Roles of Type V Collagen in the Structure and Biomechanics of TMJ Condylar Cartilage

项目摘要 颞下颌关节紊乱病的特征是下颌骨的不可逆性破坏 髁突软骨细胞外基质（ECM）。髁突软骨ECM具有特定的双层布局， 纤维软骨层覆盖第二透明软骨层。有效TMJ的开发 再生策略受到挑战，因为我们对ECM中的某些分子如何 与这种独特的纤维-透明混合组织的结构和生物力学功能有关。这个项目 将研究I型胶原纤维形成的调节因子V型胶原在髁突发育中的作用， 软骨细胞外基质。总的目的是确定胶原V在调节细胞凋亡中的作用。 颞下颌关节髁状突软骨细胞外基质在生后发育中建立我们的核心假设是， 胶原V对TMJ髁突软骨的适当生物力学功能至关重要，因为它调节关节软骨的生物力学功能。 纤维层和透明层的胶原纤维结构。 为了检验中心假设，在目标1中，我们将确定胶原V损失对形成的影响。 和髁突软骨的成熟。这将通过研究结构和生物力学来实现 髁突软骨细胞外基质的表型在整体胶原V-敲低小鼠，并在我们新建立的， 正常TMJ负荷下的软骨特异性胶原V诱导型敲除（cKO）小鼠。其次，由于 胶原蛋白V对纤维层的影响是众所周知的，在目的2中，我们将确定胶原蛋白V在纤维层中的作用。 调节透明软骨层的形成。首先，我们将测试胶原蛋白V是否影响透明层 通过改变纤维层和透明层之间的载荷传递。这将通过研究来实现， cKO小鼠中透明层的表型在体内减少的TMJ负荷下减轻。二是 测试胶原V是否调节纤维层中祖细胞的软骨形成，以及结果 生物合成和组装的透明软骨新基质在没有机械负荷。一些 将采用创新办法。使用cKO小鼠，我们将描绘胶原V活性在每个阶段， TMJ生长，并最大限度地减少与其他TMJ组织的脱靶变化相关的混杂效应。应用 原子力显微镜（AFM）-纳米力学测试，我们将量化小鼠的力学变化 髁突软骨由于胶原V缺乏。激光捕获显微切割在微流控技术中的应用 qPCR，我们将描绘纤维层与透明层中细胞的基因表达谱。成功 这项研究的完成将阐明控制髁突形成的新的分子活动， 软骨ECM，这是必要的发展组织工程和疾病干预策略， 靶向高度特化的纤维透明混合组织

PROJECT SUMMARY Temporomandibular joint (TMJ) disorder is characterized by the irreversible breakdown of the mandibular condylar cartilage extracellular matrix (ECM). The condylar cartilage ECM has a specialized bilayer layout of a fibrocartilage layer covering a secondary hyaline cartilage layer. The development of effective TMJ regeneration strategies is challenged by our incomplete understanding of how certain molecules in the ECM are linked to the structure and biomechanical functions of this unique fibrous-hyaline hybrid tissue. This project will study the roles of collagen V, the regulator of collagen I fibrillogenesis, in the development of condylar cartilage ECM in vivo. The overall objective is to determine the roles of collagen V in regulating the establishment of TMJ condylar cartilage ECM during post-natal development. Our central hypothesis is that collagen V is crucial for proper biomechanical function of the TMJ condylar cartilage, because it regulates the collagen fibril structure of both the fibrous and hyaline layers. To test the central hypothesis, in Aim 1, we will determine the impact of collagen V loss on the formation and maturation of condylar cartilage. This will be achieved by studying the structural and biomechanical phenotype of condylar cartilage ECM in global collagen V-knockdown mice, and in our newly established, cartilage-specific collagen V inducible knockout (cKO) mice under normal TMJ loading. Next, since the influence of collagen V on the fibrous layer is well known, in Aim 2, we will determine the role of collagen V in regulating the formation of the hyaline cartilage layer. First, we will test if collagen V impacts the hyaline layer by altering the load transfer between the fibrous and hyaline layers. This will be achieved by studying if the phenotype of the hyaline layer in cKO mice is mitigated under reduced TMJ loading in vivo. Second, we will test if collagen V regulates the chondrogenesis of progenitor cells in the fibrous layer, as well as the resulted biosynthesis and assembly of hyaline cartilage neo-matrix in the absence of mechanical loading. A number of innovative approaches will be utilized. Using cKO mice, we will delineate collagen V activities at each stage of TMJ growth, and minimize confounding effects related to off-target changes of other TMJ tissues. Applying atomic force microscopy (AFM)-nanomechanical tests, we will quantify the mechanical changes of murine condylar cartilage as a result of collagen V deficiency. Applying laser capture microdissection with microfluidic qPCR, we will delineate gene expression profiles of cells in the fibrous versus hyaline layers. Successful completion of this study will elucidate new molecular activities that govern the formation of the condylar cartilage ECM, which is necessary for developing tissue engineering and disease intervention strategies to target this highly specialized, fibrous-hyaline hybrid tissue.