Engineering Developmental Microenvironments: Cartilage Formation and Maturation

描述（由申请人提供）：关节软骨排列在关节表面并传递载荷产生的力。由于软骨自然愈合能力的限制，以及骨关节炎发病率的增加，对基于细胞的修复策略的需求日益增长。组织工程，特别是那些基于自体间充质干细胞（MSCs）的方法，正在发展成为一种促进软骨再生的临床相关技术。然而，形成的组织特性以及结构内表型的稳定性和异质细胞反应是目前限制该技术翻译的问题。我们基于间质干细胞的软骨修复的一般方法解决了早期快速软骨形成阶段和逐渐重塑（成熟）导致具有成人功能的组织之间观察到的差异。这种转变过程是由多种时间因素（化学的、机械的和可溶的）驱动的。我们正在进行的研究表明，基质和细胞密度、材料降解的时间、可溶性诱导因子的引入以及机械载荷（压缩和滑动接触）在指导软骨形成和成熟中的作用。在这里，我们从这些研究中引入了一个发育相关的信号，即在肢体芽发育过程中发现的通过n -钙粘蛋白的细胞-细胞相互作用，进入我们的工程水凝胶系统。在第一个目标中，MSCs将被包裹在HA水凝胶中，这些水凝胶用模拟N-cadherin细胞外结构域的肽修饰，除了肽对群体异质性和表型稳定性的影响外，还将研究肽密度对软骨发生和软骨成熟的影响。在第二个目标中，将通过引入从HA水凝胶中进行细胞介导的肽蛋白水解的连接物来研究肽的时间呈现。除了对基于软骨形成加速的机械负荷的反应性外，时间肽呈现对软骨形成、软骨成熟、种群异质性和表型稳定性的影响将在第一个Aim中进行评估。在第三个目标中，n -钙粘蛋白肽修饰的水凝胶，包括稳定和短暂的表现，将在临床相关的负重猪缺陷模型中进行研究，以评估这些相互作用在植入水凝胶中软骨缺陷修复中的作用。这些目标旨在测试我们的假设，即控制间充质干细胞微环境，包括正常发育过程中存在的对软骨形成和成熟既允许又指导的信号，将导致产生具有类似于天然组织特性和改善修复的结构。

DESCRIPTION (provided by applicant): Articular cartilage lines the surfaces of joints and transmits the forces generated with loading. Due to limitations in the natural healing capacity of cartilage, and given the increasing incidence of osteoarthritis, there exists a growing demand for cell-based strategies for repair. Tissue engineering, and particularly those approaches based on autologous mesenchymal stem cells (MSCs), is evolving as a clinically relevant technique to promote cartilage regeneration. Yet, the formed tissue properties as well as the stability of phenotype and heterogeneous cellular response within constructs are concerns that currently limit translation of this technology. Our general approach to MSC-based cartilage repair addresses the differences observed between early rapid stages of cartilage formation and the gradual remodeling (maturation) that results in a tissue capable of adult function. This transformative process is driven by a multitude of temporal factors (chemical, mechanical, and soluble). Our progress during the ongoing grant has shown a role for matrix and cellular density, the timing of material degradation, introduction of soluble inductive factors, and mechanical loading (both compression and sliding contact) in guiding cartilage formation and maturation. Here, we build from these studies by introducing a developmentally relevant signal, namely cell-cell interactions through N-cadherin that are found during limb bud development, into our engineered hydrogel systems. In the first Aim, MSCs will be encapsulated in HA hydrogels modified with peptides that mimic the extracellular domain of N-cadherin, and the influence of peptide density on chondrogenesis and cartilage maturation will be investigated, in addition to the influence of the peptide on population heterogeneity and phenotypic stability. In the second Aim, the temporal presentation of the peptides will be investigated by introducing linkers that undergo cell-mediated proteolysis of the peptides from the HA hydrogels. The influence of the temporal peptide presentation on chondrogenesis, cartilage maturation, population heterogeneity, and phenotypic stability will be assessed as in the first Aim, in addition to the responsiveness to mechanical loading based on the acceleration of chondrogenesis. In the third Aim, N-cadherin peptide modified hydrogels, including both stable and transient presentation, will be investigated in a clinically-relevant load-bearing porcine defect model to assess the role of these interactions in an implanted hydrogel in cartilage defect repair. These Aims were designed to allow the testing of our hypotheses that control over the MSC microenvironment, and inclusion of signals present during normal development that are both permissive and instructive for cartilage formation and maturation, will lead to the generation of constructs with properties akin to native tissue and improved repair.