Objectives: To explore the effect of hydrogel loaded with exosomes from Wharton's Jelly-derived mesenchymal stem cell (WJMSC) on wound healing.
Methods: Exosomes were extracted from WJMSC, and the morphology and size of WJMSC-derived exosomes (WEX) were analyzed by transmission electron microscopy and nanoparticle size analyzer, respectively. The surface markers CD9, CD81, and Calnexin of WEX were detected by Western blotting. Exosome-loaded alginate hydrogel (WEX-gel) was prepared; its morphology was studied by scanning electron microscope, and its rheological behavior was examined by a rheometer. The in vitro drug release performance of WEX-gel was investigated by BCA method. RAW264.7 cells were treated with alginate hydrogel, WEX and WEX-gel, respectively; and the expression of CD86 and CD206 in macrophages was detected by flow cytometry. A full-thickness skin wound model was established in mice; the model mice were randomly divided into blank control group, WEX control group and WEX-gel group, and PBS, WEX and WEX-gel were applied to the wound area of mice, respectively. On day 3, the skin tissue of mice was excised, and the antibacterial effect of WEX hydrogel was evaluated by plate counting. On day 15, the mice were euthanized and the percentage of residual wounds was calculated. The histological changes of the skin wound were observed after hematoxylin and eosin (HE) and Masson stainings. The expression of CD86, CD206, CD31 and vascular endothelial growth factor (VEGF) in the skin wound tissue was detected by immunohistochemistry.
Results: Exosomes were successfully extracted from WJMSC. WEX-gel presented a regular three-dimensional network structure, good rheology and controlled drug release performance. WEX-gel promoted the polarization of RAW264.7 cells from the M1 phenotype to M2 phenotype in vitro. The residual wound percentage in blank control group, WEX control group and WEX-gel group were (27.5±3.4)%, (15.3±1.2)% and (7.6±1.1)%, respectively (P<0.05). The antibacterial property of WEX-gel is better than that of WEX (P<0.05). The dermis thickness, the number of new hair follicles, and the rate of collagen deposition in the WEX-gel group were significantly higher than those in the other two groups (all P<0.05). The expression of CD206, CD31 and VEGF in skin wound tissue was higher and the expression of CD86 was lower in WEX-gel group than those in other two groups (all P<0.05).
Conclusions: WEX-gel can significantly promote wound healing in mice by regulating the polarization of macrophages.
目的: 探究负载Wharton’s Jelly衍生的间充质干细胞（WJMSC）来源的外泌体（WEX）的水凝胶对创面修复的作用。方法: 从WJMSC中提取WEX，通过透射电子显微镜和纳米粒度分析仪分析WEX的形态和大小，通过蛋白质印迹法检测外泌体表面标志物（CD9、CD81和Calnexin）鉴定WEX。通过结合WEX与海藻酸钠水凝胶来制备WEX水凝胶，扫描电子显微镜观察WEX水凝胶的形态，流式细胞仪检测WEX水凝胶的流变行为，BCA法评估WEX水凝胶的体外释药性能。海藻酸纳水凝胶、WEX和WEX水凝胶作用巨噬细胞系RAW264.7后，流式细胞术检测CD86和CD206的表达。建立小鼠全层皮肤创伤模型，将小鼠随机分为空白对照组、WEX对照组和WEX水凝胶组，按照分组分别将磷酸盐缓冲液、WEX和WEX水凝胶覆盖到创面。造模第3天，取小鼠皮肤组织，采用平板计数法评价WEX水凝胶的抗菌功效。造模后第15天处死小鼠，计算残余创面百分比，苏木精-伊红染色及Masson染色后观察皮肤创面组织学变化，免疫组织化学法检测皮肤创面组织中CD86、CD206、CD31和血管内皮生长因子（VEGF）的表达。结果: 成功从WJMSC中提取出WEX。制备的WEX水凝胶呈现规整的三维网络结构，具有良好的流变学和药物控释性能。WEX水凝胶可以促进体外RAW264.7细胞从M1表型极化为M2表型。空白对照组、WEX对照组和WEX水凝胶组残余创面百分比分别为（27.5±3.4）%、（15.3±1.2）%和（7.6±1.1）%，WEX水凝胶明显增强WEX对小鼠皮肤创面的修复作用（P<0.05），且抗菌性能优于WEX（P<0.05）。WEX水凝胶组的真皮厚度、新生毛囊数和胶原蛋白沉积率均明显高于其余两组（均P<0.05）。WEX水凝胶可抑制皮肤创面组织中CD86的阳性表达，并促进CD206、CD31和VEGF的阳性表达（均P<0.05）。结论: WEX水凝胶能够通过调节巨噬细胞极化明显促进小鼠皮肤创面修复。.
Keywords: Alginate hydrogel; Exosome; Macrophages; Mesenchymal stem cell; Mice; Wound healing.