纯钛表面载阿司匹林微球与聚多巴胺复合涂层促进成骨分化

Aspirin-loaded chitosan nanoparticles and polydopamine modified titanium sheets improve osteogenic differentiation

背景:钛作为骨替代材料已在口腔种植领域中得到广泛应用,但其生物惰性会影响植入早期与骨组织形成稳定的结合,因此探索通过表面改性来提高钛的成骨活性是很有必要的。目的:探讨钛表面载阿司匹林的壳聚糖微球与聚多巴胺复合涂层对体外成骨细胞活性的影响。方法:取纯钛片,表面分别构建聚多巴胺涂层和载阿司匹林的壳聚糖微球与聚多巴胺复合涂层,采用扫描电镜和接触角检测对改性前后钛片的表面微观形貌和亲水性进行表征,检测纯钛表面阿司匹林纳米微球涂层的体外缓释性能。将大鼠骨髓间充质干细胞分别接种于纯钛片与两种改性钛片上培养,采用细胞骨架染色观察钛片表面细胞的铺展形态,CCK-8实验测定细胞的增殖活性,碱性磷酸酶染色和免疫荧光染色评估钛片表面细胞的成骨分化能力。结果与结论:①扫描电镜显示,纯钛表面相对光滑,聚多巴胺改性后出现沉积物及颗粒状突起,阿司匹林微球呈圆球形且粒径分布均匀;聚多巴胺涂层组与阿司匹林微球涂层组钛片的亲水性均明显优于纯钛组(P<0.05);阿司匹林在微球的包裹下呈现缓慢持续释放;②细胞骨架染色显示,纯钛表面的细胞伸展不充分,聚多巴胺涂层组钛片表面的细胞伸出少量伪足,阿司匹林微球涂层组钛片表面的细胞伸展良好;③CCK-8实验结果显示,3组钛片均无明显细胞毒性,且随着细胞培养时间的延长,阿司匹林微球涂层组钛片表面的细胞增殖速率高于其他两组(P<0.05);④阿司匹林微球涂层组钛片表面细胞的碱性磷酸酶活性最高,免疫荧光显示该组细胞中成骨相关蛋白碱性磷酸酶的荧光强度最强;⑤结果表明,纯钛表面载阿司匹林的壳聚糖微球缓释涂层可以增强大鼠骨髓间充质干细胞的增殖和黏附,并促进其成骨向分化。

BACKGROUND:Titanium as a bone substitute material has been widely used in the field of oral implant devices,but its bioinert would affect the stable combination with bone tissue at the early stage of implantation.Therefore,exploring to improve the osteogenic properties of titanium through surface modification is necessary.OBJECTIVE:To investigate the effect of composite coating of aspirin loaded chitosan nanoparticles and polydopamine on the viability of osteoblasts in vitro.METHODS:The titanium sheets modified by polydopamine coating as well as aspirin loaded chitosan nanoparticles and polydopamine composite coating were constructed.The surface morphology and microstructure of the coatings were characterized by scanning electron microscopy.In vitro sustained-release properties of aspirin nanosphere coatings on pure titanium were detected.Rat bone marrow mesenchymal stem cells were seeded and cultured on the pure titanium sheet and the two kinds of modified titanium sheets.The spreading morphology of rat bone marrow mesenchymal stem cells on the surface of titanium sheets was observed by cytoskeleton staining.Cell proliferation was determined by CCK-8 assay.The osteogenic differentiation of rat bone marrow mesenchymal stem cells on titanium sheets was measured using alkaline phosphatase staining and immunofluorescence staining.RESULTS AND CONCLUSION:(1)Scanning electron microscope images showed that the pure titanium surface was relatively smooth,and deposits as well as grain appeared after the dopamine polymerization.Aspirin microspheres were spherical with uniform size distribution.The hydrophilicity of the nanoparticles immobilized titanium surfaces and the polydopamine coating modified titanium surfaces were better than that of the pure titanium sheets(P<0.05).The aspirin appeared to be slow and sustained under the encapsulation of chitosan nanoparticles.(2)As observed by cytoskeleton staining,the cells showed insufficient cell expansion on the surface of the pure titanium sheets and formed a few filopodia on the dopamine modified titanium sheets.The cells on nanoparticles modified sheets showed perfect expansion.(3)CCK-8 assay showed that the titanium sheets had no obvious cytotoxicity,and the cell proliferation rate of the nanoparticles modified titanium sheets displayed higher rates of proliferation than that of the dopamine coated and pure titanium sheets with increasing cell culture time(P<0.05).(4)Alkaline phosphatase activity was highest on the surfaces of nanoparticles modified sheets.Immunofluorescence showed that the fluorescence intensity of alkaline phosphatase was strongest.(5)The results showed that the sustained-release coating of aspirin loaded chitosan nanoparticles on the titanium surfaces could enhance the proliferation and adhesion of rat bone marrow mesenchymal stem cells and promote osteogenic differentiation.