High-Throughput Microenvironment Regulation for Chondrogenesis

 描述（由申请人提供）：肌肉骨骼组织损伤仍然是骨科研究中的一个重大挑战。例如，目前，数百万患者遭受软骨损伤，相关的年度财务成本超过1000亿美元。有几种可行的临床选择来解决这些损伤。在这种情况下，人类间充质干细胞（hMSC）是软骨组织工程的一个有前途的细胞来源，因为它们能够分化软骨形成途径，可以从骨髓中以微创方式获得，并且容易在培养中生长。尽管hMSCs的分化受可溶性分子、不溶性生化信号和机械信号的调节，但机械载荷和生物材料信号的组合效应在很大程度上是未知的。我们的中心假设是，在机械刺激下使用高通量系统（HTSs）可用于阐明单一和协同的微环境因素，用于指导hMSCs的软骨分化，从而在体外和临床相关的体内模型中形成功能性工程软骨。从HTS获得的信息将有助于开发具有增强的软骨形成的宏观结构，并且这些结构的性能将通过治疗临界尺寸的关节软骨缺损在体内进行验证。这些目标将通过实现以下具体目标来实现：（1）开发三维（3D）组合的基于HTS水凝胶的微阵列，其由不同的细胞外基质分子和生长因子组成，其可以机械变形以模拟hMSC的软骨形成微环境，（2）定量评价三维组合HTS微阵列和从这些微阵列中选择的大规模构建体中hMSC的软骨形成分化反应，和（3）确定具有HTS鉴定的组合物和机械刺激方案的载有hMSC的构建体在体内诱导软骨再生的潜力。骨科社区将受益于更好地了解这些软骨诱导微环境，最终诱导新组织形成，并将代表当前临床治疗的可行替代方案。虽然这项研究的最终目标是设计临床相关的关节软骨疗法，但这种HTS也适用于测试 其他组织的再生策略。

 DESCRIPTION (provided by applicant): Musculoskeletal tissue injuries remain a significant challenge in orthopaedics research. For example, currently, millions of patients are suffering from cartilage injuries, with associated annual financial costs of more than $100 billion dollars. There are several viable clinical options to address these injuries. In this context, human mesenchymal stem cells (hMSCs) are a promising cell source for cartilage tissue engineering as they are capable of differentiating down the chondrogenic pathway, can be obtained from bone marrow in a minimally invasive manner, and are easily grown in culture. Although differentiation of hMSCs is regulated by soluble molecules, insoluble biochemical signals and mechanical cues, the combinatorial effects of mechanical loading and biomaterial signals are largely unknown. Our central hypothesis is that the use of high-throughput systems (HTSs) under mechanical stimulation can be used to elucidate single and synergistic microenvironmental factors for directing the chondrogenic differentiation of hMSCs, and thereby functional engineered cartilage formation in vitro and in a clinically relevant in vivo model. The information obtained from the HTS will help in the development of macroscale constructs with enhanced chondrogenesis, and the performance of these constructs will be validated in vivo by treating critical-sized articular cartilage defects. These goals will be accomplished by achieving the following specific Aims: (1) to develop three dimensional (3D) combinatorial HTS hydrogel-based microarrays, consisting of different extracellular matrix molecules and growth factors, which can be mechanically deformed to mimic the chondrogenic microenvironment of hMSCs, (2) to evaluate quantitatively the chondrogenic differentiation response of hMSCs in 3D combinatorial HTS microarrays and macroscale constructs selected from these microarrays, and (3) to determine the potential of hMSC-laden constructs with HTS-identified compositions and mechanical stimulation regimes to induce cartilage regeneration in vivo. The orthopedic community would benefit from a better understanding of these chondroinductive microenvironments that will ultimately induce neo-tissue formation and will represent a viable alternative to current clinical therapies. While the ultimate objective of this research is to engineer clinically relevant articular cartilage therapies, this HTS can also be applicable to test regeneration strategies for other tissues.