Engineered Developmental Microenvironments: Cartilage Formation and Maturation

摘要 关节软骨排列在关节表面，并传递载荷产生的力；然而，软骨 可因创伤和疾病而受损，并具有有限的自然愈合能力。尽管在那里 自体间充质基质细胞在软骨治疗方面取得了许多进展 (MSCs)，为了确定合适的细胞载体和培养方式，还有很多工作要做 最能促进功能性软骨形成的环境。我们基于MSC的一般方法 软骨修复一直是重现关键发育信号的工程环境。对此， 在早期的资金周期中，我们设计了基于生物分子透明质酸(HA)的水凝胶环境， 包括受控降解、生长因子递呈和机械加载。在最近的 在资金周期中，我们捆绑并控制了在N- 钙粘附素，它在发育中的微环境中含量丰富，并介导细胞间的直接通讯。在……里面 在这次更新中，我们继续处理与发育相关的细胞-细胞信号，现在重点放在间接 沟通机制。具体地说，我们最近发现，一小部分分化的软骨细胞 改善MSCs形成基质的数量和质量，并提高其表型稳定性。我们进一步 表明这种现象是旁分泌囊泡介导的细胞间信号转导的结果。 “广播”软骨细胞给“接收者”的MSCs。在这里，我们假设，无论是制作还是接收 这些信号受微环境(基质刚性、与发育配体的相互作用以及 包埋材料的分子扩散率)。为了解决这个新的假设，第一个目标将利用我们的 最近开发的微环境筛选平台，以确定水凝胶配方的最佳 支持骨髓间充质干细胞和软骨细胞共培养中的软骨形成。这将通过空间上的 不同的多肽和包膜材料特性以及软骨形成和软骨形成的早期标志物的成像 软骨基质形成，以确定最佳环境。在第二个目标中，水凝胶配方 优化MSC在共培养中的软骨形成将在更长的时间进程中放大并进行评估 以与当前临床工作流程兼容的可注射格式实施。第三个目标是，这些 优化的配方将在临床相关的负重猪局部关节镜下应用 以评估这种细胞递送系统促进软骨缺损功能修复的效果。成功 完成这些目标将为软骨损伤患者确定新的翻译选择。

Abstract Articular cartilage lines the surfaces of joints and transmits the forces generated with loading; however, cartilage can be damaged due to traumatic injury and disease and has a limited natural healing capacity. Although there have been many advances in the development of cartilage therapies with autologous mesenchymal stromal cells (MSCs), there is still much work to be done in order to identify the appropriate cell carriers and culture environments that best promote the formation of functional cartilage. Our general approach for MSC-based cartilage repair has been to engineer environments that recapitulate key developmental signals. Towards this, in early funding cycles, we engineered hydrogel environments based on the biomolecule hyaluronic acid (HA), including controlled degradation, growth factor presentation, and mechanical loading. During the most recent funding cycle, we tethered and controlled the temporal presentation of a bioactive peptide (HAV) found in N- cadherin, which is abundant in the developing microenvironment and mediates direct cell-cell communication. In this renewal, we continue to address developmentally relevant cell-cell signaling, focusing now on indirect communication mechanisms. Specifically, we recently found that a small fraction of differentiated chondrocytes improves the amount and quality of matrix formation by MSCs and promotes their phenotypic stability. We further showed that this phenomenon was the consequence of paracrine vesicle-mediated cell-to-cell signaling from ‘broadcasting’ chondrocytes to ‘receiver’ MSCs. Here, we hypothesize that both the production and reception of these signals is regulated by the microenvironment (matrix stiffness, interaction with developmental ligands, and molecular diffusivity of the embedding material). To address this novel hypothesis, the first Aim will utilize our recently developed microenvironmental screening platform to determine the hydrogel formulation that optimally supports MSC chondrogenesis in co-cultures of MSCs and chondrocytes. This will be achieved by spatially varying peptide and encapsulating material properties and imaging early markers of chondrogenesis and cartilage matrix formation to identify optimal environments. In the second Aim, hydrogel formulations that optimize MSC chondrogenesis in co-cultures will be scaled up and evaluated over longer time courses and when implemented in an injectable format that is compatible with current clinical workflows. In the third Aim, these optimized formulations will be arthroscopically administered in clinically-relevant load-bearing porcine focal cartilage defects to assess the efficacy of this cell delivery system to promote functional repair. Successful completion of these Aims will identify new translational options for patients suffering from cartilage injuries.