长效抑菌复合材料的构建及骨诱导再生性能研究

Osteoradionecrosis of the jaw (ORNJ) is a severe complication after radiotherapy for head and neck cancer. Infection, especially caused by anaerobes has been confirmed in ORNJ and during this process, it usually accompanies with bone defects. The current treatment of ORNJ, including antibiotics medication and autologuous reconstruction, has limitations. For drug delivery system, it was difficult to achieve both antibacterial effect and osteoinductivity simultaneously. Meanwhile these systems usually have drawbacks such as high initial burst release, non-steady release rate and entrapped biomolecules partially denatured. Improving the outcome of ORNJ treatment is still chanllenging. Hence, a composite microparticle system with long term antibacterial effect and osteoinductivity will be fabricated in this proposal. Wet chemical method coupled with ion exchange will be applied to synthesize porous hydroxyapatite (HAp) microparticles with organic addtitive (casein) as modifier. Casein has the potential to promote mesenchymal stem cell differentiating into osteoblast. Since HAp microparticles are surface charged in aqueous phase, zinc (Zn) will be introduced into HAp so as to further increase the charge capacity and thus drug loading. More interestingly, Zn has antibacterial effect, especially targeting oral anaerobes; meanwhile Zn can also promote bone formation. Thus HAp microparticles self have antibacterial effect and enhanced osteoinductvity. Bone morphogenetic protein (BMP-2) will be loaded onto HAp. Before emulsion method carried out to fabricate the composite microparticles, the surface of HAp microparticles will be modified to improve the compatibility of HAp in poly(lactide-co-glycolide) (PLGA) phase and also minimize the negative effect of organic solvent on BMP-2. The antibacterial property and osteoinductivity of HAp, combined with the controlled release of BMP-2, will make the composite system a potentially effective treatment strategy for bone defects resulted from ORNJ.

颌骨放射性骨坏死（ORNJ）是放疗诱发的严重并发症。以厌氧菌为主的多种细菌已被证实在其发病中的作用且ORNJ往往伴随有骨缺损。目前，临床上治疗ORNJ，包括服用抗生素与术后重建，都有局限性。由生物材料制备的药物载体不易同时实现抑菌与促骨再生，且存在初始释放速度过快、释放速度不稳定与所负载生物大分子的失活等问题。因此，本项目拟构建同时具有长效抑菌与骨诱导性能的结构规整可控的复合微球体系。通过酪蛋白调节制备多孔性羟基磷灰石（HAp）微球，辅以离子替换引入锌，使得HAp自身具有长效抑菌特别针对口腔厌氧菌，与双重促进骨再生的潜力。负载骨形成蛋白（BMP-2），通过表面改性提高BMP-2的稳定性与HAp在有机相（PLGA）中的相容性，最终制得复合微球体系。该体系，因无机载体自身具有抑菌性和骨诱导性，综合可控的BMP-2缓释，在治疗ORNJ领域有很强应用潜力。

本项目成功制备了可促进骨再生的复合支架体系。先由湿化学法制备多孔HAp复合微球，后将其包埋在聚合物基质中。在水相中，探索了用有机质调节HAp微球成形的最优制备参数。研究了BMP-2的最佳负载条件以及表面改性的最佳方案。研究了复合支架的体外释放行为以及促进骨再生能力。经酪蛋白等有机质调节，可由CaCl2与Na2HPO4水溶液成功制备分散性良好的HAp纳米粒子，亦可成功引入Zn离子；还可先由酪蛋白调节制备CaCO3微球，再将其原位转化为HAp。为模拟骨ECM，应用静电纺丝将酪蛋白/碳酸钙微球包埋在PCL纤维中，并通过明胶接枝表面改性。为寻找复合电纺纤维支架的最佳组成，制备了含不同浓度微球的纤维膜，微球的加入并没有明显改变纤维直径。由于球霰石的存在，复合膜的生物矿化性能得到显着增强。经过FDA染色法和MTT法，以及分析Runx2、COL-I和ALP等成骨基因的表达及监测ALP的活性，发现微球的存在促进了MSCs增殖和成骨分化，特别是样品P-20表现出最优应用于骨组织工程的潜力。受酪蛋白（CA）具有天然的结合钙离子能力的启发，应用壳聚糖（CS）作为阳离子聚合物，采用层层自组装技术制备了多层膜（(CA/CS)n）。多层膜有更多的羟基磷灰石晶体沉积。将MSCs接种到这些样品上，与TCP对照组相比，多层膜显著促进了MSCs粘附、增殖及成骨分化。深入研究组装过程所获得的客观规律，有望指导其他活性表面的构建。该配方与该技术，避免了生物材料来源有限等不足，且可应用于具有任意形状骨组织工程材料的表面改性，具有普适性。为构建具有骨诱导性且负载生长因子BMP-2的可注射三维支架体系，引入肝素，与酪蛋白协同调节碳酸钙微球，将其作为生长因子缓释的载体并与纤维蛋白胶复合，制备可注射三维支架，后对该支架的骨诱导性能进行体外评价。肝素的添加有助于生长因子的组装，且引入碳酸钙，纤维蛋白胶支架能促进干细胞成骨分化，尤其以添加BMP-2后效果最为明显。