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  • Title: [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].
    Author: Chen YQ, Zhou YQ, Wei Q, Xie XY, Liu XZ, Li DW, Shen ZA.
    Journal: Zhonghua Shao Shang Yu Chuang Mian Xiu Fu Za Zhi; 2024 Apr 20; 40(4):323-332. PubMed ID: 38664026.
    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. 目的： 探究负载人脐带间充质干细胞来源的小细胞外囊泡（hUCMSC-sEV）的甲基丙烯酸酐化明胶（GelMA）水凝胶治疗小鼠全层皮肤缺损创面的效果。 方法： 该研究为实验研究。采用超速离心法提取hUCMSC-sEV，通过透射电子显微镜观察其形貌，采用蛋白质印迹法检测CD9、CD63、肿瘤易感基因101（TSG101）及钙联蛋白的表达。将人脐静脉内皮细胞（HUVEC）及第3、4代人表皮角质形成细胞（HEK）、人真皮成纤维细胞（HDF）均分为常规培养的空白对照组和在细胞培养液中加入hUCMSC-sEV培养的hUCMSC-sEV组，行细胞划痕试验并计算划痕后 6、12、24 h 的细胞迁移率，行细胞Transwell试验并计算培养12 h细胞迁移数量，行5-乙炔基-2'-脱氧尿嘧啶核苷、Hoechst染色检测培养24 h增殖细胞比例，样本数均为3。制备单纯GelMA水凝胶及负载hUCMSC-sEV的GelMA水凝胶（以下简称hUCMSC-sEV/GelMA水凝胶），通过扫描电子显微镜观察2种水凝胶微观形貌，通过激光扫描共聚焦显微镜观察hUCMSC-sEV的分布情况，采用蛋白质比色定量法测定并计算hUCMSC-sEV/GelMA水凝胶在磷酸盐缓冲液（PBS）中浸泡0（即刻）、2、4、6、8、10、12 d时hUCMSC-sEV累积释放率（样本数为3）。将24只6周龄雄性C57BL/6J小鼠按随机数字表法分为PBS组、单纯hUCMSC-sEV组、单纯GelMA水凝胶组和hUCMSC-sEV/GelMA水凝胶组（每组6只），于小鼠背部制备全层皮肤缺损创面后分别行PBS注射、hUCMSC-sEV悬液注射、单纯GelMA水凝胶覆盖、hUCMSC-sEV/GelMA水凝胶覆盖。于伤后0（即刻）、4、8、12 d观察创面愈合情况并统计伤后4、8、12 d创面愈合率，于伤后12 d取创面组织行苏木精-伊红染色后观察创面新生组织结构，样本数均为6。 结果： 提取的hUCMSC-sEV呈杯状结构，表达CD9、CD63和TSG101，几乎不表达钙联蛋白。划痕后6、12、24 h，hUCMSC-sEV组HEK（t值分别为25.94、20.98、20.04）、HDF（t值分别为3.18、5.68、4.28）、HUVEC（t值分别为4.32、19.33、4.00）的迁移率均明显高于空白对照组（P<0.05）。培养12 h，hUCMSC-sEV组HEK、HDF及HUVEC迁移数量分别为（550{L-End} ±23）、（235{L-End} ±9）、（856{L-End} ±35）个，均明显多于空白对照组的（188{L-End} ±14）、（97{L-End} ±6）、（370{L-End} ±32）个（t值分别为22.95、23.13、17.84，P<0.05）。培养24 h，hUCMSC-sEV组HEK、HDF及HUVEC增殖细胞比例均明显高于空白对照组（t值分别为22.00、13.82、32.32，P<0.05）。单纯GelMA水凝胶内部呈疏松多孔的海绵状结构且其中未见hUCMSC-sEV，hUCMSC-sEV/GelMA水凝胶具有相同海绵状结构且其中可见hUCMSC-sEV呈团块状均匀分布。hUCMSC-sEV/GelMA水凝胶浸泡2 d后hUCMSC-sEV累积释放率曲线趋于平缓，浸泡12 d时hUCMSC-sEV累积释放率为（59.2{L-End} ±1.8）%。伤后0~12 d，4组小鼠创面均不断缩小。伤后4、8、12 d，hUCMSC-sEV/GelMA水凝胶组小鼠创面愈合率均明显高于其余3组（P<0.05），单纯GelMA水凝胶组、单纯hUCMSC-sEV组小鼠创面愈合率均明显高于PBS组（P<0.05）；伤后8、12 d，单纯hUCMSC-sEV组小鼠创面愈合率均明显高于单纯GelMA水凝胶组（P<0.05）。伤后12 d，hUCMSC-sEV/GelMA水凝胶组小鼠创面上皮化程度最佳，真皮胶原排列松散有序，炎症细胞数量最少；其余3组小鼠创面均可见真皮胶原排列致密且存在不同程度的炎症细胞浸润。 结论： hUCMSC-sEV能够促进皮肤创面愈合相关细胞HEK、HDF与HUVEC迁移与增殖，并可在GelMA水凝胶内缓慢释放。hUCMSC-sEV/GelMA水凝胶作为创面敷料能够显著提高小鼠全层皮肤缺损创面愈合速度。.
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