【作者】 房维；

【导师】 龙星；

【作者基本信息】 武汉大学 ， 口腔颌面外科学， 2010， 博士

【摘要】 颞下颌关节紊乱病(temporomandibular disorders, TMDs)是临床常见病和多发病,极易发生颞下颌关节(temporomandibular joint, TMJ)髁突和关节盘的退行性改建。利用组织工程(tissue engineering)方法再生修复病变的TMJ髁突和关节盘为治疗TMDs提供了新的手段。虽然已经有了很多进展,但是距离应用组织工程化TMJ髁突和关节盘还有很远的距离。目前很多研究表明软骨调节素-1(chondromodulin-1, ChM-1)在透明软骨发生和维持软骨正常生理功能中有重意义,但是在TMJ中以及在TMJ髁突和关节盘组织工程中的表达以及意义还不清楚,因此本实验首先收集TMJ髁突以及关节盘标本,检测ChM-1在TMJ髁突和关节盘的表达和分布,然后通过体外细胞实验,分析关节软骨细胞(articular chondrocyte, AC),骨髓间充质干细胞(bone marrow-derived mesenchymal stem cell, BMSC),以及这两种细胞混合后软骨化过程中ChM-1表达的差异以及相关软骨化产物移植到体内后的不同变化,明确ChM-1在软骨化中的作用,为组织工程化TMJ髁突以及关节盘的具体应用打下基础。本研究包括以下五个部分。实验一软骨调节素-1在颞下颌关节髁突软骨和关节盘中的表达目的：检测ChM-1在TMJ髁突和关节盘软骨中的表达,探讨ChM-1在TMJ中的生理作用。方法：应用番红-O染色,免疫组化染色检测8侧日本大白兔TMJ髁突和关节盘中ChM-1的表达,同时以Lewis大鼠胫骨关节头做为阳性对照。结果：番红-O染色结果发现TMJ髁突和关节盘中均有阳性染色,但是染色强度不如胫骨关节软骨和生长板软骨。免疫组化结果显示TMJ髁突和关节盘中均有ChM-1阳性染色。结论：本实验发现ChM-1在TMJ髁突和关节盘中有表达,可能在TMJ改建中发挥作用。实验二软骨调节素-1在颞下颌穿孔关节盘中的表达目的：利用人TMJ关节盘穿孔标本,通过免疫组织化学方法,明确血管内皮细胞生长因子(vascular endothelial growth factor, VEGF)和ChM-1在穿孔TMJ关节盘中的表达情况。分析ChM-1在TMJ关节盘穿孔改变中的意义。方法：通过HE染色和免疫组化染色方法,检测3例人TMJ关节盘穿孔标本中ChM-1和VEGF的表达。结果：在所有检测标本中均发现ChM-1和VEGF的表达。ChM-1和VEGF的表达位于TMJ关节盘软骨样细胞胞浆,且两种因子的表达模式比较类似。结论：ChM-1可能在TMJ关节盘穿孔的修复中起到调节作用,阻止新生血管进入TMJ关节盘进行组织修复。实验三软骨调节素-1在细胞软骨化中的表达目的：比较AC,BMSC和两种细胞等比例混合后体外软骨化过程中ChM-1的表达差异。分析不同实验组ChM-1表达差异对体内移植后的影响。方法：将AC,BMSCs和两种细胞等比例混合后进行体外三维培养,形成细胞团。经过3周软骨化诱导后,通过HE染色,番红-O染色,免疫组化染色以及实时定量PCR分析各组细胞团软骨化水平和ChM-1的表达差异。结果：经过3周软骨化诱导后,各实验组细胞团均显示明显软骨分化,通过Bern score分析,AC组和混合细胞组细胞团软骨化水平高于BMSCs,同时免疫组化染色和实时定量PCR结果发现AC组和混合细胞细胞团ChM-1表达高于BMSC组细胞团。结论：ChM-1的表达差异可能影响软骨分化后体内移植的转归。实验四软骨缺损模型中软骨化体外实验研究目的：检测软骨分化后的AC,BMSC和等比例混合细胞的细胞团在体外软骨缺损模型中的生物学改变。方法：取正常关节软骨标本,建立软骨缺损模型,将经3周软骨分化后的AC,BMSCs和等比例混合细胞的细胞团置入该模型,继续在体外软骨化培养3周,经过HE染色、番红-O染色和Ⅱ型胶原免疫组化染色分析不同实验组细胞团的软骨化水平。结果：经过3周在软骨缺损中的软骨分化培养,HE染色发现各实验组细胞团与软骨缺损的软骨组织整合良好,番红-O染色显示各实验组细胞团均有阳性染色,Ⅱ型胶原免疫组化染色也显示各实验组细胞团均有阳性染色。结论：在该软骨缺损模型中,各实验组可以维持软骨分化。实验五软骨缺损模型体内软骨化的实验研究目的：将经过3周软骨化培养的AC组,BMSC组和混合细胞组细胞团置入软骨缺损模型并移植入严重免疫缺陷小鼠背部皮下,分析ChM-1的表达差异对该移植物体内软骨化的影响。方法：取正常软骨标本构建软骨缺损模型,置入软骨化培养3周的细胞团,用Matrixgel固定细胞团。将移植物移植到严重免疫缺陷小鼠背部皮下3周,通过HE染色,番红-O染色以及免疫组化染色,分析各实验组细胞团的软骨分化水平和ChM-1的表达。结果：经过3周体内移植,AC组和混合细胞组细胞团保持了软骨分化,没有血管进入和钙化的发生,BMSCs组细胞团完全丧失软骨分化,有大量血管进入。番红-O染色显示AC组和混合细胞组细胞团有阳性染色,而BMSC组细胞团则没有染色。ChM-1在AC组和混合细胞组细胞团有表达,在BMSC组细胞团没有表达。结论：软骨分化后的AC组和混合细胞组细胞团在体内移植后可以维持软骨化,ChM-1在其中起到重要作用。更多还原

【Abstract】 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.更多还原