attained compressive properties akin to native tissue, tensile properties still require improvement before being able to deploy engineered neomenisci as functional tissue repair or replacement options. In order to augment tensile properties, this study utilized bioactive factors known to augment matrix content in combination with a soluble factor that enhances matrix organization and anisotropy via cell traction forces. Using a bioactive factor to enhance matrix organization mitigates the need for bioreactors used to apply mechanical stimuli or scaffolds to induce proper fiber alignment.

Nanofiber-based transforming growth factor-ß3 release induces fibrochondrogenic differentiation of stem cells.

Fibrocartilage is typically found in regions subject to complex, multi-axial loads and plays a critical role in musculoskeletal function. Mesenchymal stem cell (MSC)-mediated fibrocartilage regeneration may be guided by administration of appropriate chemical and/or physical cues, such as by culturing cells on polymer nanofibers in the presence of the chondrogenic growth factor TGF-ß3. However, targeted delivery and maintenance of effective local factor concentrations remain challenges for implementation of growth factor-based regeneration strategies in clinical settings. Thus, the objective of this study was to develop and optimize the bioactivity of a biomimetic nanofiber scaffold system that enables localized delivery of TGF-ß3. To this end, we fabricated TGF-ß3-releasing nanofiber meshes that provide sustained growth factor delivery and demonstrated their potential for guiding synovium-derived stem cell (SDSC)-mediated fibrocartilage regeneration. TGF-ß3 delivery enhanced cell proliferation and synthesis of relevant fibrocartilaginous matrix in a dose-dependent manner. By designing a scaffold that eliminates the need for exogenous or systemic growth factor administration and demonstrating that fibrochondrogenesis requires a lower growth factor dose compared to previously reported, this study represents a critical step towards developing a clinical solution for regeneration of fibrocartilaginous tissues. STATEMENT OF SIGNIFICANCE: Fibrocartilage is a tissue that plays a critical role throughout the musculoskeletal system. However, due to its limited self-healing capacity, there is a significant unmet clinical need for more effective approaches for fibrocartilage regeneration. We have developed a nanofiber-based scaffold that provides both the biomimetic physical cues, as well as localized delivery of the chemical factors needed to guide stem cell-mediated fibrocartilage formation. Specifically, methods for fabricating TGF-ß3-releasing nanofibers were optimized, and scaffold-mediated TGF-ß3 delivery enhanced cell proliferation and synthesis of fibrocartilaginous matrix, demonstrating for the first time, the potential for nanofiber-based TGF-ß3 delivery to guide stem cell-mediated fibrocartilage regeneration. This nanoscale delivery platform represents an exciting new strategy for fibrocartilage regeneration.

In vitro effect of caveolin-1 as a slow-release material on bone-tendon junction healing: A comparative study.

Bone tendon junction injury is hard to cure because of its special anatomical structure, and the treatment applied for bone-tendon junction injury cannot result in the perfect vascular regeneration and restoration of the fibrocartilage zone. In this article, we aim to explore the effect of caveolin-1 as a slow-release material on bone-tendon junction healing. Seventy-two New Zealand rabbits were randomly selected and assigned into the experimental, sham-operated and control groups (n = 24). Caveolin-1 microspheres and microcapsule were developed as drug delivery system. At the 4th, 8th, and 12th weeks after surgery, quadriceps muscle patella-patellar tendon (QMPPT) was obtained from each rabbit to observe the tendon-to-bone tunnel healing, and X-ray examination, histological examination and biomechanical testing were applied for evaluating new bone formation. As the X-ray showed, caveolin-1 increased the new bone area at each time point. At the 4th and 8th weeks after surgery, the rabbit treated with caveolin-1 slow release material showed repair of fibrocartilage. According to the biomechanical results, the cross-sectional area, breaking load and ultimate tensile strength were increased along with time. At the same time point, caveolin-1 increased the ultimate tensile strength. Our study demonstrates that caveolin-1 as a slow-release material could accelerate bone-tendon junction healing by promoting the formation of the transition zone.

Kartogenin with PRP promotes the formation of fibrocartilage zone in the tendon-bone interface.

