甲基丙烯酸钠修饰光交联海藻酸盐水凝胶支架的制备和表征

Preparation and characterization of sodium methacrylate modified photocrosslinked alginate hydrogel scaffold

背景:光交联海藻酸盐水凝胶因具有良好的生物相容性、可微创注射等优势已为热门的组织工程研究材料,但是仍然存在强度不足、细胞黏附能力不足等问题。目的:构建载负电荷的光交联海藻酸盐水凝胶材料,探索其物理性能和细胞黏附性能变化。方法:利用海藻酸钠和2-氨乙基甲基丙烯酸酯盐酸盐制备甲基丙烯酸酯化海藻酸盐后,再与光引发剂和不同浓度甲基丙烯酸钠(0,20,40,60mmol/L)混合制备载负电荷光交联海藻酸盐水凝胶,利用傅里叶红外光谱仪分析水凝胶的功能基团变化情况,扫面电镜观察水凝胶的表面形态,并测量其溶胀率。将MC3T3-E1细胞与各组水凝胶共培养48h,采用活死染色与CCK-8法分析水凝胶的细胞毒性;接种MC3T3-E1细胞于4组水凝胶表面,在第4小时活死染色观察细胞早期黏附情况,第3天活死染色观察细胞伸展情况。结果与结论:①傅里叶红外光谱分析显示,甲基丙烯酸钠的引入可在水凝胶红外波普波数1600 cm-1左右处出现来自甲基丙烯酸钠的新波峰;②扫描电镜显示随着甲基丙烯酸钠浓度的增加,光交联海藻酸盐水凝胶的致密度增加,孔径减小;③溶胀率测试显示随着甲基丙烯酸钠浓度的升高,光交联海藻酸盐水凝胶的溶胀率逐渐降低;④活死染色显示4种水凝胶表面的细胞生长状态良好,细胞活性均在95%以上;CCK-8检测显示,载负电荷的光交联海藻酸盐水凝胶材料无细胞毒性;⑤随着甲基丙烯酸钠引入量的增加,载负电荷光交联海藻酸盐水凝胶表面的早期细胞黏附率逐渐增加,细胞伸展状态明显改善;⑥结果表明,甲基丙烯酸钠修饰的引入调节了光交联海藻酸盐水凝胶物理性能,并明显提高了其细胞黏附性能。

BACKGROUND:Photocrosslinked alginate hydrogel has been a popular bone tissue engineering material because of its excellent biocompatibility and minimally invasive injection,but there are still problems such as insufficient strength and poor cell adhesion.OBJECTIVE:To construct the negatively charged hydrogels by introducing sodium methacrylate into photocrosslinked alginate hydrogels,and to explore the changes in its physical performance and cell adhesion.METHODS:After preparation of methacrylated alginate by reacting sodium alginate with 2-aminoethyl methacrylate,methacrylated alginate,photoinitiator and sodium methacrylate(0,20,40,60 mmol/L)were homogeneously mixed.The negatively charged photocrosslinked alginate hydrogels were prepared under ultraviolet light.The functional groups of the hydrogels were analyzed by fourier transform infrared spectroscopy.The surface morphology of the hydrogels was observed by scanning electron microscopy and the swelling ratio was measured.MC3T3-E1 cells were cultured with each group of hydrogels for 48 hours,and the cytotoxicity of the hydrogels was investigated by cell counting kit-8 assay.MC3T3-E1 cells were seeded on the surface of each group of hydrogels.The early adhesion of the cells was observed by live/dead staining at the 4th hour,and cell spreading was observed on the 3rd day.RESULTS AND CONCLUSION:(1)Fourier transform infrared spectroscopy demonstrated that the introduction of sodium methacrylate could lead to a new peak at wavenumber of about 1600 cm-1 in the hydrogel infrared wave,which was from the sodium methacrylate.(2)Scanning electron microscope observed that the density of the negatively charged photocrosslinked alginate hydrogels increased and the pore size of the gels decreased with augment of concentrations of sodium methacrylate.(3)The swelling ratio of the hydrogel decreased with the increase of the concentration of sodium methacrylate.(4)The live/dead staining revealed that the cells grew well on the surface of each hydrogel,and the cell viability reached above 95%.The cell counting kit-8 assay results showed that the negatively charged photocrosslinked alginate hydrogels had no cytotoxicity.(5)The early cell adhesion rate increased gradually and the cell extension became better with the increase of concentration of sodium methacrylate.(6)In summary,the introduction of sodium methacryl into photocrosslinked alginate hydrogels can adjust its physical properties and significantly improve its cell adhesion.