[Effects of gelatin methacrylate anhydride hydrogel loaded with small extracellular vesicles derived from human umbilical cord mesenchymal stem cells in the treatment of full-thickness skin defect wounds in mice].

Y Q Chen, Y. Q. Zhou, Q Wei, X. Y. Xie, X Z Liu, D. W. Li, Z. A. Shen
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The human umbilical vein endothelial cells (HUVECs), the 3rd and 4th passages of human epidermal keratinocytes (HEKs) and human dermal fibroblasts (HDFs) were all divided into blank control group (routinely cultured) and hUCMSC-sEV group (cultured with the cell supernatant containing hUCMSCs-sEVs). The cell scratch test was performed and the cell migration rates at 6, 12, and 24 h after scratching were calculated, the cell Transwell assay was performed and the number of migration cells at 12 h after culture was calculated, and the proportion of proliferating cells was detected by 5-acetylidene-2'-deoxyuridine and Hoechst staining at 24 h after culture, with sample numbers being all 3. The simple GelMA hydrogel and the GelMA hydrogel loaded with hUCMSCs-sEVs (hereinafter referred to as hUCMSC-sEV/GelMA hydrogel) were prepared. Then the micromorphology of 2 kinds of hydrogels was observed under scanning electron microscope, the distribution of hUCMSCs-sEVs was observed by laser scanning confocal microscope, and the cumulative release rates of hUCMSCs-sEVs at 0 (immediately), 2, 4, 6, 8, 10, and 12 d after soaking hUCMSC-sEV/GelMA hydrogel in phosphate buffer solution (PBS) were measured and calculated by protein colorimetric quantification (n=3). Twenty-four 6-week-old male C57BL/6J mice were divided into PBS group, hUCMSC-sEV alone group, GelMA hydrogel alone group, and hUCMSC-sEV/GelMA hydrogel group according to the random number table, with 6 mice in each group, and after the full-thickness skin defect wounds on the back of mice in each group were produced, the wounds were performed with PBS injection, hUCMSC-sEV suspenson injection, simple GelMA coverage, and hUCMSC-sEV/GelMA hydrogel coverage, respectively. Wound healing was observed on post injury day (PID) 0 (immediately), 4, 8, and 12, and the wound healing rates on PID 4, 8, and 12 were calculated, and the wound tissue was collected on PID 12 for hematoxylin-eosin staining to observe the structure of new tissue, with sample numbers being both 6. Results: The extracted hUCMSCs-sEVs showed a cup-shaped structure and expressed CD9, CD63, and TSG101, but barely expressed calnexin. At 6, 12, and 24 h after scratching, the migration rates of HEKs (with t values of 25.94, 20.98, and 20.04, respectively), HDFs (with t values of 3.18, 5.68, and 4.28, respectively), and HUVECs (with t values of 4.32, 19.33, and 4.00, respectively) in hUCMSC-sEV group were significantly higher than those in blank control group (P<0.05). At 12 h after culture, the numbers of migrated HEKs, HDFs, and HUVECs in hUCMSC-sEV group were 550±23, 235±9, and 856±35, respectively, which were significantly higher than 188±14, 97±6, and 370±32 in blank control group (with t values of 22.95, 23.13, and 17.84, respectively, P<0.05). At 24 h after culture, the proportions of proliferating cells of HEKs, HDFs, and HUVECs in hUCMSC-sEV group were significantly higher than those in blank control group (with t values of 22.00, 13.82, and 32.32, respectively, P<0.05). The inside of simple GelMA hydrogel showed a loose and porous sponge-like structure, and hUCMSCs-sEVs was not observed in it. The hUCMSC-sEV/GelMA hydrogel had the same sponge-like structure, and