Engineered Developmental Microenvironments: Cartilage Formation and Maturation

摘要 关节软骨排列在关节表面并传递负荷产生的力;然而，软骨 可能由于创伤和疾病而受损，并且具有有限的自然愈合能力。虽然 在利用自体间充质基质细胞的软骨疗法的发展方面已经取得了许多进展 但是，为了找到合适的细胞载体并进行培养，还有许多工作要做 最能促进功能性软骨形成的环境。我们的基于MSC的一般方法 软骨修复是为了设计重现关键发育信号的环境。为此， 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、生长因子呈递和机械负载。在最近一 资金周期，我们拴系和控制的生物活性肽（HAV）在N- 钙粘蛋白，其在发育微环境中丰富并介导直接的细胞-细胞通讯。在 在这次更新中，我们继续讨论与发育相关的细胞间信号传导，现在重点关注间接信号传导。 沟通机制。具体来说，我们最近发现一小部分分化的软骨细胞 改善MSC形成基质的数量和质量，并促进其表型稳定性。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传导的结果， “广播”软骨细胞到“接收者”MSC。在这里，我们假设，无论是生产和接收的 这些信号受微环境（基质硬度，与发育配体的相互作用， 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， 支持MSC和软骨细胞共培养中的MSC软骨形成。这将通过空间上的 改变肽和包封材料的性质，并对软骨形成的早期标志物进行成像， 软骨基质形成，以确定最佳环境。在第二个目标中， 优化共培养物中MSC软骨形成将在更长的时间过程中扩大和评估， 以与当前临床工作流程兼容的可注射形式实施。在第三个目标中，这些 优化的制剂将在关节镜下给予临床相关的承重猪病灶， 软骨缺损，以评估这种细胞递送系统促进功能修复的功效。成功 这些目标的完成将为患有软骨损伤的患者确定新的平移选择。

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.