同种异体皮质骨表面结构化联合LL-37偶联双重修饰治疗大段骨缺损的实验研究

Currently，the long term follow-up of using cortical allograft transplantation for segmental bone regeneration is still not satisfied. Dense surface, poor osteoinductivity and in absence of cytokines are the major disadvantage. As a means to enhance osteoinductivity and osseointegration, the favorable and predictable effect of surface structuring applied to the implant surface has been well-recognized. Surface features can enhance the adhesion and proliferation of osteoblast, and also induce the express of bone morphogenetic protein 2(BMP2). Our preliminary results first demonstrated that the application of a surface structuring to the native (smooth) cortical bone surface increases local bone formation and graft integration. LL-37/hCAP-18 is the only cathelicidin-derived antimicrobial peptide found in human with a wide range of natural antimicrobial activities. In addition, it was found to be a novel multi-function peptide, which is taking an important role in the wound healing、bone regeneration and re-endothelialization. It can induce the express of over 40 kinds of cytokines including functional cytokines for bone regeneration and re-endothelialization as BMPs, Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), human acidic fibroblast growth factor (haFGF). Furthermore, LL-37 can activate angiogenic early outgrowth cells (EOCs) and Endothelial progenitor cells(EPCs) to form vessel-like structures for re-endothelialization, mediated respectively by formyl peptide receptor 2 (FPR2) and nuclear factor kB (NF-kB) pathway. The multi-function characters of LL-37 make it have a great potential for enhancing segmental bone regeneration. Based on the above and our preliminary results, we expect to screen out a best method for surface structuring cortical allograft to stimulate osteoinductivity, considering the physical and chemical properties of cortical bone. In addition, couple LL-37 on the structured bone surface. We expect the dual-modification both from structural and cytokines leaves could make cortical allograft of multiple identities as osteoinductivity, promoting re-endothelialization and anti-bacterial, a traditional and novel allograft material will be provided for enhancing segmental bone regeneration. Its function will be evaluated both in cell and animal levels, mechanism also be initially described.

表面致密、缺乏骨诱导性和细胞因子缺失是同种异体皮质骨移植治疗大段骨缺损愈后不佳的主要因素。表面结构化能激发材料骨诱导性，促进骨细胞粘附和骨形态发生蛋白2（BMP2）的表达和富集。本课题组首次发现该作用同样适用于异体皮质骨。LL-37作为唯一的人源cathelicidin抗菌肽，除抗菌特性外，在创伤修复、骨和血管再生中也扮演了关键角色：它能诱导BMP2和酸性成纤维细胞生长因子（aFGF）表达促进骨再生；上调血管内皮生长因子表达并通过甲酰肽受体2（FPR2）和核因子(NF-kB)途径激活内皮前体细胞形成血管样组织。我们推测LL-37的多功能性对于骨缺损修复也有多方面促进作用。综合上述和我们的研究基础，本课题组拟筛选一种适合皮质骨理化性质的表面结构化方法以激发其骨诱导性，并偶联LL-37，表面结构和细胞因子水平"双重修饰"赋予其骨诱导、促血管化和抗菌的多重特性，并在细胞和整体水平评价其功能。

项目的背景：表面致密、缺乏骨诱导性和细胞因子缺失是同种异体皮质骨移植治疗大段骨缺损愈后不佳的主要因素。表面结构化能激发材料骨诱导性，促进骨细胞粘附和骨形态发生蛋白2（BMP2）的表达和富集。本课题组首次发现该作用同样适用于异体皮质骨。.主要研究内容：1、从表面形态（水接触角测定、扫描电镜SEM）、生物力学、生物毒性（MTT 法）和体外骨诱导能力（共培养MG63 细胞）多角度评价不同改性方式结构化同种异体皮质骨的安全性和效率。2、偶联 LL-37 与结构化的同种异体皮质骨表面，荧光免疫、共聚焦扫描技术（CLSM）检测产物表面浓度及稳定性和时效性；分别共培养成骨细胞、内皮细胞、和流氏细胞技术观察细胞粘附型态、增值和分化情况，构建大段骨缺损、大段骨缺损+感染动物模型的构建。3、利用股骨大段骨缺损动物模型，观察双重修饰对植骨愈合过程成骨和血管化的影响及其机制：采用显微CT、SEM、免疫组化观察宿主骨骨长入和粘附以评价成骨情况。.重要结果和关键数据及其科学意义：本课题组从多角度评价不同改性方式结构化同种异体皮质骨的形态学研究，发现15%磷酸改性同种异体皮质骨1min后具有一定激发其骨表面的诱导活性和安全性。表面改性的同种异体皮质可导致其表面接触角的下降，提示经过磷酸改性后，同种异体皮质骨的表面亲水性提高。改性后的同种异体皮质生物力学无明显改变。MTT法评价15%磷酸改性的同种异体皮质骨前后的进行的细胞培养显示其生物毒性无明显改变。扫描电镜评价15%磷酸改性的同种异体皮质骨前后的进行的细胞培养显示其对细胞粘附和增殖活性增加。初步完成了偶联LL-37于结构化的骨面，初步尝试骨缺损模型的构建。活性小分子物质Icarrin（ICA）促进MC3T3和HUVC细胞增殖，并且分别抑制双氧水诱导的凋亡和细胞的死亡，并且更好的诱导MC3T3向成骨细胞分化，并促进ALP的表达，并在动物模型中，骨长入，骨愈合以及micro-CT显示骨静态参数的良好，我们的实验初步提示低浓度磷酸表面结构化同种异体皮质骨是一种合适的骨表面改性的方法，并且能提高骨表面的生物相容性并激活其骨诱导能力，加快骨长入和爬行替代速度，结合活性小分子ICA，能比较好的增加缺损骨的愈合以及骨长入，为大段骨缺损的治疗提供一种传统又新颖的植入材料可能提供实验依据。