钛表面聚乙二醇水凝胶、载庆大霉素交联淀粉微球抗感染药物控释系统构建及生物学性能

Fabrication and biological effect investigation of the antibacterial drug delivery system with PEG-hydrogel and gentamicin-loaded-CSt on titanium surface

目的构建及评价钛表面聚乙二醇水凝胶、载庆大霉素交联淀粉微球抗感染药物控释系统的表面特征、溶胀行为、体外释药、抑菌性能、体内抗感染性能与生物相容性。方法制备交联淀粉（cross-linked starch，CSt），使用CSt负载庆大霉素（gentamicin，GEN）制成载药交联淀粉微球（GEN@CSt）并加入四臂巯基聚乙二醇（polyethylene glycol，PEG）制备载药水凝胶；在钛金属表面构建聚多巴胺（poly dopamine，PDA）涂层；将载药水凝胶附着于PDA涂层表面，并覆盖聚乳酸-聚羟基乙酸（poly lactic-co-glycolic acid, PLGA）阻隔层，制成层次为Ti-PDA-PEG（GEN@CSt）-PLGA的复合涂层。体外表征复合涂层的形貌与溶胀特性，检测复合涂层的体外释药行为。使用金黄色葡萄球菌（SAU）、表皮葡萄球菌（SEP）、大肠埃希菌（ECO）菌株行体外抑菌实验。取36只新西兰大白兔制作股骨髁骨感染模型，并随机分为3组，分别植入负载庆大霉素复合涂层的钛棒、未负载庆大霉素的复合涂层钛棒与裸钛棒，评价体内抗感染效果。另取12只兔随机分为两组，分别于股骨髁植入复合涂层钛棒与裸钛棒，观察材料的体内生物相容性。结果复合涂层牢固黏附在钛金属表面，呈光滑、半透明状。不同CSt／PEG配比溶胀行为表现不同，不含淀粉的凝胶平衡溶胀度比值维持在7．4；CSt／PEG配比达到1：1后，平衡溶胀度比值维持在3．0。体外释药实验显示前8h释放速率较快，前7d的累积释放量约占药物总量的83％，总释放周期达13d。体外抑菌实验中，抑菌环平均直径SAU组为（3．6±0．13）cm，SEP组为（3．4±0．11）cm，ECO组为（3．7±0．10）cm。体内抗感染实验显示载药复合钛棒组动物伤口感染、发热、体重改变、白细胞计数与病理学炎症反应均优于未载药复合钛棒组和裸钛棒组。体内生物相容性实验显示，载药复合钛棒组和裸钛棒组在纤维组织增生和炎性反应方面变化基本一致。结论Ti-PDA-PEG（GEN@CSt）-PLGA复合涂层具有合理的释药行为，能抑制常见致病菌生长，具有优异抗感染性能，生物相容性良好，是一种有潜力的骨科抗感染局部药物控释体系。

Objective To fabricate an-antibacterial controlled drug delivery system with PEG-hydrogel and gentamicinloaded-CSt on titanium surface, and to investigate its surface characteristics, swelling behavior, drug release behavior in vitro, antiinfection performance in vivo, and tissue biocompatibility. Methods Cross-linked starch （CSt） was synthesized first and then CSt was loaded with gentamicin （GEN） as a carrier （GEN@CSt）, then 4-arm-polyethylene glycol （PEG） was added to it which was mixed by ultrasound. The surface of titanium （Ti） was covered with a layer of poly dopamine （PDA）. The drug-loaded hydrogel was fixed to the titanium surface, subsequently capped by poly lactic-co-glycolic acid （PLGA） membranes, and then the Ti-PDA-PEG （GEN@CSt）-PLGA composite coating was fabricated finally. Surface morphology of the system was observed, while the swelling behavior was characterized; release behavior of the composite coating was detected; the bacteriostatic experiments were carried out with staphylococcus aureus （SAU）, staphylococcus epidermidis （SEP） and escherichia coli （ECO） in vitro. The animal models of infected bone defect was established in 36 New Zealand white rabbits. These animals were randomly divided into three groups. Group 1 animals were implanted with drug-loaded composite coatings. Group 2 animals were implanted with drug-free composite coatings. Group 3 animals were implanted with bare titanium rods. The infection data were collected periodically to carry out antiinfection experiments in vivo. Another 12 rabbits were divided into the experimental group and the control group randomly. Biocompatibility of the materials was observed by histopathology after implantation of the corresponding materials into the femoral condyle. Results The composite coating adhered to the titanium surface firmly, presenting a smooth and translucent shape. The ratio of CSt/PEG affects swelling behavior varied, starch-free gels maintained an equilibrium swelling of 7.4, after the ratio reached 1 ： 1, the equilibrium swelling ratio remained at 3.0. In-vitro the release rate of the first 8 h was fast, and the cumulative release amount accounted for 83% of the total in the first 7 days, lasting more than 13 d. In vitro antibacterial test, the average diameter of the inhibition ring was 5.6±0.13 cm （SAU）, 3.4±0.11 cm （SEP）, 3.7±0.10 cm （ECO）. In-vivo anti-infection experiment, the infection situation of the group 1 was better than the control groups 2 and 3. The pathological results indicated that inflammatory reaction in the experimental group was basically the same as the control group. Conclusion The study successfully fabricated the antibacterial controlled drug delivery system with PEG-hydrogel and gentamicin-loaded-CSt on titanium surface. The system has a reasonable drug release behavior, and effectively inhibited the growth of bacteria in vivo and in vitro. It also has good biocompati- bility to stand a promising strategy to improve the orthopedics anti-infection.