锂剂预处理BMSCs来源的外泌体复合3D打印的BG支架促进骨缺损修复的作用及机制研究

Critical-sized bone defects are of challenge in clinical practice. It has been found that lithium has a beneficial effect on bone regeneration, however, the properties of significant toxicity, quick release and difficult aggregation in bone defect determine its impossibility in clinical therapy. Exosomes are nanovesicles which is recognized by target cells through antibody-receptor pairing mechanism and transfers information, regulates biological function of recipient cells. Exosomes are emerging promising therapeutics in the field of bone tissue engineering. Our studies confirm that lithium can activate beta-catenin/wnt pathway of maternal cells, while the exosomes secreted by lithium pretreated BMSCs can significantly promote the proliferation and osteogenic differentiation of recipient cells. The high expression of miR-433-3P was found by QPCR detection. Accordingly, we put forward the hypothesis that exosomes secreted by lithium pretreated BMSCs can activate target cell proliferation and osteogenic differentiation through overexpression of miR-433-3P activated the beta-catenin/wnt pathway in receptor cells. The aim of the study is to investigate the combined effects and molecular mechanism of exosomes from Li-pretreated BMSCs compound BG scaffolds on osteogenic differentiation,through rat BMSCs, β-catenin/GSK-3β knocking down cell model and rat calvarial critical defect model ,which is from molecular 、 cellular and whole level . Our study may provide an experimental support for a novel therapeutic strategy in bone tissue engineering.

大段骨缺损是临床常见的棘手问题。锂有很好的促成骨作用，但毒性大、难以在骨组织聚积，无法用于临床骨缺损的治疗。外泌体通过抗体－受体配对机制被靶细胞识别、内化后调控靶细胞生物学功能。近来它成为骨组织工程学领域的研究热点。我们研究发现锂剂可以激活母细胞β-catenin/wnt通路，而锂剂预处理BMSCs分泌的外泌体可促进受体BMSCs的增殖及成骨分化。通过QPCR发现该外泌体高表达miR-433-3P。据此我们提出假说：锂剂通过刺激母细胞分泌过表达miR-433-3P外泌体激活受体细胞中β-catenin/wnt通路，促进靶细胞增殖及成骨。本研究利用大鼠BMSCs、β-catenin/GSK-3β敲除细胞及大鼠颅骨临界缺损模型，通过QPCR、免疫组化、shRNA等方法，探讨锂剂预处理BMSCs分泌的外泌体复合BG支架对成骨的促进作用及机制。该研究为临床寻找一种全新的骨组织工程材料提供实验依据。

锂剂是临床上用于治疗双向情感障碍的常用药物。近年的研究发现，锂剂可激活wnt/β-catenin通路，促进BMSCs的增殖及分化，以促进成骨。大段骨缺损是临床常见的棘手问题。由于没有很好的持续促进成骨的填充材料，大段骨缺损在临床上常需要经历数次手术，大大增加了病人的痛苦。前期研究中，我们通过实验发现锂剂通过激活母细胞β-catenin/wnt通路，预处理的母细胞释放胞外囊泡可促进受体BMSCs的增殖及成骨分化。另外，我们通过构建锂剂及钙盐负载的颗粒水凝胶，将其用于大鼠颅骨临界缺损的骨修复，通过QPCR、免疫组化、shRNA等方法，证实了该锂-钙复合水凝胶可程序化促进成骨。我们在实验中发现新生骨可沿着颗粒水凝胶孔隙爬行生长，证实了该材料良好的骨诱导性和骨传导性。同时其在前期释放 Li+，促进缺损区域周边的骨髓间充质干细胞（BMSCs）的增殖，后期伴随降解释放Ca2+，参与新生骨的矿化。通过调控水凝胶内两种金属离子的有序释放，使体内骨修复 BMSCs 增殖、分化、矿化有条不紊的进行，持续在骨修复的全程发挥作用。最后我们初步证实了锂剂负载的水凝胶可以靶向调控线粒体自噬，从而保护骨缺损周边募集到的BMSCs成骨分化的功能，以促进骨缺损的修复。该研究为临床骨缺损的治疗提供了一种新的治疗方案。