纳米壳聚糖对MC3T3-E1成骨细胞生长的影响

背景：已有体内急性毒理实验证实，壳聚糖纳米微囊的半数致死量高于2 000 mg/kg，但其具体致病机制目前尚不明确。 目的：分析纳米壳聚糖作为骨替代材料对MC3T3-E1成骨细胞生长及大鼠肝、肾等器官生理功能的影响。 方法：将MC3T3-E1成骨细胞分别在含0(对照)、10 mg/L、100 mg/L、1 g/L、10 g/L纳米壳聚糖的DMEM培养液中培养，检测各组细胞A值。透射电镜观察10 g/L纳米壳聚糖溶液培养MC3T3-E1成骨细胞24 h后的细胞形态变化。采用PBS制备10 g/L纳米壳聚糖悬浮液，分别以166.67，16.67 mg/kg经腹腔注射至SD大鼠体内4周，每周3次，正常对照组注射等量生理盐水，血清生化指标分析大鼠肝、肾功能，病理切片观察组织形态学改变、炎症细胞浸润情况。 结果与结论：与对照组比较，10 mg/L、100 mg/L、1 g/L、10 g/L的纳米壳聚糖溶液均抑制MC3T3-E1细胞的生长(P < 0.05)。透射电镜见团聚的壳聚糖存在于MC3T3-E1细胞浆中，细胞表面的伪足形成，细胞膜呈波浪状起伏，细胞核变性、碎裂及固缩。与正常对照组比较，注射纳米壳聚糖悬浮液两组大鼠血尿素氮、Na+水平均有明显升高(P < 0.05)，高剂量组K+水平明显降低(P < 0.01)；肝脏、肾脏均出现组织细胞凋亡现象，高剂量组凋亡更加明显。表明纳米壳聚糖可导致细胞凋亡，超过一定剂量可造成肾功能受损，对机体生理功能造成影响。

Insight into nano chitosan effects on MC3T3-E1 cell growth

BACKGROUND:Acute toxicity in vivo experiments in previous studies has been confirmed that the median lethal dose of chitosan microcapsules is higher than 2 000 mg/kg, but the specific pathogenic mechanism is unclear. OBJECTIVE:To explore the influence of nano chitosan on MC3T3-E1 cell growth as a bone substitute material, as well as the physiological function of rats. METHODS:MC3T3-E1 cells were respectively cultured in Dulbecco’s modified Eagle’s medium with different concentrations of chitosan nanoparticles (0, 10 mg/L, 100 mg/L, 1 g/L, 10 g/L). The absorbance values were determined. Changes in MC3T3-E1 cell morphology were observed by scanning electron microscope after 24 hours culture. 10 g/L nano chitosan suspension was prepared using PBS. Two different doses of nano chitosan suspension (166.67 and 16.67 mg/kg body weight) with PBS were injected intraperitoneally into Sprague-Dawley rats, three times a week, for 4 weeks. The control group was injected with equal volume of physiologic saline. Serum biochemical markers were detected to analyze the functions of liver and kidney of rats. Moreover, histopathology slices were observed to evaluate the morphological changes of tissue and inflammatory infiltration. RESULTS AND CONCLUSION:10 mg/L, 100 mg/L, 1 g/L, 10 g/L nano chitosan suspensions were found growth inhibition in MC3T3-E1 cells as compared with the control group (P < 0.05). The reunion of chitosan was observed in the cytoplasm of MC3T3-E1 cells by transmission electron microscopy. On the cell surface, pseudopodia formed, wavy undulating membrane, nucleus degeneration, fragmentation and condensation were found. Compared with the control group, blood urea nitrogen, Na+ levels were significantly increased in rats injected with nano chitosan suspension at two dosages, but the K+ level in the high concentration group was decreased significantly (P < 0.05). Cell apoptosis was found in the liver and renal tissue in a dose-dependent manner. It suggests that apoptosis may be the possible mechanism of nano chitosan toxicity, and normal physiological function may be impacted over a certain dose.