负载褐藻多酚的丝素蛋白GBR生物膜制备及其性能的体外实验研究

Preparation and in vitro performance of silk fibroin GBR membrane loaded with phlorotannins

目的通过制备负载褐藻多酚(PT)的双层丝素蛋白(SF)膜,测试其力学性能、生物相容性及其对成骨分化的影响,研究其作为引导骨再生屏障膜的可能性。方法将不同脱胶时间处理后的SF溶液通过溶液浇筑技术、定向冷冻技术以及冷冻干燥技术处理后制备成SF双层膜,采用力学万能试验机测定湿态条件下各组膜样品(n=5)的抗拉强度。取抗拉强度最大的SF双层膜所对应的SF溶液重新制备膜样品,用扫描电镜观察其微观结构。通过在粗糙面负载PT制备PT-SF双层膜,使用傅里叶红外光谱仪分析PT与SF双层膜的相互作用。利用扫描电镜观察人牙槽骨来源的骨髓间充质干细胞(hABMSCs)在粗糙面的黏附情况,通过CCK-8及活死细胞染色探究PT-SF双层膜的生物相容性。用qRT-PCR法、ALP染色和茜素红染色检测各组hABMSCs的成骨向分化情况。结果由脱胶时间为30 min的SF溶液制备出的SF双层膜的抗拉强度最大,达(3.293±0.1228)MPa。SF双层膜的光滑面致密光滑,粗糙面呈纵向有序的疏松多孔结构。褐藻多酚通过氢键、疏水作用力等方式负载到SF双层膜的粗糙面上,空白组、SF组及PT-SF组之间的CCK-8及活死细胞染色结果均无明显差异,且与空白组和SF组相比PT-SF促进了hABMSCs的成骨向分化。结论PT-SF双层膜兼具良好的机械性能和生物学性能,可在一定程度上促进hABMSCs的成骨向分化。该研究成功制备了PT-SF双层膜。

Objective To explore the feasibility of silk fibron(SF)-phlorotannins(PT)double layer membranes as barrier membrane for guided bone regeneration(GBR)by exploring the mechanical property,biocompatibility,and the effect on osteogenic differentiation of SF-PT double layer membranes.Methods The SF solution treated with different degumming time was prepared into SF bilayer by solution cast technique,ice template method and lyophilization.The tensile strength of the membranes under hydration were measured via mechanical universal machine(n=5).The microstructures of the composite membranes with the highest tensile strength under hydration condition were observed by scanning electron microscopy.Then the rough surfaces of SF double layer membranes was loaded with PT to prepare SF-PT double layer membranes,Fourier transformed infrared spectroscopy was carried out to characterize the interaction between PT and SF double layer membranes,and the bone marrow mesenchymal stem cells of human alveolar origins(hABMSCs)adhesion was studied by SEM.CCK-8 assay and live dead staining were used to evaluated the biocompatibility of SF-PT double layer membranes.qRT-PCR,ALP staining and alizarin red stain were carried out to measure the osteogenic differentiation of hABMSCs in different groups.Results The SF membranes prepared from SF solution with a degumming time of 30 min has the highest tensile strength,reaching(3.293±0.1228)MPa.The smooth surface of SF double layer membrane was dense and smooth,while the rough surface was longitudinally ordered loose porous structure.PT was loaded onto the rough surface of the SF double layer membranes by hydrogen bond,hydrophobic interaction and other forces.There was no significant difference in the results of CCK-8 and live dead staining between blank group,SF group and PT-SF group.Compared with blank group and SF group,PT-SF double layer membranes promoted osteogenic differentiation of hABMSCs.Conclusion Thus,the PT-SF double layer membranes expressed desired mechanical,biological properties and can promote the osteogenic differentiation of hABMSCs to a certain extent.These results indicate that PT-SF double layer membranes is successfully fabricated,providing a solid foundation for future in vivo experiments.