Treatment of tendon-bone junction injuries is a challenge because tendon-bone interface often heals poorly and the fibrocartilage zone, which reduces stress concentration, at the interface is not formed. In this study, we used a compound called kartogenin (KGN) with platelet-rich plasma (PRP) to induce the formation of fibrocartilage zone in a rat tendon graft-bone tunnel model. The experimental rats received KGN-PRP or PRP injections in the tendon graft-bone tunnel interface. The control group received saline. After 4, 8 and 12 weeks, Safranin O staining of the tendon graft-bone tunnels revealed abundant proteoglycans in the KGN-PRP group indicating the formation of cartilage-like transition zone. Immunohistochemical and immuno-fluorescence staining revealed collagen types I (Col-I) and II (Col-II) in the newly formed fibrocartilage zone. Both fibrocartilage zone formation and maturation were healing time dependent. In contrast, the PRP and saline control groups had no cartilage-like tissues and minimal Col-I and Col-II staining. Some gaps were also present in the saline control group. Finally, pull-out strength in the KGN-PRP-treated group at 8 weeks was 1.4-fold higher than the PRP-treated group and 1.6-fold higher than the saline control group. These findings indicate that KGN, with PRP as a carrier, promotes the formation of fibrocartilage zone between the tendon graft and bone interface. Thus, KGN-PRP may be used as a convenient cell-free therapy in clinics to promote fibrocartilage zone formation in rotator calf repair and anterior cruciate ligament reconstruction, thereby enhancing the mechanical strength of the tendon-bone interface and hence the clinical outcome of these procedures. Copyright © 2017 John Wiley & Sons, Ltd.

Combined use of chondroitinase-ABC, TGF-ß1, and collagen crosslinking agent lysyl oxidase to engineer functional neotissues for fibrocartilage repair.

Patients suffering from damaged or diseased fibrocartilages currently have no effective long-term treatment options. Despite their potential, engineered tissues suffer from inferior biomechanical integrity and an inability to integrate in vivo. The present study identifies a treatment regimen (including the biophysical agent chondroitinase-ABC, the biochemical agent TGF-ß1, and the collagen crosslinking agent lysyl oxidase) to prime highly cellularized, scaffold-free neofibrocartilage implants, effecting continued improvement in vivo. We show these agents drive in vitro neofibrocartilage matrix maturation toward synergistically enhanced Young's modulus and ultimate tensile strength values, which were increased 245% and 186%, respectively, over controls. Furthermore, an in vitro fibrocartilage defect model found this treatment regimen to significantly increase the integration tensile properties between treated neofibrocartilage and native tissue. Through translating this technology to an in vivo fibrocartilage defect model, our results indicate, for the first time, that a pre-treatment can prime neofibrocartilage for significantly enhanced integration potential in vivo, with interfacial tensile stiffness and strength increasing by 730% and 745%, respectively, compared to integration values achieved in vitro. Our results suggest that specifically targeting collagen assembly and organization is a powerful means to augment overall neotissue mechanics and integration potential toward improved clinical feasibility.

Limited therapeutic benefits of intra-articular cortisone injection for patients with femoro-acetabular impingement and labral tear.

PURPOSE: Intra-articular (IA) hip cortisone injection is commonly performed as a therapeutic modality in patients with femoral acetabular impingement (FAI). To our knowledge, there is no published data evaluating the clinical benefit of these injections. The purpose of this study was to assess the efficacy of therapeutic IA cortisone injection in these patients. METHODS: At our institution, patients with FAI and labral tear prospectively recorded their numerical rating scale (NRS) pain scores pre-injection, during post-injection anaesthetic phase, and at 14 days post-injection. From this cohort, all patients treated with guided IA cortisone injection, no radiographic evidence of arthritis (Tönnis grade 0 or 1) and pain relief during the anaesthetic phase of the IA injection were included. An absolute change of two points on the NRS score was considered the minimal amount of clinically significant pain relief. Pain scores were compared between the different types of steroid injected. RESULTS: Fifty-four patients (35 females, 19 males) with a mean age of 32 ± 12 years were included. Average median pre-injection NRS score was 7.0 (range 2.5-10.0), post-injection anaesthetic phase was 1.0 (range 0.0-5.0), and 14 day post-injection was 5.0 (range 0.0-10.0). As a group, NRS scores significantly diminished from post-injection anaesthetic phase to 14 days post-injection (p < 0.001). At 14 days post-injection, only 20 patients (37 %) and at 6 weeks, only 3 patients (6 %) reported a clinically significant decrease in pain. Average duration of pain relief was 9.8 days. There was no difference in pain reduction between steroid preparations. CONCLUSION: In patients with symptomatic FAI and labral tear, intra-articular cortisone injection has limited clinical benefit as a therapeutic modality. However, anaesthetic-only IA injections for patients who may be candidates for hip arthroscopy can be a useful diagnostic tool.

