软骨内骨化机制研究及人工调控系统在软骨-骨共修复中的应用

Tissue engineering is promising for wide applications in future tissue restoration, by exploiting the biological principle and engineering strategies to construct complicated artificial tissues and organs. Although techniques to repair the tissues by multiple cell types were widely established , it has always been harassment for researchers to simulate cell-cell interface in a controllable manner by single cell type, due to the complication of biological interface stimulation, interface transition mechanism and fine regulation of seed cells.However, when repairing the multiple cell interface,it has always been harassment for researchers to simulate cell-cell interface in a controllable manner by single seeding cell, due to the complication of interface transition mechanism and biological precise regulation. Constructing artificial systems to regulate cell behavior has emerged as a hot field in tissue engineering nowadays. It refers to splitting the physiological process apart and designing regulation circuits and modules in synthetic biology approaches, therefore meeting the demands of controlling the gene expressions and physiological process spatially and temporally in those modified seeding cells, which contributes to in vivo co- repairation of biological interface. This grant application focused on the solution of cartilage-bone co-repairation. By introducing a controllable endochondral ossification process for seeding cells and constructing artificial system based on the synthetic biology principles, the directional differentiation could be precisely controlled from cartilagechondrocyte progenitor cells to osteoblasts through a hypertrophied hypertrophic state, providing potential solutions for cartilage-bone co-repairtion repair with single cell type. The project, for one, will dig into bone development by systematically studying in vitro endochondral ossification process, for another, apply the artificial regulation system to seeding cell modification and complex physiological process, intending for better clinical effect on cartilage-bone interface repair, and its applications in tissue engineering.

组织工程利用生物学原理与工程技术手段构建人造组织和器官，在组织修复中有广泛的应用前景。然而在对多细胞界面进行修复时，因涉及界面过渡机制认知及种子细胞的精细调控，如何用同种种子细胞可控模拟多细胞交界面一直困扰着诸多研究者。 构建人工调控系统控制细胞行为是当今研究的热点。它以合成生物学思路拆分生理现象，分阶段设计调控模块，从而精确控制种子细胞基因的时空性表达，调节生理活动，有助于解决界面共修复的问题。 本课题从软骨-骨共损伤现象入手，通过建立体外软骨内骨化模型，以合成生物学思路构建人工调控系统，实现精细调控软骨前体细胞经肥大化软骨细胞向骨细胞的定向分化过程，为单一种子细胞的软骨-骨共修复提供方法。本课题一方面进行软骨内骨化机制的系统研究，加深对骨发育的认知，另一方面将人工调控系统应用于种子细胞的改造和复杂生理过程，为软骨组织工程提供软骨-骨共修复解决方案，推动其在组织工程领域的应用。

组织工程利用生物学原理与工程技术手段构建人造组织和器官，在组织修复中有广泛的应用前景。在软骨组织工程领域，研究者尝试使用不同的生物材料、种子细胞和生长因子的组合来修复缺损的关节软骨（透明软骨），但如何理性而可控地实现透明软骨和软骨下骨的共修复一直是学界的难题。本课题从软骨发育的分子机制入手，利用单细胞技术重建了小鼠生长版中的软骨发育过程，重点关注了肥大化（软骨向硬骨转分化）过程中转录因子的变化，并在体外验证了预测得到的重要的转录因子组合。同时，我们尝试使用合成生物学手段对种子细胞进行改造，一方面构建了基于四环素诱导的二级缓释基因表达系统，并在体内实验中初步验证了效果；另一方面尝试使用启动子感应元件筛选结合转录因子的特异序列，构建人工肥大化感应系统。另外，我们对一种新型的DNA水凝胶材料在软骨-骨共修复中作为支架的性能进行了详细评估。之后，我们对现有的修复软骨-骨缺损的注射疗法得到的修复组织进行了单细胞转录组分析，结合传统的评估方法，比较了两种方法的优劣。本课题研究期间，课题组共在Cell Reports、Biomaterials等SCI杂志上发表5篇文章，另有1篇在投。通过以上探索和发现，我们软骨的内骨化过程在生理发育和再生修复过程中的作用有了更深的理解，在将合成生物学应用于再生修复方面的也取得了一定的成果，并利用单细胞组学技术解析了软骨生理发育和再生修复过程的异质性，有望进一步推动软骨组织工程修复的发展。