hUCMSCs-sEVs were uniformly distributed in clumps. The cumulative release rate curve of hUCMSCs-sEVs from hUCMSC-sEV/GelMA hydrogel tended to plateau at 2 d after soaking, and the cumulative release rate of hUCMSCs-sEVs was (59.2±1.8)% at 12 d after soaking. From PID 0 to 12, the wound areas of mice in the 4 groups gradually decreased. On PID 4, 8, and 12, the wound healing rates of mice in hUCMSC-sEV/GelMA hydrogel group were significantly higher than those in the other 3 groups (P<0.05); the wound healing rates of mice in GelMA hydrogel alone group and hUCMSC-sEV alone group were significantly higher than those in PBS group (P<0.05). On PID 8 and 12, the wound healing rates of mice in hUCMSC-sEV alone group were significantly higher than those in GelMA hydrogel alone group (P<0.05). On PID 12, the wounds of mice in hUCMSC-sEV/GelMA hydrogel group showed the best wound epithelization, loose and orderly arrangement of dermal collagen, and the least number of inflammatory cells, while the dense arrangement of dermal collagen and varying degrees of inflammatory cell infiltration were observed in the wounds of mice in the other 3 groups. Conclusions: hUCMSCs-sEVs can promote the migration and proliferation of HEKs, HDFs, and HUVECs which are related to skin wound healing, and slowly release in GelMA hydrogel. The hUCMSC-sEV/GelMA hydrogel as a wound dressing can significantly improve the healing speed of full-thickness skin defect wounds in mice.","PeriodicalId":516861,"journal":{"name":"Zhonghua shao shang yu chuang mian xiu fu za zhi","volume":null,"pages":null},"PeriodicalIF":0.0000,"publicationDate":"2024-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Zhonghua shao shang yu chuang mian xiu fu za zhi","FirstCategoryId":"0","ListUrlMain":"https://doi.org/10.3760/cma.j.cn501225-20231218-00248","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0

Abstract

Objective: To investigate the effects of gelatin methacrylate anhydride (GelMA) hydrogel loaded with small extracellular vesicles derived from human umbilical cord mesenchymal stem cells (hUCMSCs-sEVs) in the treatment of full-thickness skin defect wounds in mice. Methods: This study was an experimental study. hUCMSCs-sEVs were extracted by ultracentrifugation, their morphology was observed through transmission electron microscope, and the expression of CD9, CD63, tumor susceptibility gene 101 (TSG101), and calnexin was detected by Western blotting. The human umbilical vein endothelial cells (HUVECs), the 3rd and 4th passages of human epidermal keratinocytes (HEKs) and human dermal fibroblasts (HDFs) were all divided into blank control group (routinely cultured) and hUCMSC-sEV group (cultured with the cell supernatant containing hUCMSCs-sEVs). The cell scratch test was performed and the cell migration rates at 6, 12, and 24 h after scratching were calculated, the cell Transwell assay was performed and the number of migration cells at 12 h after culture was calculated, and the proportion of proliferating cells was detected by 5-acetylidene-2'-deoxyuridine and Hoechst staining at 24 h after culture, with sample numbers being all 3. The simple GelMA hydrogel and the GelMA hydrogel loaded with hUCMSCs-sEVs (hereinafter referred to as hUCMSC-sEV/GelMA hydrogel) were prepared. Then the micromorphology of 2 kinds of hydrogels was observed under scanning electron microscope, the distribution of hUCMSCs-sEVs was observed by laser scanning confocal microscope, and the cumulative release rates of hUCMSCs-sEVs at 0 (immediately), 2, 4, 6, 8, 10, and 12 d after