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Chinese Journal of Burns and Wounds logoLink to Chinese Journal of Burns and Wounds
. 2024 Apr 20;40(4):323–332. [Article in Chinese] doi: 10.3760/cma.j.cn501225-20231218-00248

负载人脐带间充质干细胞来源的小细胞外囊泡的甲基丙烯酸酐化明胶水凝胶治疗小鼠全层皮肤缺损创面的效果

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

Yiqi Chen 1, Yingqian Zhou 1, Qian Wei 1, Xiaoye Xie 1, Xinzhu Liu 1, Dawei Li 1, Chuan'an Shen 1,*
PMCID: PMC11630113  PMID: 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.

Keywords: Tissue engineering, Mesenchymal stem cells, Hydrogel, Extracellular vesicles, Biocompatible materials

临床上对创面的治疗方法通常为换药、负压吸引及手术治疗[1]，随着患者对创面修复要求的提高，现有的治疗方法逐渐无法满足临床需求。既往研究表明，KC和Fb是皮肤创面愈合中的关键细胞[2-3]。如何增强皮肤细胞的活性，增强其再生功能，进而促进创面愈合，是目前创面修复领域的研究热点。随着再生医学的发展，干细胞疗法为创面的修复和再生性愈合带来了新的思路[4-5]。

间充质干细胞（mesenchymal stem cell，MSC）是一种多能干细胞，近期研究显示，MSC主要依靠旁分泌产生的细胞外囊泡（extracellular vesicle，EV）发挥效应，小EV（small extracellular vesicle，sEV）是直径<200 nm的EV[6]。研究表明人MSC来源的sEV可以作用于靶细胞，发挥促进组织修复的作用[7-8]。但直接外用或浅表注射至创面的sEV在创面的有效存留时间较短，无法达到预期疗效，且提高其剂量并不能显著改善上述情况[9]。因此，如何延长sEV在创面的存留时间，使其更好地发挥疗效是目前亟须解决的问题。

水凝胶是一种有良好生物相容性的材料。甲基丙烯酸酐化明胶（gelatin methacrylate anhydride，GelMA）水凝胶含有的多肽序列有利于细胞及组织黏附[10]，且可滴加GelMA溶液至创面部位，使其在紫外光照射下原位成胶 [11]。

本研究着眼于人脐带MSC（human umbilical cord mesenchymal stem cell，hUCMSC）来源的sEV（small extracellular vesicle derived from human umbilical cord mesenchymal stem cell，hUCMSC-sEV）对创面的全层修复，详尽表征了其对创面愈合的重要细胞：人表皮KC（human epidermal keratinocyte，HEK）、人真皮Fb（human dermal fibroblast，HDF）及人脐静脉内皮细胞（human umbilical vein endothelial cell，HUVEC）的作用，并进一步开发了一种负载hUCMSC-sEV的GelMA（以下简称hUCMSC-sEV/GelMA）水凝胶，探究该水凝胶能否改善伤处皮肤细胞的功能从而促进皮肤创面愈合，以期为临床全层皮肤缺损创面治疗提供新思路。

1. 材料与方法

本实验研究遵循解放军总医院实验动物伦理审查委员会和国家有关实验动物管理使用的规定。

1.1. 动物、细胞及主要试剂与仪器来源

24只健康清洁级6周龄体重23~25 g雄性C57BL/6J小鼠购自北京科宇动物养殖中心，许可证号：SCXK（京）2018-0010。HUVEC细胞系、原代hUCMSC、原代HEK、原代HDF购自武汉普诺赛生命科技有限公司。

