软骨脱细胞基质通过ChM-I调控BMSC体内软骨修复及机制研究

The success of in vitro cartilage regeneration based on bone marrow stem cells (BMSC) provides a promising approach for cartilage repair. However, the currently BMSC-engineered cartilage is unsatisfactory for clinical application due to ectopic ossification after subcutaneous implantation. It has been widely reported that endochondral ossification mainly results from angiogenesis and thus anti-angiogenesis becomes the major obstacle for clinical application of the BMSC-engineered cartilage. Our previous studies have already showed that cartilage engineered with BMSC and acellular cartilage sheet (ACS) could maintain their cartilage phenotype in vivo. Combined with previous studies that chondromodulin-I (ChM-I), one of the anti-angiogenic factors during embryonic chondrogenesis, plays the most important role in maintaining the cartilage stability. Thus, we speculate that ACS modulate the in vivo stability of BMSC-engineered cartilage by the function of residual ChM-I. In the early stage after subcutaneous implantation of BMSC-engineered cartilage, residual ChM-I play an important role in anti-angiogenesis; on the other hand, BMSC-engineered cartilage begins to secrete new ChM-I by itself. Based on the preliminary studies, this research project will investigate the correlation, as well as the dose-effect relationship of residual ChM-I in ACS and the in vivo stability of BMSC-engineered cartilage, which might lay the foundation for the clinical application of cartilage engineering using ACS.

骨髓基质干细胞（BMSC）体外软骨构建成功，为软骨修复提供可能。然而BMSC构建软骨体内易丧失软骨表型阻碍了其临床应用。其原因主要与血管化骨化有关，解决问题的关键是抑制血管化。前期研究课题组发现应用软骨脱细胞膜片（ACS）复合BMSC构建软骨体内能维持软骨表型稳定，结合以往研究证实软骨调节素I（ChM-I）是维持软骨表型稳定最重要的抗血管化因子，我们推测ACS通过残留ChM-I调控BMSC构建软骨的体内稳定性。其一，残留ChM-I发挥早期抗血管入侵作用；其二，新生软骨体内进一步成熟，自身分泌ChM-I，发挥中后期抗血管化作用。本项目将采用三明治模型构建软骨，比较不同实验组新生软骨形态学、组织学、软骨与骨相关蛋白及基因表达情况，探索残留ChM-I与BMSC构建软骨体内稳定性之间的关联及量效关系，阐明ACS通过ChM-I调控BMSC构建软骨体内稳定性的机制，以期为ACS应用于软骨构建奠定基础。

骨髓基质干细胞（BMSC）体外软骨构建成功，为软骨修复提供可能。然而BMSC构建软骨体内易丧失软骨表型阻碍了其临床应用。其原因主要与血管化骨化有关，解决问题的关键是抑制血管化。前期研究课题组发现应用软骨脱细胞膜片（ACS）复合BMSC构建软骨体内能维持软骨表型稳定，结合以往研究证实软骨调节素I（ChM-I）是维持软骨表型稳定最重要的抗血管化因子，我们推测ACS通过残留ChM-I调控BMSC构建软骨的体内稳定性。其一，残留ChM-I发挥早期抗血管入侵作用；其二，新生软骨体内进一步成熟，自身分泌ChM-I，发挥中后期抗血管化作用。本项目采用三明治模型构建软骨，在既往对软骨脱细胞膜片（acellular cartilage sheet，ACS）的制备及相关研究的基础上，分三部分进行研究：1.ACS 中残留 ChM-I 的检测及其功能实验；2.ACS 中残留的 ChM-I 与 BMSC 构建软骨体内稳定性的相关性研究；3.ChM-I 的含量与 BMSC 构建的软骨体内稳定性的量效关系。在顺利制备ACS后，采用ELLISA法检测结果证明ACS中确实残留了一定数量的软骨调节素-I（ChM-I），并通过公认的干扰血管内皮细胞成管实验证明残留的ChM-I仍具有一定的生物学活性。通过研究ACS 复合 BMSC 构建的软骨体外诱导培养后植入体内后能否继续维持软骨表型的稳定性，取材后大体观、组织学、PCR检测等多方面证明了ChM-I 是调控 BMSC 构建软骨体内稳定性的关键因素，为ACS应用于软骨构建奠定基础。