soaking hUCMSC-sEV/GelMA hydrogel in phosphate buffer solution (PBS) were measured and calculated by protein colorimetric quantification (n=3). Twenty-four 6-week-old male C57BL/6J mice were divided into PBS group, hUCMSC-sEV alone group, GelMA hydrogel alone group, and hUCMSC-sEV/GelMA hydrogel group according to the random number table, with 6 mice in each group, and after the full-thickness skin defect wounds on the back of mice in each group were produced, the wounds were performed with PBS injection, hUCMSC-sEV suspenson injection, simple GelMA coverage, and hUCMSC-sEV/GelMA hydrogel coverage, respectively. Wound healing was observed on post injury day (PID) 0 (immediately), 4, 8, and 12, and the wound healing rates on PID 4, 8, and 12 were calculated, and the wound tissue was collected on PID 12 for hematoxylin-eosin staining to observe the structure of new tissue, with sample numbers being both 6. Results: The extracted hUCMSCs-sEVs showed a cup-shaped structure and expressed CD9, CD63, and TSG101, but barely expressed calnexin. At 6, 12, and 24 h after scratching, the migration rates of HEKs (with t values of 25.94, 20.98, and 20.04, respectively), HDFs (with t values of 3.18, 5.68, and 4.28, respectively), and HUVECs (with t values of 4.32, 19.33, and 4.00, respectively) in hUCMSC-sEV group were significantly higher than those in blank control group (P<0.05). At 12 h after culture, the numbers of migrated HEKs, HDFs, and HUVECs in hUCMSC-sEV group were 550±23, 235±9, and 856±35, respectively, which were significantly higher than 188±14, 97±6, and 370±32 in blank control group (with t values of 22.95, 23.13, and 17.84, respectively, P<0.05). At 24 h after culture, the proportions of proliferating cells of HEKs, HDFs, and HUVECs in hUCMSC-sEV group were significantly higher than those in blank control group (with t values of 22.00, 13.82, and 32.32, respectively, P<0.05). The inside of simple GelMA hydrogel showed a loose and porous sponge-like structure, and hUCMSCs-sEVs was not observed in it. The hUCMSC-sEV/GelMA hydrogel had the same sponge-like structure, and hUCMSCs-sEVs were uniformly distributed in clumps. The cumulative release rate curve of hUCMSCs-sEVs from hUCMSC-sEV/GelMA hydrogel tended to plateau at 2 d after soaking, and the cumulative release rate of hUCMSCs-sEVs was (59.2±1.8)% at 12 d after soaking. From PID 0 to 12, the wound areas of mice in the 4 groups gradually decreased. On PID 4, 8, and 12, the wound healing rates of mice in hUCMSC-sEV/GelMA hydrogel group were significantly higher than those in the other 3 groups (P<0.05); the wound healing rates of mice in GelMA hydrogel alone group and hUCMSC-sEV alone group were significantly higher than those in PBS group (P<0.05). On PID 8 and 12, the wound healing rates of mice in hUCMSC-sEV alone group were significantly higher than those in GelMA hydrogel alone group (P<0.05). On PID 12, the wounds of mice in hUCMSC-sEV/GelMA hydrogel group showed the best wound epithelization, loose and orderly arrangement of dermal collagen, and the least number of inflammatory cells, while the dense arrangement of dermal collagen and varying degrees of inflammatory cell infiltration were observed in the wounds of mice in the other 3 groups. Conclusions: hUCMSCs-sEVs can promote the migration and proliferation of HEKs, HDFs, and HUVECs which are related to skin wound healing, and slowly release in GelMA hydrogel. The hUCMSC-sEV/GelMA hydrogel as a wound dressing can significantly improve the healing speed of full-thickness skin defect wounds in mice.
