软骨特异微环境对再生软骨类型的影响及调控机制

There are three types of cartilage in human body, elastic, fibro, and hyaline cartilage, each differs from others significantly in terms of chondrocyte type, tissue structure, matrix composition, and mechanical property. Whether tissue engineering can regenerate cartilage of these three types, and what is the key regulating mechanisms are still unknown. Previous studies in our lab have demonstrated the critical role of cartilage micro-environment in regulating cartilage regeneration. However, key questions are still unanswered such as: Can specific cartilage micro-environment regulate trans-differentiation among different types of chondrocyte? Can specific cartilage micro-environment also determine the types of cartilage regenerated by stem cells? And which key factors involve in the regulation process? To address the above issues, the current project will implant different types of chondrocytes, stem cell and their engineered cartilaginous derivatives into different cartilage micro-environments in one animal; in addition, in vitro co-culture models will be used to mimic specific cartilage micro-environment to induce stem cell committed differentiation; cell tracing, long-term follow up study, traced cell sorting and phenotype evaluation, as well as comprehensive assessments of cartilage types will also be conducted to illustrate the influence of different cartilage micro-environment on chondrocyte, stem cell, and their engineered cartilaginous derivatives. Based on the above, key factors regulating the types of regenerated cartilage will also be screened through high-throughput approaches such as proteomics, so that the basic theory of micro-environment regulating tissue regeneration can be enriched, and regeneration of specific type of cartilage can also be established.

人体内软骨分为弹性、纤维及透明软骨三种类型，其在细胞种类、组织结构、基质成分及力学性能等方面存在显著差异。组织工程技术能否再生特定类型软骨及其调控机制目前尚不明确。前期研究证实软骨微环境对软骨再生具有重要调控作用。然而，特定软骨微环境能否调控不同软骨细胞横向分化再生特定类型软骨、能否调控干细胞再生指定类型软骨、哪些因素参与了调控作用等系列问题目前仍不明确。据此，本项目拟通过不同类型软骨细胞、干细胞及其构建产物在不同软骨微环境间的横向互换或定向移植，以及体外共培养模型模拟特定软骨微环境调控干细胞定向分化，结合细胞标记、长期随访、标记细胞分选回收与表型鉴定以及全面的软骨类型鉴定，阐明不同类型软骨微环境对成熟软骨细胞、干细胞及其构建产物再生软骨类型的影响，在此基础上，结合蛋白组学等高通量技术筛查微环境调控再生软骨类型的关键因子，为丰富微环境调控组织再生理论及建立特定类型软骨再生技术奠定基础。

人体内软骨分为弹性、纤维及透明软骨三种类型，其在细胞种类、组织结构、基质成分及力学性能等方面存在显著差异。实现不同类型软骨的结构和功能再生是软骨组织工程的最终目标。骨髓间充质干细胞(BMSCs)是软骨组织工程的理想种子细胞。然而利用BMSCs能否再生特定类型软骨及其调控机制目前尚不明确，从而不能达到理想的软骨结构和功能再生，最终限制其临床转化。研究发现细胞生长的微环境对其生物学行为和再生能力至关重要，而不同类型软骨具有不同的生长微环境。本项目利用特定类型的软骨脱细胞膜片（ACSs）构建的局部软骨微环境以及体内天然软骨微环境，分别研究关节软骨微环境和弹性软骨微环境对BMSCs成软骨能力及类型的调控作用；同时，分别将耳软骨细胞和关节软骨细胞与BMSCs混合共培养形成的局部微环境，交叉回植到体内关节软骨和弹性软骨微环境，进一步研究BMSCs成软骨类型。研究发现：1）BMSCs在两种软骨微环境中均能稳定再生软骨；2）BMSCs在体内关节软骨微环境中形成了透明软骨组织，BMSCs在体内弹性软骨微环境中再生成熟软骨组织，但不表达弹性软骨特异性蛋白-弹性蛋白；3）耳软骨细胞与BMSCs混合培养形成的局部弹性软骨微环境，在体内弹性软骨微环境中能够实现弹性软骨再生；4）关节软骨微环境能够完全逆转由耳软骨细胞与BMSCs混合共培养形成的局部耳软骨微环境从而再生关节软骨；耳软骨微环境能否部分逆转由关节软骨细胞与BMSCs混合共培养形成的局部关节软骨微环境再生弹性软骨样软骨组织，但是不表达弹性蛋白。上述研究为利用干细胞再生特定类型软骨提供系统的理论指导，并且为组织再生的微环境研究提供指导意义。