具有细胞募集特性的钛种植体新型缓释涂层的构建及其内源性成骨功效的研究

It is known that sustained release implant coating is the key for the long-term success of the implant in the inflammatory environment, but the shortage of timeliness and insufficient repair of bone defects still exist. Our previous research have shown that the new polylactic acid microspheres/coating and pH-sensitive chitosan nanoparticles are good at sustained release and objectives of timeliness, and capable of carrying different biological factors. Based on this, it is highly expected that the novel multilayer drug-loaded nanosparticles/coating should be achieved, which brings new hope to solve the above problem. The current project intends to prepare a novel pH-sensitive, double-layer sustained release nanoparticles combing with BMP-2 loaded poly(L-lactic acid) nanoparticles, OPG receptor-hydrazone bond-chitosan conjugate and SDF-1. By suction and infiltration method, the titanium implants functional coating with stem cell recruitment character will be prepared. With sequence release of multi-factor, it is expected that the osteoclast inhibition, stem cell recruitment and osteogenic differentiation should be achieved, which leads to repair of bone defects. Moreover, the relationship between the sequences controlled release mechanism of the novel functional coating and osteoclast and endogenous osteogenic effect in the inflammatory environment will be finally revealed. Generally, the project will be of great importance in rebuilding osseointegration in the inflammatory environment and provide new approach for the novel titanium implants coating and clinical application in the future.

种植体载药缓释涂层是提高种植体在炎性环境下远期成功率的关键，但仍存在释药时效性低以及修复骨缺损能力弱等不足。申请人及其合作团队前期的研究表明，新型聚乳酸缓释微球/涂层和pH敏感壳聚糖缓释纳米微球具有较好的缓释和目标时效性，能够搭载不同生物因子，因此基于其构建新型多层载药纳米微球和涂层可望为解决上述问题带来新的希望。本项目拟以聚乳酸缓释微球和OPG受体-pH敏感腙键-壳聚糖轭合物作为内、外层载体，包裹BMP-2和SDF-1因子，构建新型双层缓释纳米微球；通过负压吸引和渗涂法，在钛种植体表面制备具有细胞募集特性的pH敏感缓释涂层，通过多因子间的顺序控释，实现抑制破骨、干细胞募集和骨向分化，修复骨缺损；并最终揭示顺序控释机制与抑制破骨和内源性成骨功效之间的关系。该项目对于修复炎性环境下种植体局部骨缺损并重建骨整合具有重要意义，并可为钛种植体表面新型功能性涂层的研制和临床转化提供新的策略。

【背景和研究方向】如何抑制缺损区的骨吸收，促进骨再生，是种植体实现良好的骨整合、提高远期成功率的关键。【主要内容】本研究通过制备具有细胞募集特性的pH敏感新型壳聚糖双层缓释纳米微球，在钛种植体多孔涂层表面构建了新型功能性缓释涂层。分别对新型双层纳米微球和涂层的制备、释药特性、体外生物学活性和动物体内成骨特性进行了研究和评价。【重要结果和关键数据】通过单因素考察和正交试验对纳米微球的制备工艺参数进行优化，优选出制备双层载药纳米微球的最佳制备工艺参数，其粒径为（422.68±17.71）nm， rhBMP-2包封率为（72.87±0.24）%，载药量为（24.29±0.08）ng/mg；SDF-1的包封率为（81.46±0.18）%，载药量为（27.15±0.06）ng/mg；OPG的包封率为（73.69±0.23）%，载药量为（24.56±0.08）ng/mg。双层载药纳米微球在体外释放过程中呈现以初期快速释放和后期持久释放为特征的两相性。随着pH值的降低，微球的快速释放效应变得更明显。细胞学实验证明纳米微球中能够有效抑制破骨细胞的活性、诱导干细胞归巢促进骨髓间充质干细胞发生骨向分化。采用振荡渗涂交联法在微弧氧化的纯钛表面构建了载新型双层微球的功能性抗菌涂层,释药特性同双层载药纳米微球相似，且随着pH值的下降，各因子的快速释放效应更明显。进一步研究表明该涂层材料具有良好的体外生物安全性。细胞学试验表明该涂层具有一定的抑制破骨细胞成熟分化、促进干细胞募集迁移、促进干细胞成骨向分化，并促进钙盐沉积形成骨组织。动物实验结果显示，在骨缺损环境下植入种植体2个月后，新型涂层组骨-种植体接触率（55.34±7.22%）和种植体-骨结合强度（2.70±0.21MPa）较空白对照组（29.32±4.31%和1.78±0.23MPa）有显著性差异（P<0.05）。以上结果均证实新型功能性涂层具有良好的抑制破骨-促进干细胞募集、成骨的性能。【科学意义】本课题构建新型种植体表面涂层可以有效抑制破骨细胞的活性，诱导骨髓间充质干细胞归巢并发生骨向分化，为由炎症、外伤、肿瘤等原因造成的骨缺损的修复提供了一种新的思路。