The Expression Of Chondromodulin-1 In Temporomandibular Joint Cartilage And During Chondrogenesis

The pathological change of temporomandibular disorders(TMDs) in nature is degeneration remodeling of temporomandibular joint(TMJ),which including condyle and disc.Tissue engineered TMJ condyle and disc is a potential alternative method to repair degeneratived tissues.A lot of studies found that chondromodulin-1(ChM-1) played an important role in cartilage development and keeping cartilage integrity.But the roles of ChM-1 in TMJ remodeling and tissue engineering are not clear.In this study,the expression of ChM-lin TMJ tissues was determined by immunohistochemistry.And then the expression of ChM-1 in chondrogenic pellets which derived from articular chondrocyte(AC), bone marrow-derived mesenchymal stem cell (BMSC) and the equal ratio cell mixture of articular chondrocytes and BMSCs were investigated.Finally the roles of ChM-1 in keeping chondrogenesis after implantation in vivo was to be clarified.The study included 5 parts as follows:Part 1 The expression of chondromodulin-1 in temporomandibular joint condyle and discObjective:The TMJ cartilage consists of condylar cartilage and disc and undergoes continuous remodeling throughout post-natal life. To maintain the integrity of the TMJ cartilage,anti-angiogenic factors play an important role during the remodeling process. In this study, we investigated the expression of the anti-angiogenic factor, ChM-1, in TMJ cartilage and evaluate its potential role in TMJ remodeling.Methods:Eight TMJ specimens were collected from six 4-month-old Japanese white rabbits. Safranin-O staining was performed to determine proteoglycan (GAG) content. ChM-1 expression in TMJ condylar cartilage and disc was determined by immunohistochemistry. Results:Safranin-O stained weakly in TMJ compared with tibial articular and epiphyseal cartilage. In TMJ, ChM-1 was expressed in the proliferative and hypertrophic zone of condylar cartilage and chondrocyte-like cells in the disc. No expression of ChM-1 was observed in osteoblasts and subchondral bone. Conlusions: ChM-1 may play a role in the regulation of TMJ remodeling by preventing blood vessel invasion of the cartilage, thereby maintaining condylar cartilage and disc integrity.Part 2 The expression of Chondromodulin-1 in the perforated disc of temporomandibular jontObjective:In this study, we investigated the expression of ChM-1 and vascular endothelial growth factor (VEGF) in the perforated disc tissues of human TMJ and evaluated its potential role in the pathological changesof disc perforation.Methods:Three human TMJ perforated disc tissue samples were collected and ChM-1 and VEGF distribution in perforated TMJ disc were investigated by immunohistochemistry.Results:ChM-1 and VEGF were both similarly expressed in chondrocyte-like cells of the perforated disc tissues.Conlusions: ChM-1 may play a role in the regulation of TMJ disc healing by preventing blood vessel invasion of the disc, thereby maintaining disc integrity.Part 3 The expression of Chondromodulin-1 during chongenesis in vitroObjective:The aim of this study was to clarify the expression of tissue engineered cartilagous constructs derived from AC,BMSC and mixture of these two cells Methods: ACs,BMSCs,and mixed cells were cultured in 3D pellet system. After 3 week chondrogenic induction, the pellets were collected for investigation. GAG contents were determined by Safranin-O staining.gene level and protein level expression of typeⅡcollagen,ChM-1,VEGF and endostatin were determined by real time quantative-PCR and immunohistochemistry staining respectively.Results:After 3 week chondrogenic induction,AC-and Mixed derived pellets revealed upregulation of ChM-1, typeⅡcollagen in gene level and more GAG deposition compared to BMSC derived pellets. Conclusions:The difference expression of ChM-1 might affect the chondrogenesis of experimetal pelles in vivo after implantation.Part 4 Chondrogenesis in cartilage defect model in vitroObjective:The aim of this study was to analyze the chondrogenesis of experimental pellets in cartilage defect model in vitro.Methods:After 3 week chondrogenic induction, the pellets were placed into cartilage defects and then cultured in chondrgenic medium for another 3 week. The constructs were collected and analyzed by H&E staining,Safranin-O staining, immunohistochemistry staining.Results:All constrcus showed stable chondrogenesis in cartilage defect model. Safranin-O and typeⅡcollagen stained intensively in all construts.Conclusions:In cartilage defect model,all chondrogenic constrcut could their chogerogenesis in vitro.Part 5 Chondrogenesis in cartilage defect model in vivoObjective:One challenge in cartilage tissue engineering is chondrogenesis of MSC is not stable in ectopic area and calcification or degradation was occurred followed by blood vessel invasion. While tissue engineered cartilage derived from AC showed stable existence in ectopic area. The aim of this study was to analyze the correlation between ChM-1 expression and chondrogenesis in cartilage defect model in vivo.Methods:After 3 week chondrogenic induction, the pellets derived from AC,BMSC and mixture of AC and BMSC were placed into a cartilage defect respctively and then explanted into SCID mouses subcutaneously. After 3 week explantation, the samples were harvested and analyzed by H&E staining,Safranin-O staining and immunohistochemistry staining.Results:AC derived and mixed derived constructs showed stable chondrogenesis with no signs of blood vessel invasion,while BMSC derived constructs lost chondrocyte phynotype and were invaded by blood vessels. The staining of type 2 collagen and safranin-o was more intensive in AC derived and mixed derived constructs than in BMSC derived constructs.ChM-1 and VEGF expression was upregulated in AC derived and mixed derived constructs compared to BMSC derived constructs.Conclusions:The results revealed that BMSC mixed with AC will form stable ectopic cartilage by upregulation of ChM-1.