DMEM培养基、EpiLife培养基、人KC生长添加剂、胎牛血清、青霉素-链霉素溶液购自美国Gibco公司，无EV血清购自美国SBI公司，小鼠抗人CD63、CD9、肿瘤易感基因101（tumor susceptibility gene 101，TSG101）及钙联蛋白单克隆抗体购自美国Abcam公司，辣根过氧化物酶标记的山羊抗小鼠IgG多克隆抗体购自北京中杉金桥生物技术有限公司，BeyoClickTM 5-乙炔基-2'-脱氧尿嘧啶核苷（5-ethynyl-2'-deoxyuridine，EdU）-488细胞增殖检测试剂盒及结晶紫染色液购自上海碧云天生物技术有限公司，Transwell培养板购自美国Corning公司，GelMA购自苏州永沁泉智能设备有限公司。311型二氧化碳培养箱购自美国Thermo Fisher公司，GelDoc XR+凝胶成像分析系统购自美国Bio-Rad公司，CKX53型倒置荧光显微镜购自日本Olympus公司，DMI1型倒置相差显微镜、TCS SPE型激光扫描共聚焦显微镜购自德国Leica公司，LVEM 25E型透射电子显微镜购自加拿大Delong Instrument公司，Sigma 300型扫描电子显微镜购自德国ZEISS公司。

1.2. 细胞培养

用含有体积分数10%的无sEV血清和常规浓度的青霉素-链霉素溶液的DMEM培养基培养hUCMSC，用含有体积分数1%的人KC生长添加剂与常规浓度的青霉素-链霉素溶液的EpiLife完全培养基培养HEK，用含有体积分数10%的胎牛血清和常规浓度的青霉素-链霉素溶液的DMEM完全培养基培养HDF与HUVEC。将细胞置于37 ℃、含体积分数5%二氧化碳培养箱中培养，每2天更换1次培养液。于细胞生长至70%融合时进行传代。

1.3. hUCMSC-sEV的提取与鉴定

取第3~5代hUCMSC培养48 h至80%融合时，收集上清液，采用超速离心法提取hUCMSC-sEV。用无菌PBS重新悬浮沉淀后于-80 ℃冻存。取适量hUCMSC-sEV于透射电子显微镜100 000倍放大倍数下观察形态。取第5代hUCMSC及提取的hUCMSC-sEV，采用蛋白质印迹法检测CD9、CD63、TSG101及钙联蛋白的表达，其中一抗为小鼠抗人CD9、CD63、TSG101及钙联蛋白单克隆抗体（稀释比均为1∶1 000），二抗为辣根过氧化物酶标记的山羊抗小鼠IgG多克隆抗体（稀释比为1∶5 000），增强化学发光法显影，采用凝胶成像分析系统获取条带。

1.4. 细胞迁移情况

1.4.1. 细胞划痕试验

取HUVEC、第3代HEK和HDF，按每孔5×106个接种于6孔板中，将细胞分为空白对照组与hUCMSC-sEV组，空白对照组细胞加入2 mL 1.2中的相应完全培养基培养，hUCMSC-sEV组细胞加入2 mL含100 μg/mL hUCMSC-sEV的相应完全培养基培养。待细胞生长至90%融合时用容积为1 mL移液器吸头竖直向下沿孔直径划一条直线，PBS清洗3次后加入前述培养基继续培养。于划痕后0（即刻）、6、12、24 h，于倒置相差显微镜50倍放大倍数下观察划痕情况并拍照，采用Image J 2.1.0图像分析软件（美国国立卫生研究院）测量不同时间点的划痕面积并计算划痕后6、12、24 h的细胞迁移率，细胞迁移率=（划痕后0 h划痕面积-划痕后6、12、24 h划痕面积）÷划痕后0 h划痕面积×100%。该实验样本数为3。

1.4.2. 细胞Transwell试验

取HUVEC、第4代HEK和HDF，同1.4.1分组后用相应完全培养基悬浮细胞，调整细胞浓度为4×107个/mL，取125 μL细胞悬液加入Transwell培养板上室中。向各Transwell培养板下室中加入与其上室细胞培养基相同的培养基600 μL。培养12 h后进行结晶紫染色（阳性染色为紫色），于倒置相差显微镜40倍放大倍数下观察细胞迁移情况，每组取3个视野，统计迁移至下室的细胞数。该实验样本数为3。

