Collagen Assembly in Tissue-Engineered Cartilage

描述（由申请人提供）：软骨作为减震器的能力取决于构成软骨的胶原纤维网络的质量。该网络由三种不同类型的胶原蛋白（II、IX和Xi型）组装并精确交联在一起形成杂聚物组成。 尽管在软骨组织工程领域取得了许多进展，但持续的挑战是将生物工程软骨的胶原蛋白含量增加到在天然关节软骨中观察到的水平。由于胶原杂聚物是成熟纤维结构的关键模板，并且对于成熟组织的持续稳定性，我们假设II/IX/Xi型胶原杂原纤维的交联模板必须在新形成的软骨基质中顺序组装，以允许II型胶原原纤维生长，增加胶原含量，从而实现功能性关节软骨的生物力学性质。次要胶原模板（IX型和Xi型）与II型胶原的正确交联组装体是否在新软骨中形成还没有很好地表征。 我们的目标是确定透明软骨典型的胶原蛋白网络是否组装在基于软骨细胞和基于间充质干细胞的生物工程组织中。由于次要胶原蛋白调节网络的组织，（例如，由Xi/V型胶原调节的胶原原纤维直径），对胶原类型间交联的模式进行指纹识别可以提供正常基质组装的筛选和理解经历软骨形成的成体间充质干细胞中II/IX/Xi型胶原原纤维的原纤维形成中的事件顺序的有价值的工具。目的是使用蛋白质印迹、ELISA、LC-质谱和蛋白质化学中的其他先进方法来建立胶原原纤维组装的分子指纹和一组参数，以用作生物标志物，从而更好地评估正常和工程化软骨中的交联胶原网络。 最终目标是验证蛋白质组学方法，以用作组织工程软骨的结果测量，该组织工程软骨具有移植到软骨缺损中所需的关节软骨的胶原蛋白含量、质量和结构特性。这些目标还包括为评估作为愈合反应产生的软骨质量变化奠定基础。 公共卫生相关性：软骨作为减震器的能力取决于构成软骨的胶原异原纤维网络的质量。在关节炎中，这种胶原蛋白网络被破坏，导致正常关节功能丧失和严重疼痛。本研究的目的是开发和鉴定一种生物标志物，以监测生物工程软骨中组装的胶原异原纤维网络是否是正常软骨的典型特征。

DESCRIPTION (provided by applicant): The ability of cartilage to act as a shock absorber depends on the quality of the collagen fibrillar network that frames cartilage. This network is made up of three different types of collagen (types II, IX and XI) assembled and precisely cross-linked together into heteropolymers. Despite many advances in the field of cartilage tissue engineering, a continuous challenge has been to increase the collagen content of bioengineered cartilage to levels observed in native articular cartilage. Since the collagen heteropolymer is a crucial template of the mature fibrillar architecture and for the ongoing stability of the mature tissue, we hypothesize that a cross-linked template of type II/IX/XI collagen heterofibrils must sequentially assemble in the matrix of newly forming cartilage to allow type II collagen fibril growth, an increased collagen content and so achieve the biomechanical properties of functional articular cartilage. Whether correct cross-linked assemblies of the minor collagen template (types IX and XI) with type II collagen form in neo-cartilage, is not well characterized. Our goal is to determine if a collagen network typical of hyaline cartilage is assembled in chondrocyte based and mesenchymal stem cell based bio-engineered tissues. Since the minor collagens regulate the organization of the network, (e.g., collagen fibril diameter modulated by type XI/V collagen), fingerprinting the pattern of collagen inter-type cross-linking can provide a screen for normal matrix assembly and a valuable tool in understanding the sequence of events in the fibrillogenesis of the type II/IX/XI collagen fibril in adult mesenchymal stem cells undergoing chondrogenesis. The aim is to use Western blot, ELISA, LC-mass spectrometry, and other advanced methods in protein chemistry to establish a molecular fingerprint of the collagen fibril assembly and a set of parameters to use as a biomarker to better evaluate the cross-linked collagen network in normal and engineered cartilage. The ultimate goal is to validate proteomic methods to use as an outcome measure of tissue engineered cartilage that has the collagen content, quality and structural properties of articular cartilage needed to transplant into cartilage defects. The goals also include laying the foundation for evaluating the changing quality of cartilage produced as a healing response. PUBLIC HEALTH RELEVANCE: The ability of cartilage to act as a shock absorber depends on the quality of the collagen heterofibrillar network that frames cartilage. In arthritis, this collagen network is broken down leading to a loss of normal joint function and severe pain. The goal of this study is to develop and authenticate a biomarker to monitor if this collagen heterofibrillar network assembled in bioengineered cartilage is typical of normal cartilage.