[明胶甲基丙烯酸酯酐水凝胶负载从人脐带间充质干细胞提取的细胞外小泡对治疗小鼠全厚皮肤缺损伤口的影响]。
目的研究甲基丙烯酸明胶酸酐(GelMA)水凝胶负载由人脐带间充质干细胞(hUCMSCs-sEVs)提取的小细胞外囊泡治疗小鼠全厚皮肤缺损伤口的效果。研究方法超速离心提取 hUCMSCs-sEV,透射电镜观察其形态,Western 印迹检测 CD9、CD63、肿瘤易感基因 101(TSG101)和 calnexin 的表达。将人脐静脉内皮细胞(HUVECs)、人表皮角质细胞(HEKs)和人真皮成纤维细胞(HDFs)分为空白对照组(常规培养)和 hUCMSCs-sEV 组(用含 hUCMSCs-sEVs 的细胞上清液培养)。进行细胞划痕试验并计算划痕后 6、12 和 24 h 的细胞迁移率,进行细胞 Transwell 试验并计算培养后 12 h 的迁移细胞数,培养后 24 h 用 5-acetylidene-2'-deoxyuridine 和 Hoechst 染色法检测增殖细胞的比例,样本数均为 3。制备简单的 GelMA 水凝胶和负载 hUCMSCs-sEV 的 GelMA 水凝胶(以下简称 hUCMSC-sEV/GelMA 水凝胶)。然后用扫描电子显微镜观察两种水凝胶的微观形态,用激光扫描共聚焦显微镜观察 hUCMSCs-sEVs 的分布,用蛋白质比色定量法测量并计算 hUCMSCs-sEVs 在磷酸盐缓冲液(PBS)中浸泡 0(立即)、2、4、6、8、10 和 12 d 后的累积释放率(n=3)。将 24 只 6 周大雄性 C57BL/6J 小鼠按随机数字表法分为 PBS 组、单独 hUCMSC-sEV 组、单独 GelMA 水凝胶组和 hUCMSC-sEV/GelMA 水凝胶组,每组 6 只、各组小鼠背部全厚皮肤缺损创面制作完成后,分别进行 PBS 注射、hUCMSC-sEV 悬液注射、单纯 GelMA 覆盖和 hUCMSC-sEV/GelMA 水凝胶覆盖。在损伤后第 0 天(即刻)、第 4 天、第 8 天和第 12 天观察伤口愈合情况,计算第 4 天、第 8 天和第 12 天的伤口愈合率,并在第 12 天收集伤口组织进行苏木精-伊红染色,观察新生组织的结构,样本数均为 6。结果提取的 hUCMSCs-sEVs 呈杯状结构,表达 CD9、CD63 和 TSG101,但几乎不表达 calnexin。划痕后 6、12 和 24 h,hUCMSCs-sEV 组 HEKs(t 值分别为 25.94、20.98 和 20.04)、HDFs(t 值分别为 3.18、5.68 和 4.28)和 HUVECs(t 值分别为 4.32、19.33 和 4.00)的迁移率明显高于空白对照组(P<0.05)。培养 12 h 后,hUCMSC-sEV 组迁移的 HEKs、HDFs 和 HUVECs 数量分别为 550±23、235±9 和 856±35,明显高于空白对照组的 188±14、97±6 和 370±32(t 值分别为 22.95、23.13 和 17.84,P<0.05)。培养 24 h 后,hUCMSC-sEV 组的 HEKs、HDFs 和 HUVECs 增殖细胞比例明显高于空白对照组(t 值分别为 22.00、13.82 和 32.32,P<0.05)。单纯 GelMA 水凝胶内部呈疏松多孔的海绵状结构,未观察到 hUCMSCs-sEV。hUCMSC-sEV/GelMA 水凝胶具有相同的海绵状结构,hUCMSCs-sEV 呈团块状均匀分布。浸泡 2 d 后,hUCMSCs-sEV/GelMA 水凝胶中 hUCMSCs-sEV 的累积释放率曲线趋于平稳,浸泡 12 d 后,hUCMSCs-sEV 的累积释放率为(59.2±1.8)%。从 PID 0 到 12,4 组小鼠的伤口面积逐渐缩小。在 PID 4、8 和 12,hUCMSC-sEV/GelMA 水凝胶组小鼠的伤口愈合率显著高于其他 3 组(P<0.05);单独 GelMA 水凝胶组和单独 hUCMSC-sEV 组小鼠的伤口愈合率显著高于 PBS 组(P<0.05)。在 PID 8 和 12,单用 hUCMSC-sEV 组小鼠的伤口愈合率明显高于单用 GelMA 水凝胶组(P<0.05)。在 PID 12,hUCMSC-sEV/GelMA 水凝胶组小鼠的伤口上皮愈合最好,真皮胶原排列疏松有序,炎症细胞数量最少,而其他三组小鼠的伤口真皮胶原排列密集,炎症细胞有不同程度的浸润。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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