壳聚糖/藻酸盐纳米膜在促进小鼠全层损伤皮肤创面愈合中的作用

Chitosan / alginate nanomembrane promotes healing of full-thickness wounds in mice

目的评价复合生物材料壳聚糖/藻酸盐纳米膜作为组织工程的细胞支架材料,通过促进小鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)在其表面的粘附、迁移、增殖及分化,从而加速皮肤创面修复的效果。方法实验动物为BALB/c小鼠,8周龄,于各小鼠背部制备皮肤缺损创面模型。实验先分2组,实验组小鼠背部创面以壳聚糖/藻酸盐纳米膜覆盖,对照组不予该纳米膜覆盖创面,直接粘贴手术贴巾,并分别围绕创缘皮下注射等量绿色荧光蛋白(green fluorescent protein,GFP)标记的骨髓间充质干细胞(GFP-MSCs),在术后进行小动物活体成像观察绿色荧光蛋白信号强度,用流式细胞仪检测阳性GFP-MSCs比例,并取创面新生组织行冰冻切片观察GFP-MSCs生长情况;再分别设置空白对照组、纳米膜组、MSCs注射组和纳米膜+MSCs注射组,观察术后各组小鼠创面愈合情况。结果实验组小鼠术后第7天的创面GFP信号强度[(25.97±6.98)×104]明显高于对照组[(6.00±2.84)×104,P<0.05];流式细胞仪检测创面GFP-MSCs水平,术后第5天实验组阳性率[(66.78±7.00)%]明显高于对照组[(33.58±3.62)%,P<0.01],第7天实验组阳性率[(53.03±3.15)%]明显高于对照组[(34.20±7.98)%,P<0.01];术后第14天小鼠新生组织中GFP-MSCs密度实验组[(101.00±15.51)个/视野]明显高于对照组[(25.25±5.07)个/视野,P<0.05];创面愈合观察发现纳米膜+MSCs注射组创面愈合速度快于其他3组(P<0.05)。结论壳聚糖/藻酸盐纳米膜用于皮肤创面,可促进MSCs在体内粘附、生长、分化、迁移,进而促进皮肤组织修复,加速创面愈合,是一种良好的细胞支架材料。

Objective To evaluate the efficacy of the chitosan/alginate nanomembrane as cell scaffold material to enhance burn wound healing by promoting the adhesion, migration, proliferation and differentiation of mouse bone marrow mesenchymal stem cells （MSCs）. Methods BALB/c mice （aged 8 weeks） were selected as the experimental animals. A 5-mm diameter full-thickness cutaneous burn wound was inflicted on the depilated dorsal skin in each of mice. Then the animals were firstly divided into 2 groups. The wounds in experimental group were covered with the nanomembrane, while those in control group were not. Equal bone marrow-derived MSCs transduced with green fluorescent protein （GFP） gene were transplanted to the periphery of the skin wounds in both groups of mice. The intensity of the GFP signal was evaluated after surgery with an In Vivo Imaging system. Fluorescence activated cell sorting （FACS） was used to quantify the ratio of the positive GFP-MSCs. Frozen sections of the newly formed tissues were observed respectively to evaluate the survival condition of the GFP-MSCs. Then another groups of mice were divided into blank control group, nanomembrane group, MSCs injected group and nanomembrane＋MSCs injected group （n=8）. The condition of wounds healing in each group were observed and assessed at different time points after surgery. Results The expression of GFP was significantly higher in the experimental group than the control group on the 7th day after surgery [intensity of GFP signal （25.97±6.98）×10^4 vs （6.00±2.84）×10^4, P〈0.05]. Flow cytometry showed that the average percentage of the positive GFP-MSCs was obviously higher in experimental group than that in control group on the 5th day after surgery [（66.78±7.00）% vs （33.58±3.62）%, P〈0.01] and on the 7th day [53.03±3.15）% vs （34.20±7.98）%, P〈0.01]. Fluorescence microscopy indicated the density of GFP-MSCs in newly formed tissue was greater in experimental group than control group in 14 d after surgery [（101.00±15.51）/horizon vs （25.25±5.07）/horizon, P〈0.05]. The wounds of the namomembrane＋MSCs group showed significantly faster healing speed than the other 3 groups （P〈0.05）. Conclusion When applied in skin wounds, the chitosan/alginate nanomembrane promotes the adhesion, migration, proliferation and differentiation of MSCs, and can served as a good cell scaffold for reconstruction of skin tissue.