载万古霉素/聚乳酸-羟基乙酸共聚物缓释涂层多孔镁合金支架的抗菌和成骨性能研究

Antibacterial and osteogenic properties of vancomycin/PLGA-coated porous magnesium alloy scaffolds

目的制备载万古霉素/聚乳酸-羟基乙酸共聚物(PLGA)多孔镁合金支架, 并在体外评价其理化性质、降解及药物释放行为、细胞相容性和抗菌性能等。方法利用模板复制技术制备多孔镁合金(Mg-2Zn-0.3Ca)支架, 通过高温氟化处理后获得MgF_(2)表层, 再将其浸没于含有盐酸万古霉素/PLGA的二氯甲烷溶液后匀速提拉, 获得载万古霉素/PLGA的多孔镁合金载药支架。根据制备过程中各阶段产物(镁合金、氟化镁合金、PLGA涂层氟化镁合金、载万古霉素/PLGA氟化镁合金支架)分组, 分别为Mg组、MgF_(2)组、PLGA组、万古霉素/PLGA组。将各组支架制备完成后立即测定并评价其材料学表征、降解速率、药物释放速率、抑菌性能、血液相容性及促细胞增殖分化能力等。结果成功制备出载万古霉素/PLGA镁合金支架, 其孔隙率平均为66.39%, 降解速率[(0.540±0.102) mm/年]显著低于镁合金支架[(10.048±0.297) mm/年], 差异有统计学意义(P<0.05)。载万古霉素/PLGA在Hank平衡盐溶液中降解的pH值接近生理酸碱度;万古霉素在前48 h释放速度较快, 48 h后逐渐变缓, 11 d时累积药物浓度达最大值(43 mg/L), 11 d后仍缓慢释放。万古霉素/PLGA组支架的抗菌率(97.89%±0.28%)显著高于Mg组(74.92%±2.20%)、MgF_(2)组(78.46%±2.59%)、PLGA组支架(61.08%±4.21%), 差异均有统计学意义(P<0.05);其溶血率为0.55%, 远低于ISO 10993-4标准要求(5%);其浸提液培养大鼠骨髓间充质干细胞1、3、7 d, 促细胞增殖率分别为104.80%±5.13%、112.36%±2.07%、127.79%±4.61%;培养14 d时钙结节明显增多, 吸光度OD值为1.189±0.020, 显著高于Mg组(0.803±0.020)、MgF_(2)组(0.878±0.028)、PLGA组支架(0.887±0.026), 差异均有统计学意义(P<0.05)。结论载万古霉素/PLGA的多孔镁合金支架在体外表现出良好的材料学性能、抗菌性能、生物相容性及促成骨性能。

Objective:To evaluate the physicochemical properties, degradation and drug release behaviour, cytocompatibility and bacteriostatic properties in vitro of porous magnesium alloy scaffolds containing vancomycin/poly(lactic-co-glycolic acid) (PLGA). Methods:Porous magnesium scaffolds (Mg-2Zn-0.3Ca) were prepared using the template replication technique. The MgF_(2) surface layer was obtained by high temperature fluorination. The vancomycin/PLGA porous magnesium alloy drug-loaded scaffolds were obtained by homogeneous lifting after submersion in a dichloromethane solution of PLGA containing vancomycin hydrochloride. According to the products at each stage of the preparation (scaffolds of magnesium alloy, magnesium fluoride alloy, PLGA coated magnesium fluoride alloy, and vancomycin/PLGA magnesium fluoride alloy), they were divided into an Mg group, an MgF_(2) group, a PLGA group, and a vancomycin/PLGA group. Immediately after preparation, the material science characterization, degradation rate, drug release rate, antibacterial properties, hemocompatibility, and cell proliferation and differentiation ability of the scaffolds were measured and evaluated. Results:Vancomycin-loaded magnesium alloy scaffolds were successfully prepared with an average porosity of 66.39%. Their degradation rate [(0.540±0.102) mm/year] was significantly lower than that of the Mg ones [(10.048±0.297) mm/year] ( P<0.05). Their pH of degradation in Hank equilibrium salt solution was close to the physiological pH. Their release of vancomycin was fast in the first 48 h and gradually slowed down after 48 h. Their cumulative drug concentration reached a maximum of 43 mg/L at d 11;their vancomycin was still released slowly after d 11. The antimicrobial rate in the vancomycin/PLGA group (97.89%±0.28%) was significantly higher than that in the Mg group (74.92%±2.20%), the MgF_(2) group (78.46%±2.59%) and the PLGA group (61.08%±4.21%) ( P<0.05). Their hemolysis rate (0.55%) was much lower than the requirement of ISO 10993-4 (5%). The extract liquid from them promoted the proliferation of rat bone marrow mesenchymal stem cells (BMSCs), showing a gradually increased proliferation rate from d1 (104.80%±5.13%) to d3 (112.36%±2.07%) and d7 (127.79%±4.61%). The calcium nodules in BMSCs were significantly increased at d 14, with an OD value of absorbance of 1.189±0.020, significantly higher than that in the Mg group (0.803±0.020), the MgF_(2) group (0.878±0.028) and the PLGA group (0.887±0.026) ( P<0.05). Conclusion:Vancomycin/PLGA-loaded porous magnesium alloy scaffolds exhibit good material properties, antibacterial properties, biocompatibility and osteogenic properties in vitro.