聚多巴胺修饰聚己内酯电纺膜载铜涂层的制备及抗菌和细胞性能评价

Preparation of copper loaded coating on polydopamine-modified polycaprolactone electrospun membrane and antibacterial and cellular properties evaluation

背景:在口腔领域,引导骨组织再生膜被广泛用于口腔种植、牙周、颌面外科领域,用于快速有效的实现组织再生,但如何防止植入物周围感染一直是亟待解决的难题。目的:制备聚己内酯静电纺丝纤维,并通过聚多巴胺对其进行表面改性并黏附铜离子,以制备具有抗菌成骨双重功效的引导骨组织再生膜。方法:分别制备聚己内酯电纺膜、聚多巴胺修饰聚己内酯电纺膜与聚多巴胺修饰载铜(铜离子浓度分别为0.01,0.1,1 mol/L)聚己内酯电纺膜。将MC3T3-E1细胞分别接种于上述电纺膜上,通过CCK-8实验检测细胞增殖;将变形链球菌分别与上述电纺膜共培养,检测各组抑菌率。通过CCK-8与抑菌率实验结果确定合适的载铜浓度,用于后续实验。检测聚多巴胺修饰载铜聚己内酯电纺膜的体外缓释性能。将MC3T3-E1细胞分别接种于聚己内酯电纺膜、聚多巴胺修饰聚己内酯电纺膜与聚多巴胺修饰载铜聚己内酯电纺膜上,通过活/死细胞染色分析细胞活性,碱性磷酸酶染色评价细胞早期成骨分化;将变形链球菌与上述3种电纺膜共培养,活/死细菌染色评价电纺膜抗菌活性。结果与结论:①CCK-8实验结果显示,载铜(0.01,0.1 mol/L)电纺膜可促进MC3T3-E1细胞的增殖,载铜1 mol/L电纺膜抑制MC3T3-E1细胞的增殖;载铜电纺膜的抑菌率高于聚己内酯电纺膜、聚多巴胺修饰聚己内酯电纺膜,并且载铜0.1 mol/L电纺膜的抑菌率高于载铜0.01 mol/L电纺膜;结合上述实验结果选择载铜0.1 mol/L电纺膜进行后续研究;②载铜电纺膜体外第28天释放的铜离子浓度约为5.26 mg/L(82.8μmol/L),处于无细胞毒性且有效抗菌浓度范围内(10^(-4)-10^(-5 )mol/L);③活/死染色显示,载铜电纺膜具有良好的细胞相容性与抗菌性;④碱性磷酸酶染色显示,聚多巴胺修饰聚己内酯电纺膜与聚多巴胺修饰载铜电纺膜的促成骨能力高于聚己内酯电纺膜;⑤结果表明,聚多巴胺修饰载铜聚己内酯电纺膜具有良好的促成骨能力与抗菌性能。

BACKGROUND:In the field of oral cavity,the guided bone tissue regeneration membrane is widely used in oral implant,periodontal and maxillofacial surgery to achieve rapid and effective tissue regeneration.However,how to prevent the infection around the implant has always been a problem to be solved.OBJECTIVE:To prepare polycaprolactone electrospun fiber,and to modify its surface with polydopamine and adhere to copper ions,so as to prepare a guided bone tissue regeneration membrane with dual functions of antibacterium and osteogenesis.METHODS:Polycaprolactone electrospun membrane,polydopamine modified polycaprolactone electrospun membrane and copper(0.01,0.1,1 mol/L)loaded polydopamine modified polycaprolactone electrospun membrane were prepared,separately.MC3T3-E1 cells were seeded on the electrospun membrane and cell proliferation was detected by CCK-8 assay.Streptococcus mutans was co-cultured with the electrospun membrane to detect the antibacterial rate.According to the results of CCK-8 assay and inhibition rate experiment,the suitable copper loading concentration was determined for subsequent experiments.The in vitro sustained release properties of copper loaded polydopamine modified polycaprolactone electrospun membrane were detected.MC3T3-E1 cells were seeded on the three kinds of electrospun membranes,separately.The cell viability was analyzed by live/dead cell staining and the early osteogenic differentiation was evaluated by alkaline phosphatase staining.Streptococcus mutans was co-cultured with three kinds of electrospun membranes and the antibacterial activity of electrospun membranes was evaluated by live/dead bacterial staining.RESULTS AND CONCLUSION:(1)CCK-8 results showed that copper(0.01,0.1 mol/L)loaded polydopamine modified polycaprolactone electrospun membrane could promote the proliferation of MC3T3-E1 cells.Copper(1 mol/L)loaded electrospun membrane inhibited the proliferation of MC3T3-E1 cells.The antibacterial rate of copper loaded electrospun membrane was higher than that of polycaprolactone electrospun membrane and polydopamine modified polycaprolactone electrospun membrane.The antibacterial rate of copper(0.1 mol/L)loaded electrospun membrane was higher than that of copper(0.01 mol/L)loaded electrospun membrane.Based on the above experimental results,0.1 mol/L copper loaded electrospun membrane was selected for further study.(2)The concentration of copper ion released from the electrospun membrane on the 28^(th) day in vitro was about 5.26 mg/L(82.8μmol/L),which was in the range of non-cytotoxic and effective antibacterial concentration(10^(-4)-10^(-5)mol/L).(3)The results of live/dead staining showed that the copper loaded electrospun membrane had good biocompatibility and antibacterial activity.(4)Alkaline phosphatase staining showed that the osteogenic ability of polydopamine modified polycaprolactone electrospun membrane and copper loaded electrospun membrane was higher than that of Polycaprolactone electrospun membrane.(5)The results showed that copper loaded polydopamine modified polycaprolactone electrospun membrane had good bone promoting and antibacterial properties.