含锂生物玻璃在大鼠心肌样细胞中的生物相容性研究

Biocompatibility study of lithium-containing bioglass in rat cardiomyocyte-like cells

目的制备含锂生物玻璃(Li/BGs)纳米材料, 并探讨其对大鼠心肌样细胞(H9C2)和人类脐静脉内皮细胞(HUVECs)的生物相容性。方法采用溶胶-凝胶法, 将0.7 g十六烷基三甲基溴化铵、10 ml乙酸乙酯、7 ml 1 mol/L氨水、3.6 ml正硅酸四乙酯、3.57 g四水硝酸钙和0.37 g碳酸锂反应至少4 h, 制备Li/BGs。扫描电子显微镜(SEM)观察Li/BGs纳米颗粒的尺寸和形态。H9C2和HUVECs分别置于10~1 000 μg/ml浓度的Li/BGs培养基中, 细胞计数试剂盒(CCK-8)细胞毒性实验(450 nm吸光度值)评估细胞活性。细胞在10 μg/ml浓度的Li/BGs溶液中培养24 h后, 采用鬼笔环肽染色法(Phalloidin)和4’, 6-二脒基-2-苯基吲哚(DAPI)染色, 观察细胞核(蓝色)和细胞骨架蛋白(红色)的共聚焦显微镜图像, 评估Li/BGs对细胞形态的影响。组间比较采用单因素方差分析分析数据统计学差异。结果 SEM观察显示, 制备的Li/BGs颗粒近球形, 粗糙且均匀, 直径(82.86±14.28) nm。H9C2经钙黄绿素乙酰氧基甲酯/碘化丙啶(Calcein AM/PI)染色后, 大部分细胞展现出活跃状态, 未出现细胞的明显死亡。在细胞毒性试验中, 250 μg/ml组、500 μg/ml组及1 000 μg/ml组低于H9C2对照组(0.287±0.005、0.317±0.009、0.347±0.025比0.825±0.188, F=24.72, P<0.001)。Li/BGs处理HUVECs后250 μg/ml组、500 μg/ml组及1 000 μg/ml组低于HUVECs对照组(0.289±0.003、0.320±0.013、0.354±0.021比1.026±0.027, F=34.44, P<0.001)。Phalloidin和DAPI染色显示两种细胞的Li/BGs组与对照组比较, Li/BGs组细胞骨架保持正常的形状和结构, 未出现断裂、异常凝聚或形状改变等现象。结论 Li/BGs对细胞的形态和结构无明显影响, 具有良好的生物相容性。

Objective To prepare lithium-containing bioglass(Li/BGs)nanomaterials and to evaluate their biocompatibility with rat cardiomyocyte-like cells(H9C2)and human umbilical vein endothelial cells(HUVECs).Methods Li/BGs were prepared by sol-gel method by reacting 0.7 g of cetyltrimethylammonium bromide,10 ml of ethyl acetate,7 ml of 1 mol/L ammonia,3.6 ml of tetraethyl orthosilicate,3.57 g of tetrahydrated calcium nitrate,and 0.37 g of lithium carbonate for at least 4 h.The nanoparticle size and morphology of Li/BGs were observed in scanning electron microscope(SEM).size and morphology.H9C2 cardiomyocytes and HUVECs were placed in Li/BGs medium at concentrations ranging from 10-1000μg/ml,respectively,and cell activity was assessed by the cell counting kit-8(CCK-8)cytotoxicity assay(absorbance value at 450 nm),and t-tests were used for comparisons between groups.The effect of Li/BGs on cell morphology was assessed by observing confocal microscopy images of nuclei(blue color)and cytoskeletal proteins(red color)using Phalloidin staining and Phalloidin and 4′,6-diamidino-2-phenylindole(DAPI)staining after the cells were cultured for 24 h in Li/BGs solution at a concentration of 10μg/ml.One-way ANOVA was used for comparisons between groups.Results SEMobservation showed that the prepared Li/BGs particles were nearly spherical,rough and homogeneous,with an average diameter of(82.86±14.28)nm.After H9C2 was stained with calceinacetyloxymethyl ester/propidium iodide(Calcein AM/PI),most cells were active and no significant cell death occurred.In cytotoxicity assay,250μg/ml group,500μg/ml group and 1000μg/ml group were lower than H9C2 control group(0.287±0.005,0.317±0.009,0.347±0.025 vs.0.825±0.188,F=24.72,P<0.001).After treatment of HUVECs by Li/BGs.250μg/ml group,500μg/ml group and 1000μg/ml group were lower than HUVECs control group(0.289±0.003,0.320±0.013,0.354±0.021 vs.1.026±0.027,F=34.44,P<0.001).Phalloidin and DAPI staining showed Li/BGs group cytoskeleton maintained its normal shape and structure,without any break,abnormal condensation,or shape change.Conclusion Li/BGs had no significant effect on the morphology and structure of the cells and showed good biocompatibility.