载多因子缓释微球水凝胶仿生人工骨髓纳米人工骨快速血管化研究

The activation of vascularization in bone substitute, namely angiogenesis, is one of the key problems in the process of the in-growth of new bone. On the basis of our previous study funded by national natural fund, we found that due to the insufficient supply of blood flow in the internal part, both the degradation of bone substitute and the in-growth of new bone only occurred in the superficial part of artifical bone, hindered the efficient bone substitution. According to the molecular mechanism of angiogenesis, it is a promising strategy to controllably release multiple cytokines at the graft site, to promote rapid angiogenesis in the early stage and the vasculature construction further. In this reasearch, PLGA will be applied as drug carrier to prepare microspheres loading with angiogenic and osteogenic cytokines VEGF-165, PDGF-BB and BMP-2. The cytokine-loaded microspheres will then be combined with thermo-sensitive hydrogel to serve as bionic bone marrow. Under a relatively low temperature, the bionic bone marrow, using the hydrogel as matrix, shows good fluidity and can be loaded in the porous structure of artificial bone. It is then solidified under body temperature and works similarly to red marrow in cancellous bone gap filling. The difficulties on efficient and stable long-lasting combination of cytokines and artificial bone can be solved in this way. Taking advantage of the degradation property of the materials and the adjustable concentrations of cytokines, the burst release of cytokines can be avoided and a smooth and sequential controlled release can be achieved. It is a great chance to realize the substitute materials degradation and new bone in-growth in an omni-directional stereometric formula way, to solve the problems of nonunion, breakage and collapse of bone fracture or defect clinically due to incomplete bone transformation of artifical bone. It is an exploration for new ways of rapid angiogenesis in artifical bone and setting the foundation for its future clinical application.

移植骨血管化是移植骨替代过程中最关键和亟待解决的问题之一。在前期承担国自然基金研究中发现移植骨降解再生均在其浅表部位进行，内部因缺乏血供导致人工骨降解和新骨再生障碍。本课题依据血管生成机制在移植骨局部按血管和骨生成规律序贯缓释多种因子，促进人工骨早期快速血管化建立血管系统，采用PLGA载体制作促血管生成因子和成骨生长因子VEGF-165、PDGF-BB、BMP-2药物缓释微球，然后装载到温敏相变水凝胶中制作载多因子仿生人工骨髓，低温仿生人工骨髓呈液态下装载到人工骨孔隙中，复温后其固化于人工骨孔隙内达到类似于松质骨骨髓腔内骨髓填充效果，解决药物与人工骨难以高效、稳定和长期复合难题，利用材料降解规律调控各因子阶梯、平稳、序贯缓释，避免突释效应，实现人工骨全方位立体材料降解和新骨再生，解决临床上因人工骨内部骨替代不全导致的骨不愈合、断裂和塌陷问题。探索人工骨快速血管化新途径，为临床应用奠定基础。

背景：骨移植材料的再血管化是目前该领域亟待解决的重要问题之一，同时也是人工骨能否在临床上得到充分应用的关键和瓶颈。.内容和方法：本课题利用温敏水凝胶PLGA-PEG-PLGA将细胞因子（rhVEGF165，rhPDGF-BB，rhBMP-2）包埋并注射到靶位后形成原位固态结构，便可将细胞因子绑定到人工骨支架赋予其良好的骨诱导和血管诱导特性。首先，采用PLGA载体制作促血管生成因子和成骨生长因子rhVEGF165，rhPDGF-BB，rhBMP-2药物缓释微球，激光粒度分析仪检测微球的平均粒径，UV法测定吸光度并代入标准曲线计算其载药量和包封率。其次，利用凝胶色谱和流变仪对所制备的温敏水凝胶的结构和性能表征，确定其溶胶-凝胶转化相。于溶胶相时，将该水凝胶与细胞因子混合，实现对细胞因子溶解包埋，制备载多因子仿生人工骨髓。通过Elesa检测其凝胶相体外静止状态下降解缓释情况；通过RT-PCR检测该凝胶相对成骨细胞的生物学影响；于小鼠皮下注射检测其体内凝胶相降解规律和异位骨化情况。最后，利用熔融沉积制造（FDM）制备羟基磷灰石/聚己内酯解剖型仿生人工骨支架，将低温仿生人工骨髓呈液态下装载到人工骨孔隙中，复温后其固化于人工骨孔隙内达到类似于松质骨骨髓腔内骨髓填充效果，移植到所建骨缺损处进行重建和修复，并与自体骨移植对照，于术后4周、12周行大体观察、影像学和硬组织切片检测，比较四组骨骨移植体局部骨修复和重建情况。.结果和结论：1.该温敏性水凝胶是一种匀质的可逆性溶胶-凝胶热致聚合物，具有良好的相变温度（27-30℃）和良好生物可降解性，是一种良好的rhBMP-2、rhPDGF-BB、rhVEGF165等载体并能模拟自然骨修复过程相关讯息因子的释放。2.该水凝胶能减少rhBMP-2、rhPDGF-BB、rhVEGF165随体液扩散、将减少体内酶解消耗，降低药物的最小有效浓度和并发症；且rhBMP-2和rhVEGF165之间存在协同效应，rhVEGF165能诱导新骨中更多血管生成，有利于成骨细胞和骨髓间充质干细胞的迁移，促进骨生成。3.解剖仿生人工骨负载rhBMP2、rhVEGF165凝胶的复合体系能显著促进兔胫骨大段完全骨缺损早期修复和后期骨整合，具有替代临床自体骨移植的良好效果，这种骨结构和功能仿生将促进组织工程骨从实验研究向临床应用转化。