The effect of methotrexate on the bone healing of mandibular condylar process fracture: an experimental study in rats.

BACKGROUND: Methotrexate (MTX) is an anti-metabolite used in rheumatology and oncology. High doses are indicated for oncological treatment, whereas low doses are indicated for chronic inflammatory diseases. This study evaluated the effect of two MTX treatment schedules on the bone healing of the temporomandibular joint fracture in rats. METHODS: Seventy-five adult male Wistar rats were used to generate an experimental unilateral medially rotated condylar fracture model that allows an evaluation of bone healing and the articular structures. The animals were subdivided into three groups that each received one of the following treatments intraperitoneally: saline (1 mL/week), low-dose MTX (3 mg/kg/week) and high-dose MTX (30 mg/kg). The histological study comprised fracture site and temporomandibular joint evaluations and bone neoformation was evaluated by histomorphometric analysis. A biochemical parameter of bone formation was also assessed. RESULTS: When compared with saline, high-dose MTX delayed bone fracture repairs. In this latter group, after 90 days, the histological analysis revealed atrophy of the fibrocartilage and the presence of fibrous tissue in the joint space. The histomorphometric analysis revealed diminished bone neoformation. The alkaline phosphatase levels also decreased after MTX treatment. CONCLUSION: It was concluded that high-dose MTX impaired mandibular condyle repair and induced degenerative articular changes.

Effect of different doses of transforming growth factor-ß1 on cartilage and subchondral bone in osteoarthritic temporomandibular joints.

Transforming growth factor-beta (TGF-ß) plays an important part in the repair of cartilage in osteoarthritis. It has been hypothesised that intra-articular injection of TGF-ß1 promotes repair of cartilage and protects the subchondral bone from damage in osteoarthritic temporomandibular joints (TMJs). We made bilateral partial perforations of the disc to induce osteoarthritic joints in 36 rabbits. TGF-ß1 20, 40, or 80 ng were injected into the right joint, and vehicle alone was injected into the left joint. Four additional animals were used as normal controls. Microcomputed tomography was used to quantify the three-dimensional microarchitecture of subchondral bone, followed by assessment of the proteoglycan content. All joints treated with TGF-ß1 were covered by a layer of well-organised fibrocartilage, and had increased proteoglycan content and normal microarchitectural properties, whereas the joint treated by vehicle alone had typical osteoarthritis-related degradation of cartilage and sclerosis of subchondral bone. These results suggested that TGF-ß1 is an effective way of treating osteoarthritis of the TMJ.

In vitro ligament-bone interface regeneration using a trilineage coculture system on a hybrid silk scaffold.

The ligament-bone interface is a complex structure that comprises ligament, fibrocartilage, and bone. We hypothesize that mesenchymal stem cells cocultured in between ligament and bone cells, on a hybrid silk scaffold with sections suitable for each cell type, would differentiate into fibrocartilage. The section of scaffold for osteoblast seeding was coated with hydroxyapatite. A trilineage coculture system (osteoblasts-BMSCs-fibroblasts) on a hybrid silk scaffold was established. RT-PCR results and immunohistochemistry results demonstrated that BMSCs cocultured between fibroblasts and osteoblasts had differentiated into the fibrocartilaginous lineage. The morphological change was also observed by SEM observation. A gradual transition from the uncalcified to the calcified region was formed in the cocultured BMSCs from the region that directly interacted with fibroblasts to the region that directly interacted with osteoblasts. The role of transforming growth factor ß3 (TGF-ß3) in this trilineage coculture model was also investigated by supplementing the coculture system with 10 ng/mL TGF-ß3. The TGF-treated group showed similar results of fibrocartilaginous differentiation of BMSCs with coculture group without TGF-ß3 supplement. However, no calcium deposition was found in the cocultured BMSCs in the TGF-treated group. This may indicate TGF-ß3 delayed the mineralization process of chondrocytes.

