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
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摘要:
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 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。 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 3 rd and 4 th 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 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. -
参考文献
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1 hUCMSC-sEV的鉴定。1A.hUCMSC-sEV呈杯状结构 透射电子显微镜×100 000；1B.采用蛋白质印迹法检测的hUCMSC-sEV和人脐带间充质干细胞中CD9、CD63、TSG101和钙联蛋白表达情况
注：TSG101为肿瘤易感基因101，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡；条带上方1、2分别指示hUCMSC-sEV、人脐带间充质干细胞
2 划痕试验检测的2组HEK、HDF及HUVEC划痕后各时间点水平迁移情况 倒置相差显微镜×50。2A、2B、2C.分别为空白对照组HEK划痕后0（即刻）、12、24 h的划痕面积；2D、2E、2F.分别为hUCMSC-sEV组HEK划痕后0、12、24 h的划痕面积，图2D与图2A划痕面积相近，图2E、2F划痕面积分别明显小于图2B、2C；2G、2H、2I.分别为空白对照组HDF划痕后0、12、24 h的划痕面积；2J、2K、2L.分别为hUCMSC-sEV组HDF划痕后0、12、24 h的划痕面积，图2J与图2G划痕面积相近，图2K、2L划痕面积分别明显小于图2H、2I；2M、2N、2O.分别为空白对照组HUVEC划痕后0、12、24 h的划痕面积；2P、2Q、2R.分别为hUCMSC-sEV组HUVEC划痕后0、12、24 h的划痕面积，图2P与图2M划痕面积相近，图2Q、2R划痕面积分别明显小于图2N、2O
注：HEK为人表皮角质形成细胞，HDF为人真皮成纤维细胞，HUVEC为人脐静脉内皮细胞，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡；空白对照组细胞仅常规培养，hUCMSC-sEV组细胞培养液中另加入hUCMSC-sEV
3 Transwell试验检测的2组HEK、HDF及HUVEC培养12 h垂直迁移情况 结晶紫×40。3A、3B、3C.分别为空白对照组HEK、HDF、HUVEC迁移至Transwell下室的细胞情况；3D、3E、3F.分别为hUCMSC-sEV组HEK、HDF、HUVEC迁移至Transwell下室的细胞情况，图3D、3E、3F的细胞数量分别多于图3A、3B、3C
注：HEK为人表皮角质形成细胞，HDF为人真皮成纤维细胞，HUVEC为人脐静脉内皮细胞，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡；空白对照组细胞仅常规培养，hUCMSC-sEV组细胞培养液中另加入hUCMSC-sEV；细胞结晶紫阳性染色为紫色
4 2组HEK、HDF及HUVEC培养24 h增殖能力 EdU-Hoechst 33342×200。4A、4B、4C.分别为空白对照组HEK、HDF、HUVEC EdU及Hoechst 33342共染色情况，3种细胞的细胞核均完整；4D、4E、4F.分别为hUCMSC-sEV组HEK、HDF、HUVEC EdU及Hoechst 33342共染色情况，3种细胞的细胞核均完整，图4D、4E、4F EdU染色阳性细胞数量分别多于图4A、4B、4C
注：HEK为人表皮角质形成细胞，HDF为人真皮成纤维细胞，HUVEC为人脐静脉内皮细胞，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡，EdU为5-乙炔基-2'-脱氧尿嘧啶核苷；空白对照组细胞仅常规培养，hUCMSC-sEV组细胞培养液中另加入hUCMSC-sEV；各图中右上小图为EdU阳性染色（绿色）图，右下小图为Hoechst 33342阳性染色（蓝色）图，各图中左侧大图为右侧2张小图的叠加图像，绿色+蓝色双荧光染色的细胞为增殖细胞
5 2种GelMA水凝胶冻干后的微观形貌。5A.单纯GelMA水凝胶呈多孔结构且孔隙均匀 扫描电子显微镜×500；5B.为图5A中方框中图形的放大图，显示单纯GelMA水凝胶清晰的孔壁结构 扫描电子显微镜×1 000；5C.负载hUCMSC-sEV的GelMA水凝胶孔隙均匀，孔隙较图5A小 扫描电子显微镜×500；5D.为图5C中方框中图形的放大图，黄色箭头指水凝胶中的hUCMSC-sEV 扫描电子显微镜×1 000
注：GelMA为甲基丙烯酸酐化明胶，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡
6 2种GelMA水凝胶中hUCMSC-sEV分布情况 1,1'-二十八烷基-3,3,3',3'-四甲基氨基氰高氯酸盐×400。6A.单纯GelMA水凝胶中无hUCMSC-sEV；6B.负载hUCMSC-sEV的GelMA水凝胶中可见hUCMSC-sEV均匀分布