1.5. 细胞增殖情况

取HUVEC、第4代HEK和HDF，同1.4.1分组后将2组细胞均按每孔1×104个接种于24孔板中，同1.4.1培养24 h后采用EdU-488细胞增殖检测试剂盒行EdU、Hoechst染色。封片后，于倒置荧光显微镜200倍放大倍数下观察、拍照，细胞EdU染色阳性为绿色，细胞核Hoechst 33342染色阳性为蓝色，并计算增殖细胞比例。增殖细胞比例=（EdU染色阳性细胞数÷细胞核数）×100%。该实验样本数为3。

1.6. GelMA水凝胶的制备与表征

1.6.1. GelMA水凝胶的形貌

称取适量GelMA固体溶解于含2.5 g/L光引发剂的PBS中，分别添加PBS或200 μg/mL的hUCMSC-sEV悬液，制成终质量分数15%的GelMA（以下简称单纯GelMA）溶液及含终质量分数15% GelMA和终质量浓度0.2 g/L hUCMSC-sEV的hUCMSC-sEV/GelMA溶液，用波长405 nm的紫外光照射30 s制成水凝胶（光照处理下同）。

将2种GelMA水凝胶样本经真空冻干12 h，从样本中间切开，暴露水凝胶内部表面，真空喷金后分别在扫描电子显微镜500、1 000倍放大倍数下观察水凝胶的微观形貌，并采集图像。

1.6.2. hUCMSC-sEV在hUCMSC-sEV/GelMA水凝胶中的分布

将200 μg/mL的hUCMSC-sEV悬液（1 mL）与1 μmol/L的1，1'-二十八烷基-3，3，3'，3'-四甲基氨基氰高氯酸盐染液（0.1 mL）在37 ℃下避光共孵育3 h得到红色荧光染色的hUCMSC-sEV。按照1.6.1中方法制备单纯GelMA水凝胶及hUCMSC-sEV/GelMA水凝胶，其中hUCMSC-sEV/GelMA水凝胶使用荧光染色的hUCMSC-sEV制备，于激光扫描共聚焦显微镜400倍放大倍数下观察2种水凝胶中hUCMSC-sEV的分布情况。

1.6.3. hUCMSC-sEV/GelMA水凝胶中hUCMSC-sEV的释放

同1.6.1分别制备3份100 μL的hUCMSC-sEV/GelMA水凝胶及单纯GelMA水凝胶样本，并放入48孔板中，每孔加入200 μL PBS。每2天吸出上清液，另加入200 μL PBS。分别于浸泡0（即刻）、2、4、6、8、10、12 d通过蛋白比色定量法测定释放到上清液中的hUCMSC-sEV蛋白量，进而计算hUCMSC-sEV的累积释放率，各时间点hUCMSC-sEV累积释放率=浸泡各时间点内hUCMSC-sEV的蛋白释放总量÷加入的hUCMSC-sEV蛋白总量×100%。该实验样本数为3。

1.7. 动物实验

1.7.1. 创面模型建立与分组处理

将24只小鼠常规麻醉备皮，按随机数字表法分为PBS组、单纯hUCMSC-sEV组、单纯GelMA水凝胶组及hUCMSC-sEV/GelMA水凝胶组，每组6只，并使用打孔器在每只小鼠背部造1个直径为12 mm的全层皮肤缺损创面。伤后即刻采用微量注射器在PBS组小鼠创缘周围3、6、9、12点方向均匀注射PBS（共100 μL），对单纯hUCMSC-sEV组小鼠采用同样方式注射200 μg/mL的hUCMSC-sEV悬液（100 μL），对单纯GelMA水凝胶组小鼠在其创面表面滴加100 μL单纯GelMA溶液后行光照处理，对hUCMSC-sEV/GelMA水凝胶组小鼠在其创面表面滴加100 μL hUCMSC-sEV/GelMA溶液后行光照处理。

