iPSC-MSCs复合活性大孔CPC用于牙槽骨缺损修复及转归机制的研究

The repair of critical-sized alveolar bone defects remains a major challenging health issue in clinical dentistry worldwide. Bone tissue engineering using scaffolds and stem cells with induction methods provides a new promising approach for bone repair. Our research group recently found that mesenchymal stem cells derived from induced pluripotent stem cells (iPSC-MSCs), a new type of progenitor cells, had a great potential to regenerate bone tissue with more efficiency compared with BM-MSCs. Moreover, we fabricating a novel macroporous biofunctionalized calcium phosphate cement (CPC) scaffold. The CPC scaffold had good mechanical strength for craniofacial bone repair applications and the seeding cells attached and grew well on it. Recently, microRNAs (miRNAs) have become an important tool to induce both osteogenic and angiogenic differentiation of stem cells simultaneously in the field of tissue engineering. However, to date there has been no report on miRNA regulation of human iPSC-MSC for bone regeneration. Moreover, only a few investigations have investigated the mechanism of miRNA regulation of osteogenic differentiation. The present project will seed human iPSC-MSCs on the novel macroporous CPC biofunctionalized with Arg-Gly-Asp (RGD) (referred to as RGD-CPC) for repair of rat critical-sized alveolar bone defects. The key miRNA(s) related with both iPSC-MSC osteogenesis and angiogenesis will be selected and iPSC-MSCs will be induced by miRNA gene modification. The project will investigate the effects and mechanisms of miRNA regulation of iPSC-MSC proliferation and differentiation. The project will hopefully provide theoretical and experimental supports for alveolar bone defect repair.

较大范围牙槽骨缺损的修复是口腔临床亟待解决的难题，骨组织工程的发展为其提供了全新的思路和方法。骨再生包括三个关键要素：种子细胞、支架和诱导分化策略。课题组前期研究发现新兴种子细胞iPSC-MSCs比BM-MSCs更具骨再生潜能，并成功制备一种新型大孔磷酸钙骨水泥(CPC)支架材料；miRNA是近年干细胞组织工程新的强有力诱导手段，最新文献报道特定miRNA可同时促进BM-MSCs成骨分化及血管化。目前尚未见miRNA诱导人iPSC-MSCs进行骨再生的报道。本项目拟在前期研究基础上，使用新型大孔生物活性RGD-CPC为支架，接种iPSC-MSCs为种子细胞，从已知骨再生相关的重要miRNAs中挑选并转染iPSC-MSCs的最优miRNA，针对大鼠牙槽骨缺损进行修复研究，并探索特定miRNA对细胞增殖、分化的调控作用机制。结果可望为牙槽骨缺损修复提供理论和实验依据。

较大范围骨缺损的修复是临床亟待解决的难题，骨组织工程的发展为其提供了新的思路和方法。人脂肪干细胞（human adipose-derived stem cells，hASCs）因来源广泛、取材简单、避免伦理争议等优势广泛用于组织工程中。miRNA是近年干细胞组织工程新的有力诱导手段。已有研究发现 miR-20a 可上调BMP/Runx2 信号通路促进骨髓间充质干细胞生长成骨，但其在人脂肪干细胞成骨分化中的作用尚不清楚，而不同来源的干细胞在成骨分化过程中的miRNA表达谱相差甚大。本研究从体外及体内两个水平探讨miR-20a对hASCs成骨分化的调控作用，为 miRNA调控hASCs成骨分化的机制提供实验依据，也为治疗大面积骨缺损的临床应用提供理论基础。同时，项目进一步对与miRNA密切作用的lncRNA、circRNA做了初步研究。进行了hASCs成骨分化的mRNA/lncRNA表达谱、差异性表达的circRNA的生物信息学分析。项目还研究了NELL-1促hASCs软骨内成骨的作用以及相关机制。研究结果表明，miR-20a体外促进hASCs成骨分化，miR-20a促进hASCs中成骨基因ALP的表达，降低其目标基因PPARγ的蛋白表达，促进成骨相关蛋白 ALP、osteocalcin、RUNX2的表达，促进成骨过程中钙结节的形成。hASCs对HA/β-TCP黏附良好，对细胞-支架复合体成骨诱导后，miR-20a促进其ALP表达和钙结节形成，但体内结果表明miR-20a在8周观察期内未见明显促进成骨分化作用，在更长观察期内有无成骨调控作用有待进一步实验研究。项目还系统报告利用lncRNA/mRNA芯片检测hASCs成骨分化过程中差异表达的lncRNA和mRNA，并通过多种生物信息学分析方法筛选出了可能与成骨定向分化过程相关的部分lncRNA，为后续深入的机制研究提供实验基础及研究方向。项目构建了hASCs成骨分化中circRNA-miRNA交互作用网络，并对其可能的作用机制进行预测和验证。项目还发现，NELL-1具有促hASCs软骨内成骨的作用，这为模拟软骨内成骨生理过程进行大面积骨缺损修复提供参考。