载PaMZ/BMP-2的3D打印纳米HA抗结核支撑人工骨的构建及实验研究

The insufficient local drug concentration and the lack of bone-grafting materials for spinal reconstruction after focal debridement are problem in the treatment of spinal tuberculosis. The three-dimensional functional scaffold is a possible way to solve this problem. However， how to ensure the effective support of artificial bone, drugs release uniformly, and bone fusion is the key in this study. Micro regulation ability for materials of 3D printing technology find a new way to this problem. Based on the research on characteristics of n-HA composite material and the successful establishment of the model of rabbit’s spinal tuberculosis on the previous studies, this research intends to develop a 3D printing n-HA composite material scaffold with sustained-release microspheres contained new anti-TB drugs PaMZ and bone morphogenetic protein (BMP-2), develop an three-dimensional scaffold artificial bone contains anti-TB drugs. This research is aimed to complete the following things: 1.To develop n-HA composite material artificial bone of porous structure loaded PaMZ/BMP-2 using 3D printing technology; 2. The research of optimum composition and physical properties of the materials; 3. To explore the influence of PaMZ on BMP-2 performance; 4. To study the ability of anti-tuberculosis of the drug loading materials in vitro; 5. Applying the model of rabbit’s spinal tuberculosis to study the drug release , osteogenic property and toxicity to tissues in vivo. It is desired to develop an artificial bone with high osteogenic property, which is both sustainable and effective in anti-TB and excellent in characteristics of supporting grafting. This research can provide experimental data and theoretical basis for the new method in treatment of spinal tuberculosis.

病灶局部药物浓度不足及病灶清除术后植骨材料紧缺是脊柱结核治疗的难题，功能化三维支架人工骨是解决该难题的方向。但如何保证人工骨有效支撑、均匀释药及成骨融合是材料研究的关键，3D打印技术对材料结构的微观调控为此开辟了新途径。基于前期对纳米羟基磷灰石（n-HA）复合材料的特性研究及对兔脊柱结核模型的成功构建，本研究拟将抗痨新药PaMZ及骨形态发生蛋白-2（BMP-2）打印进n-HA材料支架中，研制载抗结核药的3D支架人工骨。本课题拟完成：1.研制载PaMZ/BMP-2的3D打印n-HA复合材料人工骨；2.探索载抗结核药3D人工骨的最优构成及其物理特性；3.探究PaMZ对BMP-2性能的影响；4.载药3D人工骨体外抗结核性能研究；5.应用兔脊柱结核模型进行体内释药、成骨及组织毒性研究，力图获得既具病灶高效、长程抗痨，又有优良支撑成骨性能的活性人工骨，为脊柱结核治疗的新方法提供实验支持及理论依据。

脊柱结核是严重危害人类健康的感染性疾病，结核性骨缺损的修复重建效果不佳、病灶局部药物浓度不足以及耐多药结核菌增多是脊柱结核手术治疗失败的主要原因。如何高效抗痨快速治愈病灶以及如何满意地修复结核性骨缺损重建脊柱的稳定是脊柱外科亟待解决的难题。3D打印技术、局部药物缓释系统与新型抗结核药物的发展与结合为解决这一难题提供了新的方向。本研究首先通过扫描电镜、X射线衍射观察法、差示扫描量热法等对目前对敏感结核菌和耐药结核菌都有效的抗结核新药组合Pa-824（Pa）、莫西沙星（M）、吡嗪酰胺（Z）和聚乳酸-羟基乙酸共聚物（PLGA）的最优配方进行筛选，在比较了复溶法、浸渍法和挂浆法后，采用低温3D打印技术将PaMZ- PLGA固体分散体和成骨形态发生蛋白BMP-2与纳米羟基磷灰石（nHA）生物墨水通过一定比例组合后制备出载PaMZ/BMP-2的纳米HA人工骨。该材料在体外抗压缩强度可达4.82±0.29Mpa，Pa、M、Z在84天的累积释放率分别为：（71.0±0.71）%、（96.01±1.54）%、（96.39±2.22）%。在与兔骨髓间充质干细胞（BMSCs）共培养的过程中表现出良好的成骨性能；在与敏感结核菌和耐药结核菌共培养的实验中，时间杀菌曲线提示支架在植入后第三天开始表现出明显的抑菌性能，在植入10天后已能彻底抑制结核杆菌的生长和繁殖，支架在体外释放84天时的药物浓度仍高于10MIC（最低抑菌浓度）。在体内实验中，通过将支架植入我们构建的兔脊柱骨缺损模型中表明，支架具有良好的骨传导性、骨诱导性，成骨性能与自体骨相当，无肝肾毒性。受实验条件限制我们的体内释药实验选择将支架植入SD大鼠体内，结果显示支架体内释药行为与体外相似，对血药浓度的影响小，结合体外实验结果推测3D打印载PaMZ/BMP-2的纳米HA人工骨在体内也具有良好的抗结核杆菌性能。体内外实验结果表明，该材料已具备修复结核性骨缺损理想植骨材料的基本要求，但仍有改进空间，如能进一步提升支架的抗压缩性能至与人体密质骨强度相当，那么在未来的临床应用中将具有更广阔的前景。