功能化聚羟基脂肪酸酯微球的制备及其抗菌、促成骨分化功能评价

Preparation of functional polyhydroxyalkanoate microspheres and their antibacterial activity and osteogenic effect evaluation

目的构建负载BMP-2和人β防御素3(humanβ-defensin 3,HBD3)的聚羟基脂肪酸酯(polyhydroxyalkanoate,PHA)微球,并评估其抗菌性、生物相容性以及促成骨分化效果,旨在为骨组织工程提供一种新的支架材料。方法采用大豆磷脂(soybean lecithin,SL)介导的生长因子修饰技术制备SL-BMP-2和SLHBD3,再利用微流控技术制备含有BMP-2及HBD3的功能化PHA微球(functional microsphere,f-PMS),同法制备单纯PHA微球(pure microsphere,p-PMS)作为对照。然后,通过扫描电镜观察两种微球形貌、检测吸水率,并利用ELISA试剂盒检测f-PMS中BMP-2和HBD3释放规律;采用活/死细菌染色观测两种微球在金黄色葡萄球菌以及大肠杆菌悬液中的抗菌效果;采用与人BMSCs共培养,通过Transwell和细胞计数试剂盒8检测微球生物相容性,以及Ⅰ型胶原(collagen typeⅠ,COL-1)免疫荧光染色和ALP浓度测定来判定微球促成骨分化作用。结果实验成功构建f-PMS及p-PMS两种微球。扫描电镜观察示p-PMS表面粗糙,分布直径1~3μm的孔隙;而f-PMS表明光滑,且存在白色颗粒状物质;两组吸水率差异无统计学意义(P>0.05);f-PMS中BMP-2和HBD3释放曲线基本一致,均呈现早期突释、后期缓释规律。f-PMS能抑制金黄色葡萄球菌及大肠杆菌生长,且抗菌性高于p-PMS,差异有统计学意义(P<0.05);但两种微球生物相容性差异无统计学意义(P>0.05)。免疫荧光染色示f-PMS成骨特异性蛋白COL-1荧光强度高于p-PMS,ALP浓度高于p-PMS,差异均有统计学意义(P<0.05)。结论p-PHA具有良好的抗菌能力和生物相容性,并且能促进hBMSCs成骨分化,有望作为骨组织工程支架材料。

Objective To construct polyhydroxyalkanoate(PHA)microspheres loaded with bone morphogenetic protein 2(BMP-2)and humanβ-defensin 3(HBD3),and evaluate the antibacterial activity of microspheres and the effect of promoting osteogenic differentiation,aiming to provide a new option of material for bone tissue engineering.Methods The soybean lecithin(SL)-BMP-2 and SL-HBD3 were prepared by SL-mediated introduction of growth factors into polyesters technology,and the functional microsphere(f-PMS)containing BMP-2 and HBD3 were prepared by microfluidic technology,while pure microsphere(p-PMS)was prepared by the same method as the control.The morphology of microspheres was observed by scanning electron microscopy and the water absorption was detected;the release curves of BMP-2 and HBD3 in f-PMS were detected by ELISA kit.The antibacterial effect of microspheres in Staphylococcus aureus and Escherichia coli was tested with the LIVE/DEADTM BacLightTM bacterial staining kit;the biocompatibility of microspheres was tested using Transwell and cell counting kit 8(CCK-8).The effect of microspheres on osteogenic differentiation was determined by collagen typeⅠ(COL-1)immunofluorescence staining and alkaline phosphatase(ALP)concentration.Results In this experiment,the f-PMS and p-PMS were successfully constructed.Morphological characteristics showed that p-PMS surface was rough and distributed with micropores of 1-3μm,while f-PMS surface was smooth and existed white granular material.There was no significant difference in water absorption between the two groups(P>0.05).The release curves of BMP-2 and HBD3 in the f-PMS and p-PMS were basically the same,showing both early sudden release and late slow release.The antibacterial activity of f-PMS was significantly higher than that of p-PMS in the test that against Staphylococcus aureus and Escherichia coli(P<0.05),but there was no significant difference in biocompatibility between the two groups(P>0.05).The results of osteogenic differentiation of human BMSCs showed that the fluorescence intensity of osteogenic specific protein COL-1 of f-PMS was significantly higher than that in p-PMS,and the activity of ALP in f-PMS was also significantly higher than that in p-PMS(P<0.05).Conclusion The p-PHA have good antibacterial activity and biocompatibility,and can effectively promote the osteogenic differentiation of human BMSCs,which is expected to be applied to bone tissue engineering in the future.