3D打印材料复合miR-30b修饰在关节内环境中的促软骨再生作用机制研究

Cartilage defects seriously affect joint functions, which, however, fail to be effectively repaired via the common therapies currently used in clinical practice. The development of biological materials promises a new prospect for cartilage functional repair, but biomaterial implants for functional repair prove to be a complex process involving multiple factors. Not until the key scientific issues covered in this process, including cellular and molecular mechanisms as well as their influencing factors, are clarified can the functional repair effect of cartilage tissue be effectively improved. Our previous study has suggested that miR-30b affects the secretion of characteristic cartilage extracellular matrix through regulating the target gene SOX9 and preliminarily discussed the involvement of miR-30b in the regulatory process of cartilage differentiation progress. According to findings of the previous study, the cell printing ink prepared by sodium alginate/gelatin composite material is suitable for the 3D printing of mesenchymal stem cells (MSCs). Through investigating the role and mechanism of 3D printing material modified by miR-30b-regulated Sox9 in promoting MSCs chondrogenic differentiation under different environmental conditions like cellular stress and apoptosis, hypoxia, TGF-β3 induction, Collagen II coating and stress, this paper is designed to provide a new idea and new strategy for the functional design of biological materials to better promote the cartilage differentiation.

软骨缺损严重影响关节功能，但目前临床上常用的治疗方法仍不能有效修复。生物材料的发展为软骨功能性修复带来希望，但生物材料植入体内达到功能性修复是一个多因素影响的复杂过程，只有弄清这一过程所发生的细胞和分子机制及其影响因素的关键科学问题，才能有效提高软骨组织的功能性修复效果。我们前期研究提示miR-30b通过调控靶基因SOX9影响软骨特征型胞外基质的分泌，初步探讨了miR-30b参与了软骨分化进程的调控过程。前期研究还发现，海藻酸钠/明胶复合材料适合制备成细胞打印墨水，适合间充质干细胞(mesenchymal stem cells，MSCs)的3D打印。本课题通过研究探索miR-30b通过调控Sox9修饰3D打印材料在细胞应激、低氧环境、TGF-β3诱导、Collagen Ⅱ包被、应力等内环境条件下，促进MSCs成软骨分化的作用及机制，为功能化设计生物材料能更好促软骨分化提供新思路及新策略。

软骨缺损严重影响关节功能，目前临床常用修复方法都无法得到满意效果。生物材料发展、表观遗传学及关节内环境的不断深入研究为软骨功能化修复带来新的希望，但生物材料运用于关节内环境进行软骨修复是一个多因素影响的复杂过程，具体机制尚不清楚。本项目首先通过构建稳定表达miR-30b的兔骨髓间充质干细胞（rBMSC），利用免疫印迹实验等证明了miR-30b通过Wnt/β-catnein/SOX9信号轴调控rBMSC成软骨分化。探索miR-30b在不同关节内环境下rBMSC成软骨分化的作用机制发现：miR-30b能缓解氧化应激诱导对rBMSC的成软骨分化的抑制作用；在Collagen II包被下，经miR-30b修饰的rBMSC成软骨分化能力减弱；在低氧条件下，miR-30b能抑制rBMSC成软骨分化。其次，我们构建兔股骨软骨缺损模型，并利用3D打印技术成功制备出含细胞活力的海藻酸钠/明胶生物支架用于体内实验，研究结果发现加入磷酸盐离子控制后的海藻酸钠/明胶生物支架具有良好的机械性能、生物活性及成骨分化能力，为进一步构建软骨缺损所需的软骨及软骨下骨复合材料提供前期实验基础，但表观遗传学修饰生物材料体内成软骨分化功能尚待优化实验方案后进一步探讨。总之，本研究重点阐明了miR-30b在氧化应激、Collagen II包被、低氧三种内环境下通过Wnt/β-catnein/SOX9信号轴调控rBMSC成软骨分化的具体机制，为软骨损伤的功能化修复提供了分子生物学依据。

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