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

描述（由申请人提供）：组织工程方法已被探索为韧带重建手术创造移植物的手段，而不会造成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骨-韧带-骨移植物，在体内完全复制插入处的组织结构。我们认为细胞外基质（ECM）对齐和控制ECM异质性是这种自体植入物长期成功的关键因素。然而，利用患者来源的骨髓基质细胞（MSCs）产生具有这些特征的移植物，由于缺乏对韧带-骨界面附近发生的直接排列和分化的清楚了解而受到阻碍。因此，作为我们实现长期目标的第一步，本应用程序的目的是使用一种独特的系统，结合一种新颖的分层，酶敏感的水凝胶载体和宏观负载参数的精确控制，以确定细胞外环境的两个重要特征，环境的物理化学性质以及与成骨细胞的共培养，如何影响MSCs的排列和表型表达。该建议的中心假设是，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)微环境的物理化学特征和2)附近成骨细胞的存在以可预测的方式调节。我们的总体目标将通过在以下两个特定目标中测试我们的中心假设来实现：1)确定水凝胶微环境的生化特性（粘附配体浓度）和物理特性（聚合物网络的酶降解）对细胞排列和成纤维细胞表型表达程度的影响，将包封的兔间充质干细胞暴露在循环拉伸载荷下超过21天。2)测定成骨细胞的存在对包封兔间充质干细胞在21天的循环拉伸载荷下向成纤维细胞/成纤维软骨细胞分化的时间和程度的影响。这项工作是创新的，因为结合了一种新的、明确的三维细胞环境，包括允许间充质干细胞和成骨细胞共同培养的层状结构，以及宏观机械负荷的精确控制，为骨-韧带界面附近对间充质干细胞分化的影响的可控研究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间充质干细胞成纤维分化的影响。这些关键信息将指导未来患者特异性组织工程移植物生产策略的设计，以替代受损的韧带并恢复关节的全部功能。

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.