仿生软骨细胞周基质的构建及其成分和硬度对MSC软骨分化的调控研究

Clinical treatment of chondral lesions remains to be a big challenge owing to the limited self-repair capacity of articular cartilage. Mesenchymal stem cells (MSCs) that have the potential to undergo chondrogenic differentiation have been widely used as a cell source for cartilage regeneration. However, due to the environmental factors, MSCs often suffer from some issues, such as insufficient chondrogenesis and hypertrophy, which greatly hamper their therapeutic efficiency. Therefore, construction of bio-mimetic microenvironment for MSCs to promote their chondrogenesis is highly desired. It has been reported that the unique component (Col VI) and stiffness of pericellular matrix (PCM) of chondrocytes play a critical role in facilitating their chondrogenic differentiation and tissue regeneration. In this project, a biomimetic PCM with tunable components and stiffness on the surface of MSCs will be constructed, and their influences on chondrogenesis of MSCs will be systematically studied. The main contents of this proposal involve: (i) fabrication of biomimetic PCM with tunable components and stiffness; (ii) investigation of the influence of biomimetic PCM on chondrogenesis and hypertrophy of MSCs both in vitro 3D culture and in vivo implantation; (iii) exploring the sole or synergistic mechanisms of the component and stiffness of biomimetic PCM on chondrogenesis and hypertrophy of MSCs. This project will propose a feasible method to fabricate PCM and elucidate the effects of pericellular microenvironment on chondrogenesis of MSCs, thus providing scientific evidence for the clinical application of MSCs in cartilage tissue engineering.

间充质干细胞（MSC）因其良好的成软骨分化能力而被广泛应用于软骨组织工程研究以期解决关节软骨损伤修复的难题。然而，MSC分化受微环境影响大，容易发生软骨分化不足和肥大等问题严重影响了软骨修复效果。因此，构建促软骨分化的微环境是当前研究的热点和难点。软骨组织中，软骨细胞周基质（PCM）的特异性成分VI型胶原（Col VI）和适宜硬度对软骨分化和再生具有重要促进作用。本项目拟在MSC表面构建成分和硬度可控的仿生PCM，系统研究仿生PCM成分和硬度微环境对MSC软骨分化的调控机制。研究内容包括：仿生PCM成分和硬度的构建策略和方法工艺；体内外3D微环境下仿生PCM促MSC软骨分化的效果；仿生PCM成分和硬度对MSC软骨分化及肥大化的单独和协同作用机制。本项目的顺利实施有助于开辟PCM的仿生构建方法，阐明细胞周围微环境对MSC软骨分化的影响机制，从而为MSC在软骨组织工程中的临床应用提供科学依据。

间充质干细胞（MSCs）因其良好的分化潜力被广泛应用于软骨和骨组织工程。细胞外基质（ECM）可以通过生物力学信号及信号的动态变化调控干细胞命运。因此，设计和构建新型促MSCs分化的可控周围力学微环境是当前研究的热点和难点课题之一。本项目其中一个方面，通过对软骨细胞周基质（PCM）的仿生构建，系统研究仿生PCM硬度微环境对MSCs成骨和软骨分化的影响作用。创造性的采用LbL沉积阻聚层来降低光聚合水凝胶PCM的交联程度，进而降低PCM的基质硬度。基于此体系研究了非均质力学微环境对MSCs成骨和成软骨分化的影响作用。证明了PCM硬度的降低增强了成骨和成软骨分化的能力，其中涉及到的机械信号通路正在进一步探究中。另一方面，对于软骨和骨组织在生长过程中基质硬度逐渐增加这一力学现象，基于GelHPAGMA进行酶和光次序引发交联实现了3D微环境动态硬化力学微环境的仿生构建。结果发现动态硬化水凝胶允许MSCs在第二次交联之前进行一定程度的铺展，并在水凝胶硬化后MSC感知高硬度力学信号，从而实现成骨分化的进一步增强。创造性地，该系统可以自主调控硬化时间点，研究证实动态水凝胶尤其是晚期硬化的水凝胶更加有利于成骨ECM分泌、基因表达和YAP/TAZ核定位。动物实验评价进一步表明，动态硬化具有增强间充质干细胞成骨分化的作用，并可显著促进骨重塑。这项工作揭示了3D动态硬化微环境作为一个关键的生物物理因素，不仅可以在体外调节干细胞的命运，而且可以增强体内的骨修复。本项目从体内非均质空间和时间动态变化力学微环境上进行仿生构建，并研究了复杂力学微环境对MSCs成软骨和成骨分化的影响作用和机制，为MSCs在软骨-骨组织工程中的临床应用提供科学依据。