1.7.2. 创面愈合情况

于伤后0（即刻）、4、8、12 d观察所有小鼠创面愈合情况并拍照，采用Image J 2.1.0图像分析软件测量创面面积并计算伤后4、8、12 d的创面愈合率，创面愈合率=（伤后0 d创面面积-伤后4、8、12 d创面面积）÷伤后0 d创面面积×100%。

1.7.3. 创面组织病理学情况

伤后12 d，于观察创面愈合情况后每组取6只小鼠麻醉后断颈处死并取背部创面及距创缘0.5 cm内的皮肤组织常规制作切片后行HE染色，于倒置相差显微镜20倍放大倍数下观察创面新生组织结构。

1.8. 统计学处理

采用SPSS 20.0统计软件进行数据分析，计量资料数据均符合正态分布，以x±s表示。2组间比较行独立样本t检验，单一时间点多组间总体比较行单因素方差分析，多个时间点多组间总体比较行重复测量方差分析，组间多重比较行Bonferroni校正。P<0.05为差异有统计学意义。

2. 结果

2.1. hUCMSC-sEV的鉴定

提取的hUCMSC-sEV呈杯状结构；hUCMSC-sEV表达CD9、CD63和TSG101，几乎不表达钙联蛋白。hUCMSC不表达CD9、CD63和TSG101，表达钙联蛋白。综上，hUCMSC-sEV鉴定成功。见图 1。

图 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

The horizontal migration of HEKs, HDFs, and HUVECs in the two groups at each time point after scratching detected by scratch test

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

图 2

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图 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

The identification of hUCMSC-sEVs

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

图 1

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2.2. 细胞迁移情况

2.2.1. 细胞划痕试验

划痕后6、12、24 h，hUCMSC-sEV组HEK、HDF、HUVEC的迁移率均明显高于空白对照组（P<0.05）。见图 2、表 1。

表 1.

2组HEK、HDF、HUVEC划痕后各时间点迁移率比较（%，x±s）

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
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
空白对照组 3 0.9±0.5 14.0±5.8 58.1±2.9 9.3±3.4 19.5±3.5 47.8±5.7 7.2±2.9 10.1±1.7 39.1±7.2
hUCMSC-sEV组 3 51.4±3.3 88.2±2.0 95.7±1.4 19.8±4.6 35.4±3.4 62.2±1.0 15.1±1.2 38.9±0.6 56.7±2.6
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
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2.2.2. 细胞Transwell试验

培养12 h，hUCMSC-sEV组HEK、HDF及HUVEC迁移数量分别为（550±23）、（235±9）、（856±35）个，均明显多于空白对照组的（188±14）、（97±6）、（370±32）个（t值分别为22.95、23.13、17.84，P<0.001）。见图 3。

图 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

The vertical migration of HEKs, HDFs, and HUVECs in the two groups at 12 h after culture detected by Transwell assay

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

图 3

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2.3. 细胞增殖情况

培养24 h，hUCMSC-sEV组HEK、HDF及HUVEC增殖细胞比例分别为（48.7±0.8）%、（35.9±2.2）%、（63.9±0.9）%，均明显高于空白对照组的（18.4±2.3）%、（5.6±3.1）%、（27.4±1.7）%（t值分别为22.00、13.82、32.32，P<0.001）。见图 4。

图 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

The proliferation ability of HEKs, HDFs, and HUVECs in the two groups at 24 h after culture

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

图 4

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2.4. GelMA水凝胶的表征

2.4.1. GelMA水凝胶的形貌

2种GelMA水凝胶内部呈疏松多孔的海绵状结构，凝胶成分均匀光滑。其中在放大后的hUCMSC-sEV/GelMA水凝胶微观结构图像中可见负载在水凝胶中的hUCMSC-sEV，这些sEV呈团块状分散在水凝胶的孔隙和骨架中。见图 5。

图 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

The micromorphology of two kinds of GelMA hydrogels after freeze-drying

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

图 5

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2.4.2. hUCMSC-sEV在hUCMSC-sEV/GelMA水凝胶中的分布

