Engineering Developmental Microenvironments: Cartilage Formation and Maturation

描述(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越来越大。组织工程学，尤其是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，形成的组织属性以及构建物内表型和异质细胞反应的稳定性是目前限制这项技术翻译的问题。我们的基于MSC的软骨修复的一般方法解决了软骨形成的早期快速阶段和导致组织具有成人功能的逐渐重塑(成熟)之间的差异。这一变革性的过程是由多种时间因素(化学、机械和可溶性)驱动的。我们在正在进行的资助过程中取得的进展表明，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)在引导软骨形成和成熟方面发挥了作用。在这里，我们在这些研究的基础上，将一种与发育相关的信号，即在肢芽发育期间发现的通过N-钙粘附素的细胞-细胞相互作用，引入到我们的工程水凝胶系统中。在第一个目标中，将MSCs包裹在模拟N-钙粘蛋白胞外区的多肽修饰的HA水凝胶中，研究多肽密度对软骨形成和软骨成熟的影响，以及多肽对群体异质性和表型稳定性的影响。在第二个目的中，将通过引入连接体来研究多肽的时间呈现，这些连接体经历了来自HA水凝胶的细胞介导的多肽的蛋白分解。在第一个目标中，除了基于软骨形成的加速对机械负荷的响应外，还将评估时间肽呈现对软骨形成、软骨成熟、群体异质性和表型稳定性的影响。第三个目的是研究N-钙粘素多肽修饰的水凝胶，包括稳定呈现和瞬时呈现，在临床相关的负重猪模型中进行研究，以评估这些相互作用在植入水凝胶中在软骨缺损修复中的作用。这些目的是为了测试我们的假设，即控制MSC微环境，并包括正常发育过程中存在的对软骨形成和成熟既允许又有指导作用的信号，将导致产生具有类似于天然组织的属性的结构并改善修复。

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.