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  • Title: [Effects of applying human umbilical cord mesenchymal stem cell exosomes through different pathways to treat full-thickness skin defect wounds in mice].
    Author: Wang HY, Ba T, Zhou B, Yan ZQ, Wang RJ, Liu LY.
    Journal: Zhonghua Shao Shang Yu Chuang Mian Xiu Fu Za Zhi; 2024 Apr 20; 40(4):314-322. PubMed ID: 38664025.
    Abstract:
    Objective: To investigate the effects of human umbilical cord mesenchymal stem cell (hUCMSC) exosomes in the treatment of full-thickness skin defect wounds in mice through local wound application, subcutaneous injection at the wound margin, and tail vein injection, and to explore the optimal administration route of hUCMSC exosomes for wound treatment. Methods: This study was an experimental study. hUCMSC exosomes were extracted from the discarded umbilical cord tissue of three normal delivery women aged 25-35 years in the Department of Obstetrics and Gynecology of Baogang Hospital of Inner Mongolia and successfully identified. Totally 120 male BALB/c mice aged 6-8 weeks were selected, and full-thickness skin defect wounds were prepared on the back of them. According to the random number table, the injured mice were divided into control group (without drug administration), local wound application group, wound margin subcutaneous injection group, and tail vein injection group (with 30 mice in each group). Mice in the latter three groups were given 0.2 mL phosphate buffer solution containing 200 μg hUCMSC exosomes by local wound application, subcutaneous injection at the wound margin, and tail vein injection, respectively. On post injury day (PID) 7, 14, and 21, the general condition of the wound was observed, and the wound healing rate was calculated; the wound tissue was collected, the pathological changes and collagen fibers were observed respectively by hematoxylin-eosin staining and Masson staining, the number of new microvessels was observed by CD31 immunohistochemical staining, and the content of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) was detected by enzyme-linked immunosorbent assay. The sample number was 10 in each group at each time point. Results: On PID 7, 14, and 21, the wounds of mice in the 4 groups all healed gradually, and the wound healing of the mice in wound margin subcutaneous injection group was the best; the wound healing rates of mice in the three administration groups were significantly higher than those in control group (P<0.05), the wound healing rates of mice in wound margin subcutaneous injection group and tail vein injection group were significantly higher than those in local wound application group (P<0.05), and the wound healing rates of mice in wound margin subcutaneous injection group were significantly higher than those in tail vein injection group (P<0.05). On PID 7, 14, and 21, the growth and epithelialization speed of the wound tissue of mice in the three administration groups were significantly accelerated, and the collagen fibers in the wounds of mice in the three administration groups were larger in number and more neatly arranged in comparison with the control group. On PID 7, 14, and 21, under every 200-fold visual field, the number of new microvessels in the wound