基于层-层/多相支架结构剖析细胞间相互作用介导工程化骨软骨组织构建的基础研究

Osteochondral tissue engineering aims at mimicking native tissue interface by fabricating integrated cartilage and bone tissues to improve tissue integration and repair. However, in order to generate the two tissues in one system, which essentially is to carry out spatial control over cell differentiation, it is prerequisite not only to perform a systemic investigation on how to design scaffolds, but also both a careful selection of appropriate cells and a thorough understanding of how cell-cell interactions affect tissue morphogenesis. In this proposal, by following tissue engineering principles, layered/multiphasic tissue engineering scaffolds will be designed and fabricated by using a 3D printing technology. Then, a systemic investigation on applying different cell combinations of mesenchymal stem cells (MSCs) and chondrocytes as well as subpopulations of chondrocytes to engineer osteochondral tissue constructs will be performed. In order to understand the mechanisms of cell-cell interactions, different coculture methods are to be established to evaluate effects of key variables such as cell combinations, cell ratio/density, layer/phase thickness, culture conditions (time and culture medium) and so on. In the end, by establishing animal models (i.e., full-thickness articular defects in rabbits), in-situ tissue repair capability of engineered osteochondral equivalents will be studied. Findings of this study will help understand whether cell-cell interactions drive engineered osteochondral.tissue devlopment and how. In addition, a new perspective of osteochodral tissue engineering by modulating cell-cell interactions can be proposed. Overall, this would expedite the transition of current osteochondral tissue engineering from simply replicating tissue structures to passively guiding tissue development and thus the translation of cartilage tissue engineering technologies into clinical settings.

骨软骨组织工程基于构建一体化骨/软骨组织的思路，本质上要求实现同一空间内细胞各向异性分化。当前研究主要侧重于支架设计，而对于选择何种细胞以及细胞间相互作用是否会影响骨软骨组织形成的问题都没有清晰认识。本项目立足骨软骨组织构建过程，拟以3D打印技术制备层-层/多相结构支架为技术手段，系统考察包括间充质干细胞、软骨细胞及其各亚群在内的各种细胞及组合构建骨软骨组织的效果，并重点以共培养方法基于细胞组合、细胞比例、支架层/相厚度、培养条件等参数变化剖析细胞间作用方式及机制，最后将结合动物模型检验工程化组织原位修复效果。基于本项目研究，将有助于认识和理解细胞间相互作用作为骨软骨组织结构形成的内在驱动机制，从而确立通过调节细胞间相互作用优化工程化骨软骨组织构建过程的新思路，推动骨软骨组织工程从当前对天然组织结构的简单复制过渡到对组织形态发生过程的理性引导的新台阶，加速软骨组织工程方法的临床转化。

组织工程利用生物材料、细胞及细胞因子的有机组合以构建具有部分或全部生物学功能的人体组织器官来促进缺损器官修复再生。本项目聚焦回答组织工程领域的基础问题，核心是通过解构工程化组织形成过程中细胞组份、生物材料等关键要素的相互作用的动态演变，来认识工程化组织构建的关键因素，为体外组织构建提供理性指导。具体而言，项目以工程化骨软骨组织构建为目标，来研究如何实现细胞在支架空间结构中各向异性分化，从而获得成熟稳定的骨软骨组织结构。项目团队围绕软骨组织构建和再生的目标，以细胞、材料和培养技术为核心手段，开展了一系列研究工作：（a）对应用于软骨组织工程的细胞进行体外培养研究，包括骨髓和脂肪等不同来源的间充质干细胞和软骨组织中不同区域的软骨细胞；（b）建立和优化3D打印技术应用于支架制备及基于脱细胞基质修饰的方法；（c）通过体外共培养手段，系统探讨间充质干细胞与软骨细胞的共培养作用效果；（d）基于转瓶方式的动态共培养，考察影响间充质干细胞和软骨细胞自组装行为；（e）通过材料制备手段，建立不同尺寸纤维材料的制备方法，探讨纤维尺寸及脱细胞基质修饰对细胞行为的影响。我们成功建立了几种具有鲜明特色和应用前景的适用于软骨组织构建的生物活性支架材料制备方法，并通过强化对细胞间相互作用的理解，并提出将支架特性与细胞间相互作用协同，对软骨组织构建具有极强的指导意义。具体来讲，一方面，基于3D打印技术可以构筑理性设计的支架微结构，基于静电纺丝方法可以制备具有不同尺寸的纤维，结合细胞培养和去细胞技术，可以对支架材料进行细胞基质的生物活性修饰，这显然可以对细胞行为起到引导作用；另一方面，软骨细胞与间充质干细胞之间存在显著的调节作用，而且可以呈现自发的组装结构，这对软骨组织形成也具有积极的意义；通过对这两方面的结合，必将为软骨组织构建提供一种更为完善的一种思路。