注：GelMA为甲基丙烯酸酐化明胶，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡；hUCMSC-sEV阳性染色为红色
7 负载hUCMSC-sEV的GelMA水凝胶浸泡各时间点的hUCMSC-sEV累积释放率（样本数为3，
注：GelMA为甲基丙烯酸酐化明胶，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡
8 4组全层皮肤缺损小鼠伤后各时间点创面愈合情况。8A、8B、8C、8D.分别为PBS组、单纯hUCMSC-sEV组、单纯GelMA水凝胶组和负载hUCMSC-sEV的GelMA水凝胶组伤后4 d创面愈合情况，图8D创面面积小于图8A、8B、8C；8E、8F、8G、8H.分别为PBS组、单纯hUCMSC-sEV组、单纯GelMA水凝胶组和负载hUCMSC-sEV的GelMA水凝胶组伤后12 d创面愈合情况，图8H创面面积小于图8E、8F、8G
注：PBS为磷酸盐缓冲液，GelMA为甲基丙烯酸酐化明胶，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡；对PBS组、单纯hUCMSC-sEV组、单纯GelMA水凝胶组和hUCMSC-sEV/GelMA水凝胶组小鼠创面分别行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；直尺刻度为1 mm
表1 2组HEK、HDF、HUVEC划痕后各时间点迁移率比较（%，
表1. Comparison of the migration rates of HEKs, HDFs, and HUVECs between the two groups at each time point after scratching
组别 样本数 HEK HDF HUVEC 6 h 12 h 24 h 6 h 12 h 24 h 6 h 12 h 24 h 空白对照组 3 0.9 14.0 58.1 9.3 19.5 47.8 7.2 10.1 39.1 hUCMSC-sEV组 3 51.4 88.2 95.7 19.8 35.4 62.2 15.1 38.9 56.7 t值 25.94 20.98 20.04 3.18 5.68 4.28 4.32 19.33 4.00 P值 <0.001 <0.001 <0.001 0.033 0.004 0.013 0.012 <0.001 0.016 注：HEK为人表皮角质形成细胞，HDF为人真皮成纤维细胞，HUVEC为人脐静脉内皮细胞，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡；空白对照组细胞仅常规培养，hUCMSC-sEV组细胞培养液中另加入hUCMSC-sEV；HEK、HDF、HUVEC处理因素主效应， F值分别为1 387.01、40.65、66.89， P值分别为<0.001、0.003、0.001；时间因素主效应， F值分别为381.30、209.70、200.13， P值均<0.001；两者交互作用， F值分别为51.20、0.93、5.90， P值分别为<0.001、0.430、0.027 
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表2 4组小鼠伤后各时间点全层皮肤缺损创面愈合率比较（%，
表2. Comparison of the wound healing rates of full-thickness skin defects of mice in the four groups at each time point post injury
组别 样本量 4 d 8 d 12 d PBS组 6 58.3±1.3 63.7±0.4 77.8±1.3 单纯GelMA水凝胶组 6 65.4±4.4 69.2±1.8 92.8±0.5 单纯hUCMSC-sEV组 6 67.8±0.9 75.7±0.4 97.2±0.6 负载hUCMSC-sEV的GelMA水凝胶组 6 80.2±2.6 97.9±0.6 100 F值 70.00 1 332.52 1 001.81 P值 <0.001 <0.001 <0.001 P 1值 <0.001 <0.001 <0.001 P 2值 <0.001 <0.001 <0.001 P 3值 <0.001 <0.001 <0.001 P 4值 0.076 <0.001 <0.001 P 5值 <0.001 <0.001 <0.001 P 6值 <0.001 <0.001 0.031 注：PBS为磷酸盐缓冲液，GelMA为甲基丙烯酸酐化明胶，hUCMSC-sEV为人脐带间充质干细胞来源的小细胞外囊泡；对PBS组、单纯GelMA水凝胶组、单纯hUCMSC-sEV组和hUCMSC-sEV/GelMA水凝胶组小鼠创面分别行PBS注射、单纯GelMA水凝胶覆盖、hUCMSC-sEV悬液注射、hUCMSC-sEV/GelMA水凝胶覆盖；处理因素主效应， F=461.49， P=0.001；时间因素主效应， F=1 714.92， P<0.001；两者交互作用， F=76.83， P=0.001； P 1值、 P 2值、 P 3值分别为单纯GelMA水凝胶组、单纯hUCMSC-sEV组、hUCMSC-sEV/GelMA水凝胶组与PBS组各时间点比较所得； P 4值、 P 5值分别为单纯hUCMSC-sEV组、hUCMSC-sEV/GelMA水凝胶组与单纯GelMA水凝胶组各时间点比较所得； P 6值为单纯hUCMSC-sEV组与hUCMSC-sEV/GelMA水凝胶组各时间点比较所得
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