单纯GelMA水凝胶中未见hUCMSC-sEV，hUCMSC-sEV/GelMA水凝胶中可见hUCMSC-sEV均匀分布。见图 6。

图 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

The distribution of hUCMSCs-sEVs in the two kinds of GelMA hydrogels

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

图 6

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2.4.3. hUCMSC-sEV/GelMA水凝胶中hUCMSC-sEV的释放

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±1.8）%。见图 7。

图 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，x±s）

The cumulative release rates of hUCMSCs-sEVs from the GelMA hydrogel loaded with hUCMSCs-sEVs at each time point after soaking

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

图 7

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2.5. hUCMSC-sEV/GelMA水凝胶对小鼠全层皮肤缺损创面愈合的影响

2.5.1. 创面愈合情况

伤后0~12 d，4组小鼠创面均未见明显感染迹象，且随时间延长，创面均不断缩小。伤后12 d，hUCMSC-sEV/GelMA水凝胶组小鼠创面已完全愈合，其余3组小鼠均可见残余创面。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组创面愈合率均明显高于单纯GelMA水凝胶组（P<0.05）。见图 8、表 2。

图 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

Wound healing of the mice with full-thickness skin defects in the four groups at each time point post injury

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

图 8

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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（%，x±s）

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
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；P1值、P2值、P3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；P4值、P5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；P6值为单纯hUCMSC-sEV组与hUCMSC-sEV/GelMA水凝胶组各时间点比较所得
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
P1值 <0.001 <0.001 <0.001
P2值 <0.001 <0.001 <0.001
P3值 <0.001 <0.001 <0.001
P4值 0.076 <0.001 <0.001
P5值 <0.001 <0.001 <0.001
P6值 <0.001 <0.001 0.031
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2.5.2. 创面组织病理学情况

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，未见明显炎症细胞浸润；其余3组小鼠创面可见真皮胶原排列致密且存在不同程度的炎症细胞浸润。

3. 讨论

创面愈合是一个复杂的过程，局部感染、氧化应激、炎症反应和血管生成受损是导致创面愈合困难的因素[12]。MSC是目前再生医学研究的焦点之一，常被用于修复包括皮肤创面在内的各种类型的组织损伤[13-14]，其旁分泌产生的纳米级囊泡被认为是其发挥修复功能的关键。

为探究hUCMSC-sEV对创面的全层修复效果，本研究通过对提取物形貌以及表面标志物进行测定后证实成功提取hUCMSC-sEV并进行体外实验，详尽探究了其对于HDF、HUVEC以及HEK的迁移与增殖的作用，结果显示hUCMSC-sEV在细胞水平和垂直迁移以及增殖方面均显示出了促进作用，与以往相关文献中MSC来源的sEV能够加速创面愈合的结论[15-18]一致。然而，单纯的sEV难以在需修复的组织中持续缓慢释放，因此其促进创面愈合的作用在一定程度上受到限制。

水凝胶作为一种传统的生物材料，常作为封闭创面的普通敷料或作为支架材料被用于组织工程研究。本研究中制备的hUCMSC-sEV/GelMA水凝胶可均匀负载hUCMSC-sEV并使其缓慢释放。研究表明，sEV的抗氧化能力随浓度的升高而提高，当sEV浓度达到100 μg/mL时，抗氧化效率约为50%[19]。在本实验中，课题组测得在浸泡12 d时hUCMSC-sEV/GelMA水凝胶中的hUCMSC-sEV累积释放率约为60%。为了保证创面处sEV浓度能够达到促进创面修复以及抗氧化的作用，本研究选择在水凝胶溶液中加入200 μg/mL的hUCMSC-sEV。

单纯GelMA水凝胶组与hUCMSC-sEV/GelMA水凝胶组小鼠创面愈合过程中炎症反应不明显，这是由于具有良好组织相容性的GelMA水凝胶能够覆盖并保护创面，减轻炎症程度。但单纯的GelMA水凝胶缺乏生物活性，故而单纯GelMA水凝胶组小鼠创面愈合速度慢于hUCMSC-sEV/GelMA水凝胶组和单纯hUCMSC-sEV组。hUCMSC-sEV/GelMA水凝胶组小鼠创面愈合速度最快，这进一步证明了水凝胶良好的生物相容性以及在水凝胶中载入sEV可以增强sEV的稳定性，并有助于sEV在所需部位的持续释放。