Protection of articular cartilage by intra-articular injection of NEL-like molecule 1 in temporomandibular joint osteoarthritis.

Several studies have proven the ability of NEL-like molecule-1 (Nell-1) to induce chondrogenesis and make it as a potential candidate for articular cartilage repair. In the current study, the chondroprotective effect of Nell-1 on osteoarthritis (OA) of the temporomandibular joint (TMJ) was investigated by intra-articular injection. Bilateral partial discectomy was performed in 24 rabbits to induce TMJOA. Four weeks later, the right TMJ was treated with Nell-1 as the experimental group, whereas the left was treated with physiologic saline as the control group. Twelve rabbits each time were randomly killed at 12 and 24 weeks after injection, respectively. Histologic observation and metabolic analysis by reverse transcription-polymerase chain reaction were used for evaluation. All TMJs appeared as OA-like histologic changes after intra-articular injection. However, the degree of osteoarthritis in the experimental group was less severe than that in the control group during the experimental time. The expression of type II collagen and aggrecan messenger RNA was significantly higher than the control group at 12 weeks after injection. However, no difference in the expression of aggrecanase or interleukin 1 messenger RNA was observed. The results suggest that intra-articular injection of Nell-1 may be a good alternative for the treatment of cartilage degeneration in OA.

The effect of magnesium ion concentration on the fibrocartilage regeneration potential of goat costal chondrocytes.

Magnesium has recently been explored as a potential biomaterial for degradable orthopedic implants but its effect on fibrocartilage remains unknown. The objective of this study was to assess the effect of high concentrations of magnesium ions on the matrix production of goat costal fibrochondrocytes in vitro. Cells were cultured using a scaffoldless approach with media containing magnesium chloride (MgCl(2)) or magnesium sulfate (MgSO(4)) at concentrations of 20, 50, and 100 mM in addition to the baseline magnesium concentration of 0.8 mM MgSO(4). At 4 weeks, there were no significant differences in compressive tangent modulus and total matrix production between constructs cultured in 20 mM Mg(2+) and the 0.8 mM Mg(2+) control (435 ± 47 kPa). There was a significant decrease in compressive tangent modulus compared to the 0.8 mM Mg(2+) constructs in the 50 mM MgCl(2) and MgSO(4) groups, while the 100 mM groups were not mechanically testable (p < 0.05). The collagen and glycosaminoglycan (GAG) content of the 50 and 100 mM MgCl(2) and MgSO(4) constructs was significantly lower than the control (6.9 ± 0.5% and 16.5 ± 1.3% per dry weight, respectively) (p < 0.05). The results show that goat costal fibrochondrocytes exhibit a high degree of resiliency to magnesium ion concentrations up to 20 mM in vitro.

Preliminary evaluation of histological changes found in a mechanical arthropatic temporomandibular joint (TMJ) exposed to an intra-articular Hyaluronic acid (HA) injection, in a rat model.

OBJECTIVE: This study investigates the histological effects of Hyaluronic acid injections in the treatment of induced temporomandibular joint (TMJ) osteoarthritis in rats. STUDY DESIGN: Twenty-four male Wister rats were subjected to induced mechanical osteoarthritis by manual hypermobility for 10 successive days. Animals were then divided into two groups; group I (control) and group II (experimental). Ten days after the induction of hypermobility, the right TMJ of the experimental animals was injected with a dose of 0.12 mg HA intra-articularly and 0.12 mg saline was injected into the left joint; while animals in the control group were left without any treatment. Two rats from group I were killed at one, two and six weeks; while 6 animals from group II were killed at one, two and four weeks post injection. RESULTS: The disk of the right joints in the experimental animals was of normal thickness and there was an increase in the thickness of the fibrocartilagenous layer. In the left joint; ulcerative changes in the disk were evident where the fibres were not well oriented and scalloped areas in the temporal bone area were present denoting osteoclastic activity. CONCLUSIONS: Repeated intra-articular TMJ injection of Hyaluronic acid appears to be a safe and effective way of inhibiting the progression of osteoarthritic changes in the joint through development of articular cartilage and reducing fibrous tissue proliferation.