tissue of mice in local wound application group was 24.1±2.5, 50.7±4.1, and 44.2±2.3, respectively, the number of new microvessels in the wound tissue of mice in wound margin subcutaneous injection group was 32.2±2.9, 67.5±4.9, and 53.6±3.7, respectively, and the number of new microvessels in the wound tissue of mice in tail vein injection group was 27.8±2.4, 59.1±3.7, and 49.6±2.6, respectively, which was significantly more than 20.6±1.7, 46.7±3.4, and 40.9±2.8 in control group (P<0.05); the number of new microvessels in the wound tissue of mice in wound margin subcutaneous injection group and tail vein injection group was significantly more than that in local wound application group (P<0.05); the number of new microvessels in the wound tissue of mice in wound margin subcutaneous injection group was significantly more than that in tail vein injection group (P<0.05). On PID 7, 14, and 21, the content of TNF-α and IL-6 in the wound tissue of mice in the three administration groups was significantly less than that in control group (P<0.05), the content of TNF-α and IL-6 in the wound tissue of mice in wound margin subcutaneous injection group and tail vein injection group was significantly less than that in local wound application group (P<0.05), and the content of TNF-α and IL-6 in the wound tissue of mice in wound margin subcutaneous injection group was significantly less than that in tail vein injection group (P<0.05). Conclusions: Local wound application, subcutaneous injection at the wound margin, and tail vein injection of hUCMSC exosomes can all promote the wound healing of full-thickness skin defects in mice through alleviating excessive inflammatory response and promoting angiogenesis. Among them, subcutaneous injection at the wound margin has a better therapeutic effect, indicating subcutaneous injection at the wound margin is the optimal administration route for hUCMSC exosomes in wound treatment. 目的： 探讨通过创面局部涂抹、创缘皮下注射和尾静脉注射人脐带间充质干细胞（hUCMSC）外泌体治疗小鼠全层皮肤缺损创面的效果，探究应用hUCMSC外泌体治疗创面的最佳给药途径。 方法： 该研究为实验研究。从3名25~35岁于内蒙古包钢医院妇产科正常分娩产妇弃用脐带组织中提取hUCMSC外泌体并成功鉴定。选用120只6~8周龄雄性BALB/c小鼠，于其背部制备全层皮肤缺损创面后，按随机数字表法分为对照组（不进行给药处理）、创面局部涂抹组、创缘皮下注射组、尾静脉注射组（每组30只小鼠），分别通过创面局部涂抹、创缘皮下注射、尾静脉注射给予后3组小鼠0.2 mL含200 μg hUCMSC外泌体的磷酸盐缓冲液。伤后7、14、21 d，观察创面的大体情况并计算创面愈合率；采集创面组织，分别通过苏木精-伊红和Masson染色观测病理学变化和胶原纤维情况，行CD31免疫组织化学染色观测新生微血管数量，采用酶联免疫吸附测定法检测肿瘤坏死因子α（TNF-α）和白细胞介素6（IL-6）的含量。各组各时间点样本数均为10。 结果： 伤后7、14、21 d，4组小鼠创面均逐步愈合，其中创缘皮下注射组小鼠创面愈合情况最佳；3个给药组小鼠创面愈合率均显著高于对照组（P<0.05），创缘皮下注射组及尾静脉注射组小鼠创面愈合率均显著高于创面局部涂抹组（P<0.05），创缘皮下注射组小鼠创面愈合率均显著高于尾静脉注射组（P<0.05）。伤后7、14、21 d，与对照组比较，3个给药组小鼠创面组织生长、上皮化的速度显著加快，创面胶原纤维生成数量更多且排列更整齐。伤后7、14、21 d，在每200倍视野下，创面局部涂抹组小鼠创面组织新生微血管数量分别为（24.1±2.5）、（50.7±4.1）、（44.2±2.3）根，创缘皮下注射组小鼠创面组织新生微血管数量分别为（32.2±2.9）、（67.5±4.9）、（53.6±3.7）根，尾静脉注射组小鼠创面组织新生微血管数量分别为（27.8±2.4）、（59.1±3.7）、（49.6±2.6）根，均显著多于对照组的（20.6±1.7）、（46.7±3.4）、（40.9±2.8）根（P<0.05）；创缘皮下注射组及尾静脉注射组小鼠创面组织中新生微血管数量均显著多于创面局部涂抹组（P<0.05）；创缘皮下注射组小鼠创面组织中新生微血管数量均显著多于尾静脉注射组（P<0.05）。伤后7、14、21 d，3个给药组小鼠创面组织中TNF-α和IL-6的含量均较对照组显著减少（P<0.05），创缘皮下注射组及尾静脉注射组小鼠创面组织中TNF-α和IL-6的含量均显著少于创面局部涂抹组（P<0.05），创缘皮下注射组小鼠创面组织中TNF-α和IL-6的含量均显著少于尾静脉注射组（P<0.05）。 结论： 创面局部涂抹、创缘皮下注射及尾静脉注射hUCMSC外泌体均可通过减轻过度炎症反应、促进血管新生发挥促进小鼠全层皮肤缺损创面愈合的作用，其中，创缘皮下注射具有更好的治疗效果，即创缘皮下注射是采用hUCMSC外泌体治疗创面的最佳给药途径。.
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