Co-Culture & Cyclic Tension to Direct Differentiation at Bone-Ligament Interface

描述(由申请人提供)：组织工程学方法已经被探索为一种方法，以创建韧带重建过程中的移植物，而不会潜在的供体部位并发症。我们的长期目标是创造一种组织工程化的骨-韧带-骨移植，完全复制体内植入时的组织结构。我们认为，细胞外基质(ECM)的排列和受控的ECM异质性都是这种自体植入物长期成功的关键因素。然而，由于缺乏对韧带-骨界面附近发生的事情的清楚了解，患者来源的骨髓基质细胞(MSCs)用于产生具有这些特征的移植物是受到阻碍的，这些细胞的直接排列和分化。因此，作为迈向我们长期目标的第一步，这项应用的目标是使用一种独特的系统，结合新型分层的酶敏感水凝胶载体和精确控制宏观负载参数来确定细胞外环境的两个重要特征，即环境的物理化学性质和与成骨细胞的共培养，如何影响MSCs的排列和表型表达。这一建议的中心假设是，在循环张力下，MSCs的成纤维细胞表型的表达可以通过改变1)微环境的理化特征和2)附近成骨细胞的存在而以一种可预测的方式调节。我们的总体目标将通过在以下两个具体目标中检验我们的中心假设来实现：1)确定水凝胶微环境的生化属性(粘附性配体浓度)和物理属性(聚合物网络的酶降解)对细胞排列的影响以及包裹的兔MSCs在21天的循环拉伸载荷下的成纤维细胞表型表达的程度。2)检测成骨细胞对兔MSCs在21d以上的循环拉伸载荷下向成纤维细胞/纤维软骨细胞分化的时间和程度的影响。这项工作具有创新性，因为新的、明确的三维细胞环境，包括允许MSCs和成骨细胞共培养的层状结构，以及对宏观机械载荷的精确控制，为在骨-韧带界面附近控制对MSC分化的影响提供了一个独特的平台。这些研究的完成有望区分微环境的物理化学性质以及与邻近细胞的相互作用对MSCs成纤维细胞分化的影响。这些关键信息将指导未来生产针对患者的组织工程化移植物的策略，以取代受损的韧带并恢复完整的关节功能。 公共卫生相关性：这项建议研究了1)微环境的化学性质和2)与邻近细胞相互作用对骨髓基质细胞成纤维细胞分化的影响，以创造一种组织工程化的骨-韧带-骨移植，再现体内韧带-骨植入时的组织结构。

DESCRIPTION (provided by applicant): Tissue engineering approaches have been explored as a means to create grafts for ligament reconstruction procedures without potential donor-site morbidity. Our long-term goal is the creation of a tissue-engineered bone-ligament-bone graft that fully reproduces the tissue architecture found at the insertions in vivo. We believe that both extracellular matrix (ECM) alignment and controlled ECM heterogeneity are crucial elements to the long-term success of such an autologous implant. However, use of patient-derived marrow stromal cells (MSCs) to produce grafts with these characteristics is hampered by a lack of clear understanding of what occurs near the ligament-bone interface to direct alignment and differentiation of these cells. Therefore, as a first step toward our long-term goal, the objective of this application is to use a unique system combining a novel layered, enzyme-sensitive hydrogel carrier and precise control of macroscopic loading parameters to determine how two important characteristics of the extracellular environment, physicochemical properties of the surroundings and co-culture with osteoblasts, influence alignment and phenotypic expression by MSCs. The central hypothesis of this proposal is that, under cyclic tension, expression of the fibroblastic phenotype by MSCs can be modulated in a predictable manner by altering 1) physicochemical characteristics of the microenvironment and 2) the presence of nearby osteoblasts. Our overall objective will be accomplished by testing our central hypothesis in the following two specific aims: 1) Determine the effect of biochemical properties (adhesive ligand concentration) and physical properties (enzymatic degradation of the polymeric network) of the hydrogel microenvironment on cellular alignment and the extent of fibroblast phenotypic expression by encapsulated rabbit MSCs exposed to cyclic tensile loading over 21 days. 2) Determine the effect of the presence of osteoblasts on the timing and extent of fibroblastic/fibrochondrocytic differentiation by encapsulated rabbit MSCs under cyclic tensile loading over 21 days. The proposed work is innovative because the combination of a novel, well-defined three- dimensional cellular environment, including the laminated structures allowing for the co-culture of MSCs and osteoblasts, and the precise control of macroscopic mechanical loading provide a unique platform for controlled study of the influences on MSC differentiation near the bone-ligament interface. Completion of these studies is expected to distinguish the effects of the physicochemical properties of the microenvironment and the interplay with neighboring cells on the fibroblastic differentiation of MSCs. Such key information will direct the design of future strategies for production of patient-specific tissue-engineered grafts to replace damaged ligaments and restore full joint function. PUBLIC HEALTH RELEVANCE: This proposal examines the effects of 1) chemical properties of the microenvironment and 2) interplay with neighboring cells on fibroblastic differentiation of marrow stromal cells in order to create a tissue-engineered bone-ligament-bone graft that reproduces the tissue architecture found at ligament-bone insertions in vivo.