Enhanced regeneration of the ligament-bone interface using a poly(L-lactide-co-ε-caprolactone) scaffold with local delivery of cells/BMP-2 using a heparin-based hydrogel.

Recently, the ligament-bone (LTB) junction has been emphasized for the effective transmission of mechanical force and the reduction in stress concentration between the soft ligament and hard bone tissue. The aim of this study was to regenerate an integrated LTB interface by inoculating LTB-relevant cells, isolated from fibrocartilage (FC) or ligament (LIG), separately into each designated region in a single porous cylindrical PLCL scaffold. An injectable, heparin-based hydrogel that has proved to be effective in the culture of chondrocytes as well as the sustained release of growth factor was employed to locally deliver fibrochondrocytes and osteoinductive bone morphogenetic protein-2 (BMP-2) into the FC region, to promote FC regeneration. In in vitro experiments the hydrogel-combined FC systems produced significantly larger amounts of calcium and glycosaminoglycans (GAGs), but less collagen and DNA than FC samples without the hydrogel and all LIG samples. After in vivo subcutaneous implantation in mice for 8 weeks the secreted calcium and GAG contents of the hydrogel-containing FC samples were superior or similar to those of the in vitro hydrogel-containing FC samples at 6 weeks. As a result of the enhanced production of calcium and GAG, the in vivo hydrogel-containing FC samples produced the highest compressive modulus among all samples. Histological and immunofluorescence analysis as well as elemental analysis also confirmed a denser and more homogeneous distribution of calcium, GAG, osteocalcin and neovascularization marker in the in vitro/in vivo hydrogel-containing FC systems than those without hydrogel. These results also show the beneficial effects of BMP-2 added using the hydrogel. In summary, the use of a heparin-based hydrogel for the local delivery of fibrochondrocytes and BMP-2 could accelerate the maturation and differentiation of LTB-specific FC tissues, and it was also possible to recreate the unique stratification of calcified FC and LIG tissues in a single porous PLCL scaffold in terms of both biochemical and biomechanical properties.

Assessment of growth factor treatment on fibrochondrocyte and chondrocyte co-cultures for TMJ fibrocartilage engineering.

Treatments for patients suffering from severe temporomandibular joint (TMJ) dysfunction are limited, motivating the development of strategies for tissue regeneration. In this study, co-cultures of fibrochondrocytes (FCs) and articular chondrocytes (ACs) were seeded in agarose wells, and supplemented with growth factors, to engineer tissue with biomechanical properties and extracellular matrix composition similar to native TMJ fibrocartilage. In the first phase, growth factors were applied alone and in combination, in the presence or absence of serum, while in the second phase, the best overall treatment was applied at intermittent dosing. Continuous treatment of AC/FC co-cultures with TGF-ß1 in serum-free medium resulted in constructs with glycosaminoglycan/wet weight ratios (12.2%), instantaneous compressive moduli (790 kPa), relaxed compressive moduli (120 kPa) and Young's moduli (1.87 MPa) that overlap with native TMJ disc values. Among co-culture groups, TGF-ß1 treatment increased collagen deposition ∼20%, compressive stiffness ∼130% and Young's modulus ∼170% relative to controls without growth factor. Serum supplementation, though generally detrimental to functional properties, was identified as a powerful mediator of FC construct morphology. Finally, both intermittent and continuous TGF-ß1 treatment showed positive effects, though continuous treatment resulted in greater enhancement of construct functional properties. This work proposes a strategy for regeneration of TMJ fibrocartilage and its future application will be realized through translation of these findings to clinically viable cell sources.

Protocol on induction of TMJ articular disc degeneration in rats by utilization of botulinum toxin.

