水凝胶负载源自牙髓间充质干细胞的外泌体可增强小鼠的伤口愈合。
Hydrogel loaded with exosomes from Whartons Jelly-derived mesenchymal stem cells enhances wound healing in mice.
机构信息
Department of Anorectal Surgery, Taizhou Central Hospital & Affiliated Hospital of Taizhou College, Taizhou 318000, Zhejiang Province, China.
出版信息
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2023 Dec 10;52(6):766-776. doi: 10.3724/zdxbyxb-2023-0316.
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 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 . 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 (<0.05). The antibacterial property of WEX-gel is better than that of WEX (<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 <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 <0.05).
CONCLUSIONS
WEX-gel can significantly promote wound healing in mice by regulating the polarization of macrophages.
目的
探讨水凝胶负载牙髓间充质干细胞（WJMSC）来源的外泌体对伤口愈合的影响。
方法
从 WJMSC 中提取外泌体，通过透射电子显微镜和纳米颗粒粒径分析仪分别分析 WJMSC 来源的外泌体（WEX）的形态和大小。通过 Western blot 检测 WEX 的表面标志物 CD9、CD81 和 Calnexin。制备载外泌体的藻酸盐水凝胶（WEX-gel）；通过扫描电子显微镜研究其形态，通过流变仪研究其流变行为。通过 BCA 法研究 WEX-gel 的药物释放性能。用藻酸盐水凝胶、WEX 和 WEX-gel 分别处理 RAW264.7 细胞，流式细胞术检测巨噬细胞中 CD86 和 CD206 的表达。建立小鼠全层皮肤伤口模型；将模型小鼠随机分为空白对照组、WEX 对照组和 WEX-gel 组，分别向小鼠伤口区域涂抹 PBS、WEX 和 WEX-gel。第 3 天，切取小鼠皮肤组织，通过平板计数法评价 WEX 水凝胶的抑菌效果。第 15 天，处死小鼠，计算残余伤口百分比。苏木精和伊红（HE）及 Masson 染色观察皮肤伤口的组织学变化。免疫组织化学法检测皮肤伤口组织中 CD86、CD206、CD31 和血管内皮生长因子（VEGF）的表达。
结果
成功从 WJMSC 中提取外泌体。WEX-gel 呈现出规则的三维网络结构，具有良好的流变性能和可控的药物释放性能。WEX-gel 促进了 RAW264.7 细胞从 M1 表型向 M2 表型的极化。空白对照组、WEX 对照组和 WEX-gel 组的残余伤口百分比分别为（27.5±3.4）%、（15.3±1.2）%和（7.6±1.1）%（<0.05）。WEX-gel 的抑菌性能优于 WEX（<0.05）。WEX-gel 组真皮厚度、新生毛囊数量和胶原沉积率均明显高于其他两组（均<0.05）。WEX-gel 组皮肤伤口组织中 CD206、CD31 和 VEGF 的表达较高，CD86 的表达较低，均低于其他两组（均<0.05）。
结论
WEX-gel 通过调节巨噬细胞的极化，显著促进小鼠伤口愈合。
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