含镁微球-凝胶复合体对MC3T3-E1细胞黏附和增殖的影响

Effects of Mg-containing microsphere-gel complex on adhesion and proliferation of MC3T3-E1 cells

目的:将载有镁的PLGA微球添加到藻酸盐凝胶中,形成新型微球-凝胶复合体,检测其理化性质并观察MC3T3-E1细胞在其表面的黏附、增殖情况。方法:将MgCO_(3)和MgO按1∶1载入PLGA微球,制备平均密度分别为1×10^(-3)kg/m^(3)、3×10^(-3)kg/m^(3)、5×10^(-3)kg/m^(3)、7×10^(-3)kg/m^(3)的B、C、D、E组Ca^(2+)-微球悬浊液作为实验组(Ca^(2+)作为凝胶交联剂),单纯的Ca^(2+)溶液作为对照组A组,分别与海藻酸钠溶液交联形成水凝胶。观察各组凝胶的表面形貌,检测降解率、Mg^(2+)析出浓度及溶胀率。再将MC3T3-E1细胞接种于凝胶表面,使用扫描电镜(scanning electronmicroscope,SEM)观察黏附形态并计算黏附率,AM/PI染色法观察活/死细胞数量,并使用CCK-8法测定增殖活性。采用GraphPad Prism 8.0.2软件包对数据进行统计学分析。结果:扫描电镜观察发现随着微球量的增加,新型微球-凝胶复合体展现出更加致密的结构;其降解率与对照组相比明显减缓,而溶胀率却增大;离子析出测定结果显示Mg^(2+)的析出浓度较为均匀稳定,改善了单纯载镁微球浸泡液Mg^(2+)突释的现象;细胞实验结果表明,与对照组相比,实验组细胞的黏附、增殖行为被明显促进,其中C组细胞黏附效果最佳,D组细胞增殖效果最佳。结论:新型微球-凝胶复合体能够促进细胞黏附、增殖,同时可通过改变载镁微球含量调整凝胶致密性、降解速率、离子析出速率等理化性能,可作为一种新型可注射骨组织工程支架。

Objective:To evaluate the physiochemical and biological properties of a novel injectable tissue engineering system including composite PLGA microspheres and alginate hydrogel.Methods:MgCO_(3) and MgO were loaded into PLGA microspheres at 1∶1 ratio.Then,different average densities of microspheres(1×10^(-3)kg/m^(3)、3×10^(-3)kg/m^(3)、5×10^(-3)kg/m^(3)、7×10^(-3)kg/m^(3))were mixed into Ca^(2+)suspension and employed as crosslinking agents for alginate hydrogel(group B,C,D,E).The pure Ca^(2+)suspension was used as alginate crosslinking agent in the control group(group A).The influence of PLGA microspheres on the physiochemical properties of injectable scaffold were investigated,such as surface morphology,degradation rate,Mg^(2+)precipitation concentration and swelling rate.Then,MC3T3-E1 cells were seeded on the hydrogels to evaluate the effect of resultant alginate on the osteoblastic attachment and proliferation.Statistical analysis was performed using GraphPad Prism 8.0.2 software package.Results:According to the SEM observation,a more compact structure was found in the novel microsphere-hydrogel complex with the increasing quantity of microspheres.Compared with the control group,the degradation rate of the experimental group significantly decreased,while the swelling rate increased.Meanwhile,the Mg^(2+)precipitation concentration of novel hydrogel was stable,which would be helpful to settle the problem of sudden release of Mg^(2+)in the immersion solution of only microspheres.The results of CCK-8 and AM/PI staining showed that the cell attachment and proliferation were significantly improved in the experimental group compared with the control group.Conclusion:This study indicated that PLGA microsphere at an appreciate average density could significantly improve the physiochemical and biological properties of alginate hydrogel as an injectable tissue engineering scaffold.