纳米改性聚己内酯微球的构建及对牙髓细胞的生物学效应

Manufacturing of nano-modified polycaprolactone microspheres and its biological effects in dental pulp cells

背景:聚己内酯具有良好的热稳定性、生物相容性、降解速率可调等优点,有作为组织工程支架应用于牙髓组织工程的潜能,但其亲水性和生物活性较差。目的:制备聚多巴胺及纳米羟基磷灰石改性的多孔聚己内酯(polycaprolactone-polydopamine-nano-hydroxyapatite,PCL-PDA-nHA)微球,探索其物理性能及对牙髓细胞增殖和矿化的影响。方法:采用双乳化法制备多孔聚己内酯微球,通过聚多巴胺进行表面改性,得到PCL-PDA微球,提高其亲水性和结晶能力;通过模拟体液在PCL-PDA微球表面原位形成纳米羟基磷灰石涂层(分反应7 d组与反应14 d组),得到PCL-PDA-nHA微球。通过扫描电镜、X射线光电子能谱技术和蛋白吸附实验检测各组微球的理化性质,通过溶血实验、凝血因子激活实验和血小板凝集实验检验各组微球的血液相容性。将4组微球分别与人牙髓细胞共培养,CCK-8法检测细胞增殖,碱性磷酸酶、茜素红与天狼星红染色分析微球的矿化诱导能力。结果与结论:①扫描电镜显示,4组微球为均匀疏松的多孔结构,微球直径无明显差异,微球孔隙率为87.4%,孔隙直径在20-50μm之间;X射线光电子能谱显示,聚多巴胺和纳米羟基磷灰石有效修饰到聚己内酯表面;蛋白吸附实验显示,改性后的材料可增强血清白蛋白的吸附能力;②溶血实验显示,4组微球的溶血率在1%以下,不会造成明显的溶血;凝血因子激活实验显示,各组改性聚己内酯微球对凝血影响较小;扫描电镜显示,各组微球表面无明显的血小板聚集;③CCK-8检测显示,各组改性微球表面的细胞增殖均快于聚己内酯微球;④碱性磷酸酶、茜素红与天狼星红染色显示,各组改性微球的矿化诱导能力均强于聚己内酯微球,其中PCL-PDA-nHA-14 d微球与PCL-PDA-nHA-7 d微球的矿化诱导能力强于PCL-PDA微球;⑤结果表明,PCL-PDA-nHA纳米微球的血液相容性良好,可促进人牙髓细胞的增殖和矿化。

BACKGROUND:Polycaprolactone based scaffolds have potential in dental pulp regeneration since its good thermal stability,excellent biocompatibility and tunable degradation rate.However,its hydrophilicity and bioactivity are poor.OBJECTIVE:To prepare microspheres with polycaprolactone-polydopamine-nano-hydroxyapatite(PCL-PDA-nHA),and explore its physical properties and the effects on the proliferation and mineralization of dental pulp cells.METHODS:The PCL microspheres were prepared by double emulsification method.The hydrophilicity and crystallinity were improved by the surface modification of dopamine and the PCL-PDA particles were obtained.Nano-hydroxyapatite coating was formed in situ on the surface of PCL-PDA microspheres by simulating body fluids(reaction 7-day group and reaction 14-day group)to obtain PCL-PDA-nHA microspheres.The physical and chemical properties of each group of microspheres were detected by scanning electron microscopy,X-ray photoelectron spectroscopy and protein adsorption experiment.Hemolysis test,coagulation factor activation test and platelet agglutination test were used to test the hemocompatibility of each group of microspheres.Four groups of microspheres were co-cultured with human dental pulp cells.Cell proliferation was detected by CCK-8 assay.The mineralization induction ability of the microspheres was analyzed using alkaline phosphatase,alizarin red and sirius red staining.RESULTS AND CONCLUSION:(1)Scanning electron microscopy showed that the four groups of microspheres were uniform and porous structure,and there was no significant difference in the diameter of microspheres;the porosity was 87.4%with pore diameter ranging from 20μm to 50μm.X-ray photoelectron spectroscopy showed that polydopamine and hydroxyapatite particles were successfully decorated on the microsphere surface.Protein adsorption experiment showed that the functional microspheres could enhance the adsorption of serum albumin.(2)Hemolysis experiment showed that the hemolysis rate of microspheres was below 1%,without obvious hemolysis.The coagulation factor activation experiment showed that each group of modified polycaprolactone microspheres had little effect on blood coagulation.Scanning electron microscopy showed that there was no obvious platelet aggregation on the surface of each group of microspheres.(3)CCK-8 assay demonstrated that cell proliferation on the surface of each group of modified microspheres was faster than that of PCL microspheres.(4)Alkaline phosphatase,alizarin red and sirius red staining exhibited that the mineralization induction ability of each group of modified microspheres was stronger than that of PCL microspheres,and the mineralization induction ability of PCL-PDA-nHA-14 day microspheres and PCL-PDA-nHA-7 day microspheres was stronger than that of PCL-PDA microspheres.(5)The results suggested that PCL-PDA-nHA nanospheres had good hemocompatibility,and could promote the proliferation and mineralization of human dental pulp cells.