细胞坏死通过TLR4诱导颅骨再生的机制研究

Currently, autologous bone graft, with its proven osteogenic, osteoinductive, and osteoconductive properties, remains the gold standard for the repair of large bone defects, as bone substitutes like demineralized bone matrix, calcium phosphate granules, and various ceramics lack comparable effectiveness. Limited availability and a high incidence of morbidity associated with the acquisition of autologous bone, however, make alternative graft materials still highly desirable. .In order to improve the design of different bio-material based reconstructive therapies, it is first necessary to determine what components of autologous bone graft are responsible for its superior osteogenic properties. Besides extracellular bone matrix, the cellular component of autologous bone is thought to play a role. However, few evidence of donor cell survival exists, which raises doubts concerning the cellular contribution to graft survival. It has only been appreciated recently that cell necrosis can induce inflammation response which has an established role in promoting tissue repair. In our previous studies, we observed that local exposure of bone components to murine calvarial defects can trigger local inflammatory response and TLR4 signal pathway was required. Furthermore, implant of cellular and acellular bone components within the defects showed enhanced bone healing at designed time points postoperatively. Thus, we hypothesize that the cellular component of bone graft promotes bone regeneration by virtue of an inflammatory response induced by donor cell necrosis and TLR4 signal pathway is involved. This hypothesis, and a larger goal to identify bone components which might be used to improve upon the design of bio-material based bone reconstructive therapy, will require the completion of two specific aims: 1) Identify the roles of bone cellular and acellular component in the repair of calvarial defects within a murine model; 2) Indentify cellular component of bone graft promotes calvarial healing through an inflammatory response induced by donor cell necrosis and TLR4/MyD88 or TLR4/TRIF signal pathway is involved..In order to evaluate the hypotheses and specific aims, gel implant will be supplemented with cellular and acellular (matrix) bone components，live mMSCs cells and necrotic mMSCs cells prior to engraftment within C57BL/6J, TLR4-mutant, MyD88-mutant and TRIF-mutant murine calvarial defects, and repair will be measured at designed multiple time points by live micro-CT radiography, computed tomography, and histology. Inflammatory molecule expression will be quantified in parallel by means of RT-PCR and LCM. Completion of these goals will provide a superior graft material for craniofacial reconstructive surgery.

颅面骨骨组织工程研究一直致力于研发出能与"自体骨"相匹配的生物材料，以解决两岁后颅骨缺损难以出现自发愈合的难题。研究表明具有骨再生潜能的多源干细胞能诱导骨再生，但移植后细胞不易存活、作用持续性差，限制其发挥骨修复作用。近年来宿主对细胞坏死产生的炎症反应有可能作用于组织修复的理论开始受到关注。前期研究中我们发现：①骨组织混悬液植入小鼠颅骨缺损后引起局部炎症反应，并可能通过TLR4介导骨缺损修复；②坏死的骨组织细胞成分植入颅骨缺损，同样激发了局部炎症反应并显示了良好的成骨效果。因此我们提出假说：细胞坏死可能通过TLR4作用于骨再生修复，并设计从整体动物、细胞和分子水平上，系统比较骨组织的细胞成分及基质成分对颅骨缺损愈合的影响，重点研究细胞坏死通过TLR4信号通路诱导骨修复的机制, 从而进一步丰富炎症反应对骨愈合的作用理论，为开发具有"自体骨"修复效果的组织工程骨提供新思路。

组织创伤中的受损基质、应激细胞及坏死细胞，具有诱导炎症及组织再生的作用。在对创伤后炎症反应的研究中，发现了 Toll样受体家族(Toll-like receptors, TLRs)。Toll样家族被认为能感知多种微生物成分、坏死细胞及应激细胞，在激活先天免疫系统方面发挥了重要作用。由于Toll受体在树突状细胞、巨噬细胞及骨细胞上高表达，学者开始关注Toll受体能否通过介导炎症而影响骨修复。. 本项目1）建立了 TLR4、TLR2全敲除（TLR4 /TLR2 KO)及髓系细胞TLR4敲除（Lyz-TLR4 KO）小鼠模型。2）在异体骨移植动物模型中，将同种异体骨、坏死骨细胞和骨基质与凝胶结合，植入WT、TLR2 KO及TLR4 KO颅骨缺损。通过影像学、组织学及免疫组化检测，分析异体骨不同成分对颅骨缺损修复的影响及TLR4受体信号在其中的作用。3）由于髓系细胞是巨噬细胞及破骨细胞的共同来源细胞，通过建立髓系细胞TLR4敲除小鼠，进一步研究TLR4介导炎症信号对骨修复再生的影响及可能机制。. 本项目研究发现：1）异体骨植入WT及TLR2 KO颅骨缺损模型中，其基质成分显示了与异体骨整体移植相类似的骨修复效果，而坏死细胞成分仅在移植术后初期有加速骨愈合的效果。而异体骨移植入TLR4 KO小鼠颅骨缺损中，术后巨噬细胞及破骨细胞浸润减少，植体重建及新骨生成均受到抑制，表明了基质是异体骨移植修复骨缺损的主要成分并且需要TLR4信号的介导。2）颅骨缺损模型中，WT及TLR2 KO小鼠骨愈合相似，而Lyz-TLR4 KO与 TLR4 KO均表现为颅骨修复加速，且术后破骨细胞浸润增加及破骨分化相关基因表达增强。从Lyz-TLR4 KO小鼠提取的前体细胞，其破骨细胞分化及吞噬能力也强于WT小鼠。因此破骨前体细胞即髓样细胞上TLR4缺失，能增强其向破骨细胞分化，从而加速颅骨缺损修复。. 本项目通过异体骨移植修复及颅骨缺损两个动物模型，及建立基因全敲除和细胞特异性基因敲除的小鼠模型，发现TLR4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。