组织病理学结果显示，在伤后12 d，单纯GelMA水凝胶组和单纯hUCMSC-sEV组小鼠创面处仍有少量炎症细胞浸润，而hUCMSC-sEV/GelMA水凝胶组小鼠创面处未见明显炎症细胞浸润。这表明在创伤初期，hUCMSC-sEV可以在一定程度上抑制创面炎症，但随着创面处免疫系统的激活以及小鼠的活动，单纯的hUCMSC-sEV很快被分解、失活，而hUCMSC-sEV/GelMA水凝胶在小鼠创面上牢固附着、保护创面、缓释hUCMSC-sEV，最终起到持续抗炎、促进创面快速愈合的作用。

将水凝胶作为载体促进组织修复是近年来的研究热点。近期研究显示hUCMSC-sEV/GelMA水凝胶可有效改善皮肤激光损伤小鼠的早期炎症反应，从而促进创面愈合，但该研究并未分析hUCMSC-sEV对皮肤创面愈合相关细胞的影响[20]。目前国内外对于负载sEV的GelMA水凝胶的研究大多仅关注sEV对皮肤或血管的某种细胞的作用。本研究团队拟在后续的研究中进一步完善hUCMSC-sEV/GelMA水凝胶对糖尿病或大面积烧伤创面的作用及其机制的相关研究，完善Masson染色、免疫组织化学染色等实验结果。

综上，本研究成功构建了hUCMSC-sEV/GelMA水凝胶，以GelMA水凝胶作为支架，在创面处缓慢释放hUCMSC-sEV，延长了hUCMSC-sEV作用于创面的时间，促进了创面愈合相关的3种细胞的增殖与迁移，从而促进全层皮肤缺损创面愈合。本研究结果为皮肤创面的临床治疗提供了一定的理论依据，为进一步采用新型生物材料治疗临床创面提供了新思路。

Funding Statement

国家自然科学基金面上项目（82272279）；北京市自然科学基金青年项目（7244411）；解放军总医院第四医学中心自主创新科学基金（2024-4ZX-MS-06）

General Program of National Natural Science Foundation of China (82272279); Beijing Natural Science Foundation Youth Project (7244411); Independent Innovation Science Fund of the Fourth Medical Center of PLA General Hospital (2024-4ZX-MS-06)

本文亮点

(1) 证实人脐带间充质干细胞来源的小细胞外囊泡（hUCMSC-sEV）对皮肤创面愈合相关的3种细胞的水平和垂直迁移以及增殖均有促进作用。

(2) 证实所构建的负载hUCMSC-sEV的甲基丙烯酸酐化明胶水凝胶能够延长hUCMSC-sEV作用于小鼠全层皮肤缺损创面的时间，从而促进创面愈合。

Highlights

(1) It was confirmed that the small extracellular vesicles derived from human umbilical cord mesenchymal stem cells (hUCMSCs-sEVs) had promoting effects on horizontal and vertical migration and proliferation of three types of cells related to skin wound healing.

(2) It was confirmed that the constructed gelatin methacrylate anhydride hydrogel loaded with hUCMSCs-sEVs could prolong the time of treatment with hUCMSCs-sEVs on the full-thickness skin defect wounds in mice, thereby promoting wound healing.

利益冲突  所有作者均声明不存在利益冲突

作者贡献声明  陈祎琦：酝酿并设计实验、实施研究、采集数据、分析数据、撰写论文；周莹芊：采集数据、分析数据、修改论文；魏茜、谢晓烨、刘馨竹、李大伟：分析数据、修改论文；申传安：指导研究、修改论文、获取研究经费

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Articles from Chinese Journal of Burns and Wounds are provided here courtesy of Chinese Medical Association

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