损伤信号HMGB1/TLR4促进破骨细胞分化增强异体骨骨重建的研究

Clinical reconstruction of large bone defects is a challenging issue that can be potentially addressed by studying the role of damage signal high-mobility group protein 1 (HMGB1) in graft-mediated bone repair. Presently, allografts are commonly utilized for treatment of large bone defects, however its performance is still inferior to autografts due to little-to-no remodeling following implantation. One of the important differences that may account for this discrepancy is that product preparation renders allografts acellular whereas freshly harvested autografts contain cell components that subsequently release damage-assoicated molecular pattern (DAMP) molecules. DAMPs, including HMGB1, are released by damaged tissue or cells, which can play an essential role for inducing the inflamamtory response and mediating tissue regeneration. As such, damage signals including HMGB1 are vital for bone repair but are absent in allografts. Our previous study demonstrated an essential role of HMGB1/TLR4 signaling in allograft-mediated calvarial bone repair. Specifically, when HMGB1 or TLR4 signals were depleted, macrophage and osteoclast infiltration was reduced and less graft remodeling occurred, resulting in decreased bone repair. When HMGB1 was added to demineralized bone matrix (DBM; clinical-grade allograft), increased calvarial bone repair was observed compared to DBM implantation alone. Together, these phenomena led us to hypothesize that increased HMGB1 signaling will improve DBM-based graft remodeling and subsequent bone repair. Our proposed project aims to elucidate the role of HMGB1 in allograft remodeling and its potential application in bone tissue engineering. In the first two parts, we seek to understand the role of HMGB1 in DBX-based grafting therapy, via μCT, fluorescence imaging, bio-informatics, gene expression, cellular infiltration, protein synthesis and activity of ECM remodeling enzymes. To appreciate the underlying mechanisms, we will utilize mouse models of global knockout and cell-specific knockout of TLR4, one of HMGB1 receptors, to understand the downstream pathway of HMGB1 signaling. Furthermore, we will perform in vitro studies to understand the effect of HMGB1 receptor and MAPK/NF-κB signaling on cytokine expression and osteoclastogenesis differentiation, thus mapping cellular behavior to the underlying molecular mechanisms in a greater detail. In the third part, we will utilize fibrin gel and PLGA to deliver HMGB1 with DBX graft, and analyze its effect on bone healing. Upon completion of this project, we will have achieved an improved understanding of how HMGB1 signaling affects allograft remodeling at the cellular and molecular level, thus advancing our knowledge on the relationship between the innate immune system and the skeletal system. In addition, the results obtained from this study will directly inform current allograft-associated tissue engineering for craniofacial reconstruction.

异体骨移植后的重建性能是影响其骨修复的一个关键因素。研究发现自体骨的细胞成分释放损伤信号DAMPs能诱导炎症及组织再生，而异体骨为避免免疫排斥常为无细胞状态，因此DAMPs可能是异体骨遗失而影响其骨修复效果的重要因素。本课题组发现，DAMPs成员HMGB1通过受体TLR4，在异体骨移植后能诱导炎症及破骨细胞浸润分化，而由于破骨细胞分化是骨移植物重建再生的重要启动环节，由此提出假说：炎症损伤信号HMGB1/TLR4能通过增强破骨细胞分化而促进异体骨骨重建。本课题拟通过建立受体TLR4全敲除及髓系细胞TLR4敲除的小鼠模型、利用CT结合荧光成像共同分析植体重建和新骨再生，及初步构建组织工程骨等方法，旨在探讨HMGB1在异体骨移植修复中的重要作用及分析HMGB1/TLR4促进破骨分化增强骨重建的可能机制，以期全新认识损伤信号在异体骨修复骨缺损中的作用并辅助骨移植材料的开发应用。

研究显示临床上常运用的各种同种异体骨材料与自体骨在成分上具有相似性，然而异体骨移植后缺乏有效的骨重建（remodeling），是其难以与自体骨修复效果相匹配的瓶颈之一。因此骨组织工程学领域常面临的科学问题是，寻找异体骨与自体骨差异的重要因素，并用以提高异体骨重建和引导骨再生的生物活性。研究表明，组织创伤中应激细胞、坏死细胞释放的损伤相关分子模式（damage-assoicated molecular patterns, DAMPs），具有诱导炎症反应及创伤后组织再生修复的重要作用。因此研究具有调控炎症和组织再生特性的损伤信号家族DAMP成员，通过其受体信号通路对异体骨重建的作用及相关机制，并以此为基础构建组织工程骨是探索骨修复材料的一条新途径。.本项目1）深入探索了HIV蛋白酶SQV抑制剂通过调节DAMPs家族成员HMGB1及其受体间的相互作用，调节破骨细胞的活性从而促进颅骨修复的作用及可能机制；2）探索DAMPs部分家族成员包括HMGB1在内的多个损伤修复因子在骨髓间充质干细胞成骨分化过程中的作用；3）探索不同异体骨和多个DAMPs的复合植体在颅骨修复缺损中的作用。本项目研究发现：1）在小鼠颅骨极限缺损模型中，短期SQV治疗对骨修复有积极作用，而长时间的SQV治疗将抑制骨修复。进一步机制研究发现，SQV可通过靶向TLR4/MyD88信号通路，调节骨缺损修复后的破骨分化，从而影响骨修复效果。2）DAMPs家族的多个成员能促进骨髓来源的干细胞的成骨分化；3）在小鼠颅骨极限缺损及异体骨修复模型中，DAMPs成员HMGB1可能通过促进再生的免疫调节以增强骨修复。但具体作用及可能机制还需要进一步深入实验研究及探讨。.本项目通过细胞实验及异体骨移植修复和颅骨缺损两个动物模型，发现HMGB1/TLR4相关信号通路作为介导免疫反应的受体信号，能通过调节炎症反应及破骨细胞分化而影响骨修复再生。调节炎症反应以促进骨修复是近年国内外骨组织工程学研究的新方向。本项目的实验结果，有利于深入理解炎症反应在骨缺损修复中的作用机制，为炎症在骨组织工程学中的应用积累经验与数据。