氧化石墨烯-海藻酸钠-壳聚糖复合支架对颅骨缺损的修复作用

Repair effect of graphene oxide-sodium alginate-chitosan composite scaffold on cranial defect

目的探讨氧化石墨烯(GO)-海藻酸钠(SA)-壳聚糖(CS)复合支架对小鼠颅骨缺损模型的修复效果及可能的作用机制。方法采用冷冻干燥技术制备GO-SA-CS复合支架,选择雄性ICR小鼠建立颅骨缺损模型,随机分为3组,空白对照组不植入材料,另两组分别植入SA-CS支架(SA-CS组)和GO-SA-CS复合支架(GO-SA-CS组)。术后4 w及8 w取材,Micro-CT扫描检测颅骨缺损面积及骨代谢相关指标,HE染色观察骨组织形态,实时荧光定量PCR(RT-qPCR)法检测Runx2和ColⅠmRNA表达水平,免疫组织化学染色检测Runx2和ColⅠ蛋白表达水平。结果Micro-CT扫描显示GO-SA-CS组颅骨缺损部位边缘及中心均有新骨形成,新生骨组织较空白对照组及SA-CS组多,骨缺损面积明显小于空白对照组及SA-CS组(P<0.05),BMD和Tb.Th升高(P<0.05),Tb.Sp降低(P<0.05);HE染色显示空白对照组骨缺损部位仅存在薄层疏松网状纤维组织,SA-CS组和GO-SA-CS组可见部分未完全降解的支架材料,且GO-SA-CS组残留支架内部有大量成骨细胞长入,少量新生血管形成;RT-qPCR和免疫组化染色结果显示,与空白对照组及SA-CS组相比,GO-SA-CS组Runx2和ColⅠmRNA及蛋白表达水平均明显升高(P<0.05)。结论GOSA-CS复合支架可能通过诱导Runx2和ColⅠ表达上调促进小鼠颅骨缺损部位骨组织再生修复。

Objective To investigate the effect and possible mechanism of graphene oxide(GO)-sodium alginate(SA)-chitosan(CS)composite scaffold in repairing cranial defect in mice.Methods GO-SA-CS composite scaffolds were prepared by freeze-drying technique,and male ICR mice were selected to establish cranial defect models.They were randomly divided into three groups:blank control group without material implantation,the other two groups were implanted with SA-CS scaffold(SA-CS group)and GO-SA-CS composite scaffolds(GO-SA-CS group).At 4 and 8 weeks after operation,the area of cranial defect and the related indexes of bone metabolism were detected by Micro-CT scanning,the bone morphology was observed by HE staining,the expression of Runx2 and ColⅠmRNA was detected by real-time PCR(RT-qPCR),and the expression of Runx2 and ColⅠprotein was detected by immunohistochemical staining.Results Micro-CT scan showed that there were new bone formation at the edge and center of cranial defect in GO-SA-CS group,the number of new bone tissue was more than that in blank control group and SA-CS group,the area of bone defect was significantly smaller than that in blank control group and SA-CS group(P<0.05),BMD and Tb.Th increased(P<0.05)and Tb.Sp decreased(P<0.05).HE staining showed that there was only a thin layer of loose reticular fibrous tissue in the bone defect in the blank control group,and some undegraded scaffolds could be seen in the SA-CS and GO-SA-CS groups.In the GO-SA-CS group,a large number of osteoblasts grew into the residual scaffolds and a small amount of neovascularization was formed.The results of RT-qPCR and immunohistochemical staining showed that the mRNA and protein expression of Runx2 and ColⅠin GO-SA-CS group were significantly higher than those in blank control group and SA-CS group(P<0.05).Conclusion GO-SA-CS composite scaffold may promote bone regeneration and repair of mouse cranial defect by up-regulating the expression of Runx2 and ColⅠ.