新型可注射温敏性骨形态发生蛋白2活性肽长效缓释传递系统的研究

The key of the realization of long-term and slow-release delivery of bone-morphogenetic protein (BMP) is to resolve the problems of complexity of the preparation process, protection of activity of protein, burst effect, and incomplete release. There are two ways to resolve these problems, one is to replace BMP with BMP-derived peptide, and the other is to develop new slow-release materials, mechanism and technology. In this research, we will replace BMP-2 with BMP-2-derived peptide, and develop new slow-release mechanism and technology. Firstly, the BMP-2-derived peptide is modified with polyglutamate sequence, which can react with the (002) face of hydroxyapatite crystal on the surface of nano-hydroxyapatite/collagen (nHAC),and the peptide-binding efficiency and drug loading will be improved by the imitation of the combined mechanism of HA with bone sialoprotein and osteopontin. Secondly, the peptide will be injected to the areas needed using the former developed temperature-sensitive delivery system of chitosan/nHAC,which is injectable,biodegradable,biocompatible and osteoconductive. This procedure is designed for the following purposes: the local drug delivery, the prevention or reduction of burst and the protection of activity of protein. Thirdly，the polyglutamate sequence will be degraded by enzymolysis, and the BMP-2-derived peptide will be slow-released. This research will focus on the combined and adsorption mechanism of nHAC with BMP-2-derived peptide, and reveal the degradation rule of this new system, the biodegration and release mechanism of the BMP-2-derived peptide, the variation of biological activity, and the heterogenous ossification ability of this new system.

实现骨形态发生蛋白（BMP）长效缓释的关键是解决制备工艺复杂、蛋白活性保持、突释及不完全释放四大难题。解决途径是用BMP活性肽替代BMP或开发新的缓释材料、机制和技术。本课题拟用BMP-2活性肽替代BMP-2，并开发新的缓释机制和技术。通过模仿骨涎蛋白和骨桥蛋白与HA的结合机制，用多聚谷氨酸多肽序列修饰BMP-2活性肽，利用多聚谷氨酸多肽与纳米晶人工骨（nHAC）表面的HA组分的（002）晶面反应形成特异性生物结合，加强活性肽与nHAC载体的结合，提高载药量。通过前期开发的具有可注射性、生物降解性、生物相容性和骨传导性的温敏性壳聚糖/nHAC将活性肽注射入所需部位，实现局部释放、防止突释和保持蛋白活性的目的。通过多聚谷氨酸多肽的酶解实现BMP-2活性肽的缓释，重点研究活性肽与nHAC之间的作用机制和吸附机理，揭示新系统的降解规律、多肽降解与释放机制和生物活性变化规律及异位成骨诱导活性。

目前由创伤、肿瘤切除、或先天性畸形等引起的复杂骨缺损的修复仍缺乏满意的骨修复材料，研制能以可注射的方式应用于骨缺损部位形状和功能重建的骨修复材料已成为临床应用的迫切需要。本研究从骨的成分出发，采用仿生的方法制备了掺入Sr2+, HPO42- 和CO32- 离子的纳米晶人工骨（SrnHAC），并制备了一类具有仿生特性的可注射的并且可用于混合细胞应用于骨组织工程的温敏性智能水凝胶支架材料。通过模仿骨涎蛋白和骨桥蛋白与HA 的结合机制，用多聚谷氨酸多肽序列修饰BMP-2 活性肽（EBMP-2），实验证实多聚谷氨酸修饰的BMP-2活性多肽与SrnHAC表面的HA 组分的（002）晶面反应形成了特异性生物结合，加强了活性肽与SrnHAC 载体的结合，提高了载药量。通过具有可注射性、生物降解性、生物相容性和骨传导性的温敏性水凝胶将结合EBMP-2的SrnHAC复合材料注射入所需部位，实现了BMP-2活性肽局部释放、防止突释和保持蛋白活性的目的。同时体系能够在室温下保持流动性和可注射性，在体温时迅速转变为具有一定强度的物理凝胶，并且在溶胶—凝胶转变过程中，体系的温度和pH值变化都保持在人体生理可接受的范围内。大鼠异位成骨实验表明新体系具有较好的生物相容性，并且材料能够在体内完全降解，并促进新生胶原生成。本研究解决了BMP-2活性肽缓释制剂制备工艺复杂、蛋白活性保持、突释及不完全释放四大难题，实现了BMP-2活性肽的长效缓释。