钛合金抗结核药物微球控释系统的研制及其骨整合效应的基础研究

Spinal tuberculosis (STB), as the most common extra-pulmonary tuberculosis, can cause bone destruction, deformity, paralysis and even death. Applicants have been long engaged in surgical treatment of STB and basic research on dosage form of anti-TB, and found that the STB would lead to poor efficacy and easy relapse after surgical debridement and titanium alloy fixation, due to low local drug concentration. In the previous study, we reviewed the use of coaxial electrostatic spraying technology to prepare the core/shell structure microspheres of multiple first-line anti-TB drugs. In vitro tests, it was confirmed that the controlled-released microspheres could maintain minimum inhibitory concentration for 28 days. Titanium alloy instrumentation being derectly planted in lesion, makes it possible that titanium alloy fixation will be combined with controlled-released microsphere of anti-TB drugs for treatment of STB. The study is to envisage that the surfaces of the titanium plants are prepared to micro-nanometer modified pore, where wrapped with controlled-released microsphere of multiple first-line anti-TB drugs, for reconstruction of spinal stability and local release of anti-TB drugs at the same time. The titanium alloy fixation after surface modification are implanted into STB model of rabbits, which will induce and promote osteogenic differentiation, adhesion of bone marrow mesenchymal stem cells (BMSCs) for enhancing the osseointegration between implant-bone interface. Meanwhile, we will explore the molecular biological regulation of osseointegration related of titanium alloy plant after surface modification.

脊柱结核是最常见的肺外结核，可引起椎体骨质破环、脊柱畸形、截瘫甚至死亡。申请人长期从事脊柱结核手术治疗及抗结核药物剂型方面的研究，发现脊柱结核在病灶清除+钛合金内固定术后，因局部药物浓度低，常导致疗效差且易复发。在前期研究中我们率先利用同轴静电喷雾技术制备出了多种一线抗结核药物壳核结构缓释微球，体外实验证实缓释微球能维持最低抑菌浓度达28天。因钛合金内植物直接接触病灶，使得钛合金内植物联合抗结核药物微球缓释系统治疗脊柱结核成为可能。本项目设想对钛合金内植物进行表面改性，制备出微纳米级孔洞，然后将包裹有多种一线抗结核药物的缓释微球负载至微孔内，在重建脊柱稳定性的同时局部缓释抗结核药物。本研究将表面改性之后的钛合金内植物置入新西兰大白兔脊柱结核模型，引导并促进骨髓间充质干细胞黏附、增殖、成骨分化以增强内植物-骨界面间的骨整合性，并探讨经表面改性之后的钛合金内植物对骨整合的相关分子生物学调控。

中国作为全球结核病高发国家，结核防治形势异常严峻。骨关节结核是最常见的肺外结核，而抗结核药物化疗和清除病灶后的骨修复是治疗骨关节结核的关键举措。然而，目前临床上的抗结核药物化疗存在患者依从性差，毒性反应大，病灶血药浓度低等缺陷；而常用于骨缺损修复（特别是承重部位修复）的钛及其合金为生物惰性材料，缺少足够的骨传导和骨诱导活性。因此，研究具有抗结核和骨修复作用的功能材料对于进一步提升骨关节结核治疗具有重要的意义。.因此，本项目成功构建了钛合金支架联合药物微球控释系统，该系统由3D打印的多孔钛合金支架和核壳结构的PLGA/PLLA药物控释微球组成，具有在病灶处局部缓释药物和填补骨缺损的双重作用。.由于脊柱结核模型的构建中新西兰大白兔的存活率较低，我们调整研究方案为构建具有促成骨性能的钛合金支架联合药物微球控释系统。项目首先制备了负载双生长因子（VEGF和BMP-2）的药物控释微球，通过调节微球制备参数及生长因子的包埋方式，初步筛选出了具有最佳促成骨性能的微球，且负载的生长因子缓释期达27天。随后，本项目通过3D打印制备了多孔钛合金支架并进行表面活化处理，该支架具有与骨组织相接近的机械性能和适宜组织长入的孔径和孔隙率。最后利用明胶将微球包裹黏附在支架上，构建出了钛合金支架联合微球控释系统。通过体外实验发现，该系统可以促进成骨细胞和血管内皮细胞的分化，明显上调一系列成骨和成血管相关分子的表达。动物实验也验证了这一结论，钛合金联合微球控释系统内部新生骨量更多，与支架材料表面接触更紧密，促成骨分化和骨整合的能力更强。.此外，本研究还制备了多联一线抗结核药物壳核结构控释微球，将四联一线抗结核药物，异烟肼、盐酸乙胺丁醇、利福平和吡嗪酰胺负载于控释微球中，获得了四种药物释放进度一致的微球样品，且样品在27天释放时间内药物释放累计达80%以上。随后的体内和体外实验证实，抗结核药物控释微球对BMSCs细胞的成骨分化和体内骨组织的修复无明显抑制作用，生物相容性好。