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。由于软骨的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不断增长。组织工程，特别是那些基于自体间充质干细胞（MSC）的方法，正在发展成为促进软骨再生的临床相关技术。然而，所形成的组织特性以及构建体内的表型和异质细胞应答的稳定性是目前限制该技术的转化的问题。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逐渐重塑（成熟）之间观察到的差异，导致组织能够发挥成人功能。这一转变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期间取得的进展表明，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中发挥了作用。在这里，我们从这些研究中建立了一个发育相关的信号，即通过在肢芽发育过程中发现的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，将MSC封装在用模拟N-钙粘蛋白胞外结构域的肽修饰的HA水凝胶中，并且除了肽对群体异质性和表型稳定性的影响之外，还将研究肽密度对软骨形成和软骨成熟的影响。在第二个目标中，将通过引入接头来研究肽的时间呈递，所述接头经历来自HA水凝胶的肽的细胞介导的蛋白水解。除了基于软骨形成加速的机械负荷反应性之外，将如第一个目的中那样评估时间肽呈递对软骨形成、软骨成熟、群体异质性和表型稳定性的影响。在第三个目标中，将在临床相关的承重猪缺损模型中研究N-钙粘蛋白肽修饰的水凝胶，包括稳定和瞬时呈递，以评估这些相互作用在软骨缺损修复中植入水凝胶中的作用。这些目的旨在允许测试我们的假设，即控制MSC微环境，并包含正常发育期间存在的对软骨形成和成熟既允许又有指导意义的信号，这将导致产生具有类似于天然组织和改善修复的特性的构建体。

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.