OBJECTIVE: To develop a model of experimental degeneration in the articular disc of the TMJ of rats through the use of botulinum toxin that can be used in future studies of degenerative diseases on fibrocartilage. METHODS: Aiming at the above-mentioned objective, 12 Lewis male rats were used and divided into two groups: CG, control group and DG, group of animals to which the botulinum toxin was administered (6 units/kg). The morphological analysis was carried out utilizing histological cuts stained with hematoxyline-eosine, toluidine blue and Picrosirius; the biochemical analysis was made by SDS-polyacrylamide gel electrophoresis. RESULTS: The DG showed peculiar characteristics regarding a degeneration joint disk, compatible with those described in literature as: reduction of cells number, general disorganization of cells direction and extracellular matrix, increase in glycosaminoglycans content and degradation of the tissue collagen. CONCLUSIONS: Based on the morphological and biochemical results, it was concluded that the proposed degeneration model showed to be satisfactory for futures studies of injuries and fibrocartilage regeneration processes.

Micromass culture of human anulus cells: morphology and extracellular matrix production.

STUDY DESIGN: Micromass culture was assessed as a cell culture microenvironment for anulus cells from the human intervertebral disc. OBJECTIVE: To determine whether the micromass culture technique might be useful for the culture of human anulus cells. SUMMARY OF BACKGROUND DATA: Culture of cells in micromass has been traditionally used as a method to culture chondrocytes in a three-dimensional (3D) microenvironment with specialized chondrocyte media which allows expression of the chondrocytic phenotype. Recently it has also been used for disc cell 3D culture. METHODS: Following approval of our human subjects Institutional Review Board, cells isolated from human anulus intervertebral disc tissue was cultured in micromass culture under control conditions or with addition of 5 ng/mL transforming growth factor-beta (TGF-beta). Cultures were grown for 7 days, and then analyzed for morphology with light microscopy, for extracellular matrix (ECM) production with transmission electron microscopy and quantitative measurement of total sulfated proteoglycan production. Immunohistochemistry was also performed to assess types I and II collagen, decorin, keratan sulfate, and chondroitin sulfate content of ECM. RESULTS: Human anulus cells form multilayered colonies when cultured with minimal media and 20% fetal bovine serum in the micromass methodology. Stimulation of ECM production occurs when 5 ng/mL TGF-beta was added to the micromass media. TGF-beta also significantly increased the production of sulfated proteoglycans (P = 0.026). Under both control and TGF-beta-supplementation, the resulting micromass formed by anulus cells is not as compact as the micromass which results when stem cells cultured in chondrogenic media. Ultrastructural studies showed the presence of apoptotic cells and the presence of peroxisomes within cells. Immunohistochemical studies on production of type I collagen, decorin and keratan sulfate showed that there was localized production of these ECM components in focal regions; chondroitin sulfate and type II collagen, however, showed a more uniform overall production by cells within the micromass. CONCLUSION: Human anulus cells were successfully cultured under micromass conditions in nonchondrogenic media and with TGF-beta supplementation which increased ECM production. The resulting anulus cell micromass, however, was not as rounded or compact as that which occurs with routine chondrocyte micromass or stem cells induced into chondrocyte differentiation. The presence of peroxisomes noted on ultrastructural studies may reflect cell stress or uneven distribution of nutrition within the micromass during the 7-day micromass culture period. Immunohistochemical studies showed nonuniform ECM gene expression and production within the micromass, suggesting variable gene expression patterns with this culture method.

Protection of articular cartilage from degeneration by injection of transforming growth factor-beta in temporomandibular joint osteoarthritis.

OBJECTIVE: This study aimed to examine the effect of injection of transforming growth factor beta (TGF-beta) on articular cartilage in osteoarthritic temporomandibular joint (TMJ). STUDY DESIGN: Disc perforation was performed bilaterally in 24 rabbits to induce osteoarthritis (OA). The right joint was injected with TGF-beta as experimental groups, and the left joint with physiologic saline as control. Animals were killed at different intervals. Histology and reverse transcriptase polymerase chain reaction was performed for comparison. RESULTS: All joints showed OA-like changes, but the degree in the experimental was significantly less severe than in the control. At 12 weeks a significantly greater expression of aggrecan and collagen type II was found in the experimental compared with the control joints. However, no difference in either anabolic or catabolic genes was found between the two groups at 24 weeks. CONCLUSIONS: Transforming growth factor beta may have a potential benefit in protecting articular cartilage during the development of TMJ OA.

