摘要
目的构建钛金属表面聚多巴胺、聚乙二醇、聚乳酸-聚羟基乙酸、异烟肼抗结核控释涂层,评价其表面特征、体内外释药行为、体外抑菌性能及生物相容性。方法合成四臂巯基聚乙二醇(polyethylene glycol,PEG);通过迈克尔加成反应在钛金属(titanium,Ti)表面构建聚多巴胺(poly dopamine,PDA)涂层;利用多孔淀粉及四臂巯基PEG水凝胶负载异烟肼(isoniazid,INH),通过浇铸法或浸渍-提拉法附着于PDA涂层表面,同法再覆盖一层聚乳酸-聚羟基乙酸[poly lactic-co-glycolic acid,PLGA],制成Ti-PDA-PEG-PLGA-INH复合涂层;核磁氢谱图(proton nuclear resonance spectroscopy, HNMR)表征四臂巯基PEG末端官能团;扫描电镜(scanning electron microscope, SEM)观察其表面特征,高效液相色谱法(high per-formance liquid chromatography,HPLC)检测Ti-PDA-PEG-PLGA-INH的体外释药行为,并进行体外抑菌实验。取25只新西兰大白兔制作股骨髁骨缺损模型,植入表面构建PDA-PEG-PLGA-INH的钛棒,采用HPLC法于术后检测静脉血以及植入材料周围肌肉组织、骨组织中INH浓度。另取12只兔随机分为实验组和对照组,实验组于背部椎旁肌内及左侧股骨髁分别植入表面构建PDA-PEG-PLGA-INH的钛片和钛棒;对照组于相同部位植入空白钛片及钛棒,术后通过病理组织学观察材料的体内生物相容性。结果 Ti-PDA-PEG-PLGA-INH控释涂层在钛金属表面均匀铺展,呈半透明状,表面光滑。体外药物释放前8 h突释明显,累积释放量占总药量65%,第9天时累积释放量达90%,随后释放趋于平缓,总释放周期达20 d以上。体内释药实验显示材料植入后兔静脉血、材料周围肌肉及骨组织中INH浓度平稳上升,于第28天左右达最高峰;且在第56天时肌肉及骨组织中INH浓度仍高于最低抑菌浓度;体外抑菌实验显示构建Ti-PDA-PEG-PLGA-INH控释涂层的钛片周围形成明显抑菌环。体内生物相容性实验显示材料植入4周后肌纤维间及骨组织周围可见少量炎细胞浸润,材料周围形成疏松纤维包膜结构;植入8周后未见明显炎性细胞浸润,纤维包膜更为致密,并明显增厚。结论成功构建Ti-PDA-PEG-PLGA-INH抗结核控释涂层,体内、外均具有合理的释药行为,体外对结核分枝杆菌具有良好的抑菌效果,生物相容性良好,是一种有潜力的脊柱结核局部药物控释体系。
Objective To fabricate an anti-tuberculosis controlled drug release coating with Ti-PDA-PEG-PLGA-INH and to investigate its surface characteristics, in vivo and in vitro drug release behavior, and tissue biocompatibility. Methods 4-arm-polyethylene glycol (PEG) was synthesized first. Then cover the surface of titanium (Ti) with a layer of poly dopamine (PDA) by Michael addition reaction. Use porous starch and 4-arm-PEG as a carrier, load with isoniazid (INH), then attach to the surface of titanium by casting or sol-gel dip coating methods, and then cover with a layer of poly lactic-co-glycolic acid (PLGA) by the same method, to fabricate the Ti-PDA-PEG-PLGA-INH composite coating finally. The functional group of 4-arm-PEG was charac-terized by proton nuclear resonance spectroscopy (HNMR). The surface characteristics of Ti-PDA-PEG-PLGA-INH were evaluated by scanning electron microscope (SEM), while drug release behaviors were detected by high performance liquid chromatography (HPLC) and the cumulative release rate was calculated, and carry out the antibacterial performance in vitro. The animal model of femoral condyle bone defect was established in 25 New Zealand white rabbits. Titanium rods covered with PDA-PEG-PLGA-INH coating were implanted into defect area. INH concentrations were detected by HPLC in venous blood, muscle and bone tissue at each time point postoperatively. Another 12 rabbits were randomly divided into experimental group and control group, the experi-mental group was implanted with titanium tablets and titanium rods coated with PDA-PEG-PLGA-INH in the paraspinous muscle and left femoral condyles respectively, while the control group was implanted with a blank sheet of titanium tablets and titanium rods in the same place. Hematoxylin and Eosin Staining were used to observe the biocompatibility of the composite system in vivo at 28 and 56 days postoperatively. Results Ti-PDA-PEG-PLGA-INH controlled drug release coating uniformly distributed on the surface of plates and rods, with translucent form and smooth surface. In vitro INH release kinetics exhibited a short-burst release during the first 8h, and the cumulative release of the INH was about 65%. On the 9th day, the cumulative release of the INH was about 90%, and then the release tended to be flat, and the drug release behavior in vitro continued more than 20d. In vivo release test showed that the concentration of INH in vein blood, muscle and bone tissue around the composite system was increased steadi-ly postoperatively. On about the 28th day, the concentration reached the max. However, the INH concentrations in muscle and bone tissue around the composite system were still higher than the minimum inhibitory concentration (MIC) on the 56th day. The antibacterial test in vitro showed that the titanium tablets coated with PDA-PEG-PLGA-INH formed obvious bacterial inhibition zones. The pathological results indicated that mild inflammatory reaction was seen in the 4th week postoperatively, and the reac-tive capsule formed with loose connective tissue. In the 8th week postoperatively, there's no obvious inflammation occurred, and the reactive capsule became more dense and thicker. Conclusion The study successfully fabricated the Ti-PDA-PEG-PLGA-INH anti-tuberculosis controlled drug release coating, with reasonable release behavior both in vivo and in vitro, effective antibac-terial effect of Mycobacterium tuberculosis in vitro and good tissue biocompatibility, which is a potentially effective drug delivery system for spinal tuberculosis.
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2016年第11期725-734,共10页
Chinese Journal of Orthopaedics
基金
国家自然科学基金面上项目(81071454)
解放军第三○九医院课题重点项目(2014ZD-001)
关键词
结核
异烟肼
乙二醇
迟效制剂
Tuberculosis
Isoniazid
Ethylene glycol
Delayed-action preparations