Up-regulation in receptor for advanced glycation end-products in inflammatory circumstances in bovine coccygeal intervertebral disc specimens in vitro.

STUDY DESIGN: This study investigated whether or not the receptor for advanced glycation end-products (RAGE) was up-regulated in inflammatory circumstances and consequently associated with aggrecan content in nucleus pulposus in vitro. OBJECTIVE: To investigate the activation of AGEs-RAGE complex by the irritation of IL-1beta in bovine intervertebral disc (IVD). SUMMARY OF BACKGROUND DATA: Although we have demonstrated that the accumulation of AGEs contributed to disc degeneration in human, it may be that acceleration in the AGEs-RAGE complex might be more important, mediated by expression levels of RAGE that increase in inflammatory mediators including IL-1beta in some tissues. Therefore, we investigated, in this study, the correlation if any between IL-1beta and AGEs-RAGE complex in bovine IVD. METHODS: Samples of bovine coccygeal IVDs were harvested (n = 6). The presence of AGEs and RAGE were identified by immunohistochemistry. Real-time polymerase chain reaction (PCR) was used to quantify the messenger RNA levels of aggrecan after 6 days' stimulation of AGEs. Real-time PCR and immunofluorescein cytochemistry were performed to analyze the expression of RAGE after 2 days' stimulation of IL-1beta. The aggrecan expressions were evaluated by real-time PCR after 2 days' stimulation of combination of AGEs and/or IL-1beta. RESULTS: Immunohistochemical analysis revealed that AGEs and RAGE were localized within the bovine IVDs. AGEs significantly decreased the aggrecan expression in bovine IVD as in human IVD. The RAGE expression was significantly increased by 2 days' stimulation of IL-1beta. The aggrecan expression was decreased by stimulated AGEs and IL-1beta together, although not decreased by stimulated AGEs or IL-1beta separately. CONCLUSION: This is the first report to show the correlation between IL-1beta and AGEs-RAGE complex in IVD. Our results suggested that the increased RAGE expression in inflammatory circumstances and interaction with AGEs are risk factors in decreasing of aggrecan content in nucleus pulposus.

Matrix metalloproteinase induction by relaxin causes cartilage matrix degradation in target synovial joints.

Our long-term goal is to understand the mechanisms by which relaxin and estrogen potentially contribute to joint diseases, particularly those afflicting the fibrocartilaginous temporomandibular joint (TMJ). Previously, we showed that relaxin produces a dose-dependent induction of tissue-degrading enzymes of the matrix metalloproteinase (MMP) family, specifically MMP-1 (collagenase-1), MMP-3 (stromelysin-1), MMP-9 (92-kDa gelatinase), and MMP-13 (collagenase-3) in cell isolates and tissue explants from TMJ fibrocartilage. The induction of these MMPs is accompanied by loss of collagen and glycosaminoglycans (GAGs), which was blocked by a pan-MMP inhibitor. We also found the targeted in vivo loss of collagen and GAGs in TMJ discs of ovariectomized rabbits treated with beta-estradiol, relaxin, or both hormones together. Progesterone attenuated the induction of MMPs and matrix loss by relaxin and estrogen. The modulation of matrix composition in TMJ fibrocartilage by these hormones was similar to that observed in the pubic symphysis and differed from that of the knee meniscus. The two target tissues showing the greatest modulation of MMPs and matrix loss, namely, the TMJ disc and pubic symphysis, had similar expression profiles of the estrogen receptors alpha and beta, relaxin-1 receptor (RXFP1, LGR7), and insulin-like peptide 3 receptor (RXFP2, LGR8) and these profiles differed from those in cells from the knee meniscus. These findings suggest a novel model for targeted tissue turnover of cartilage of specific joints through hormone-mediated induction of select MMPs.