Influence of skin organoid-derived extracellular vesicles composite hydrogels on wound healing of full-thickness skin defects in mice
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摘要:
目的 探讨皮肤类器官来源细胞外囊泡（SOEV）复合水凝胶对小鼠全层皮肤缺损创面愈合的影响。 方法 该研究为成组设计和重复测量设计实验研究。取HaCaT细胞、人皮肤成纤维细胞与人脐静脉血管内皮细胞（HUVEC）,按2∶1∶1的数量比混合后于6孔超低吸附培养板中进行三维培养以制备皮肤类器官,观察培养1、3、7、14 d皮肤类器官形成情况。培养7 d,采用免疫荧光法检测皮肤类器官中表皮标志物细胞角蛋白14（CK14）、血管标志物CD31、真皮标志物波形蛋白的表达。培养7 d,采用顺序差速超速离心法从皮肤类器官培养上清液中分离提取SOEV,采用透射电子显微镜观察SOEV超微结构,采用纳米颗粒跟踪分析仪检测SOEV粒径。取HaCaT细胞和HUVEC,将2种细胞都分为加入30 μg/mL SOEV培养的SOEV组和常规培养的对照组,行划痕试验,计算划痕后24 h即培养24 h细胞的迁移率（样本数为6）。制备甲基丙烯酸酐化明胶（GelMA）水凝胶和含30 μg/mL SOEV的GelMA水凝胶（即SOEV复合水凝胶）。取18只6周龄雄性C57BL/6J小鼠,采用随机数字表法分为对照组、GelMA组和SOEV@GelMA组（每组6只小鼠）,在所有小鼠背部造成1个全层皮肤缺损创面,伤后0 d（即刻）,分别于对照组、GelMA组、SOEV@GelMA组小鼠创面施加磷酸盐缓冲液、GelMA水凝胶、SOEV复合水凝胶。观察伤后0、3、7、10、14 d创面愈合情况并计算伤后3、7、10、14 d创面愈合率；伤后14 d,取创面组织,行苏木精-伊红染色观察创面上皮再生程度,行Masson染色观察创面胶原纤维沉积情况并计算胶原纤维阳性面积占比。 结果 培养1~14 d,皮肤类器官逐渐成熟,结构逐渐紧缩致密,球体边界清晰。培养7 d,皮肤类器官表达CK14、CD31及波形蛋白。从培养7 d皮肤类器官培养上清液中提取的SOEV呈现典型的盘状囊泡样结构,平均粒径为70.1 nm。划痕后24 h,SOEV组HaCaT细胞、HUVEC的迁移率均明显高于相应的对照组（t值分别为16.73、7.71,P<0.05）。伤后0~14 d,3组小鼠创面面积均逐渐减小。伤后3、7、10、14 d,SOEV@GelMA组小鼠创面愈合率分别为（56.47±9.26）%、（73.87±6.02）%、（92.62±3.92）%、（98.92±0.26）%,均明显高于对照组的（28.18±15.63）%、（49.21±11.96）%、（72.53±7.93）%、（86.73±2.34）%（P<0.05）；伤后3、7、10 d,SOEV@GelMA组小鼠创面愈合率均明显高于GelMA组[（34.51±14.43）%、（58.30±8.00）%、（79.16±4.15）%,P<0.05]。伤后14 d,GelMA组与SOEV@GelMA组小鼠创面新生上皮覆盖范围均大于对照组；GelMA组与对照组小鼠部分创面仍存在表皮与真皮分离现象,而SOEV@GelMA组小鼠创面已基本实现再上皮化。伤后14 d,3组小鼠创面组织中均可见胶原纤维沉积,其中SOEV@GelMA组小鼠创面组织中胶原纤维在真皮层中排列较为整齐；GelMA组与SOEV@GelMA组小鼠创面组织中胶原纤维阳性面积占比均明显高于对照组（P<0.05）,SOEV@GelMA组小鼠创面组织中胶原纤维阳性面积占比明显高于GelMA组（P<0.05）。 结论 SOEV复合水凝胶能够促进小鼠全层皮肤缺损创面胶原纤维沉积并加速创面修复进程,显著提升愈合疗效,改善愈合质量。 Abstract:Objective To investigate the influence of skin organoid-derived extracellular vesicles (SOEVs) composite hydrogels on wound healing of full-thickness skin defects in mice. Methods This study was an experimental study using a group design and a repeated-measures design. HaCaT cells, human skin fibroblasts, and human umbilical vein endothelial cells (HUVECs) were mixed at a ratio of 2∶1∶1 in number and cultured three-dimensionally in 6-well ultra-low attachment plates to generate skin organoids. The formation of skin organoids was observed on days 1, 3, 7, and 14 of culture. On day 7 of culture, the expression of epidermal marker cytokeratin 14 (CK14), vascular marker CD31, and dermal marker vimentin in skin organoids was detected by immunofluorescence method. On day 7 of culture, SOEVs were isolated from the culture supernatant of skin organoids by sequential differential ultracentrifugation. The ultrastructure of SOEV was observed using transmission electron microscopy, and the particle size of SOEV was measured using a nanoparticle tracking analyzer. HaCaT cells and HUVECs were collected, and both of the two kinds of cells were divided into SOEV group cultured with 30 μg/mL SOEV and control group with conventional culture. A scratch test was performed, and cell migration rate was calculated at 24 h post-scratching, i.e., 24 h of culture (n=6). Gelatin methacrylate anhydride (GelMA) hydrogel and GelMA hydrogel containing 30 μg/mL SOEV (i.e. SOEV composite hydrogel) were prepared. Eighteen 6-week-old male C57BL/6J mice were divided into control group, GelMA group, and SOEV@GelMA group using a random number table method, with 6 mice in each group. A full-thickness skin defect wound was created on the back of all mice. Immediately after injury (day 0), phosphate-buffered saline, GelMA hydrogel, and SOEV composite hydrogel were applied to the wounds of mice in control group, GelMA group, and SOEV@GelMA group, respectively. Wound healing was observed at post-injury day (PID) 0, 3, 7, 10, and 14, and the wound healing rate was calculated at PID 3, 7, 10, and 14. At PID 14, wound tissue was collected. Hematoxylin-eosin staining was performed to observe the degree of wound epithelial regeneration; Masson staining was performed to observe collagen fiber deposition, and the percentage of collagen fiber positive area was calculated. Results From day 1 to 14 of culture, the skin organoids gradually matured, with the structure becoming increasingly compact and dense, and the spheroids exhibiting clear boundaries. On day 7 of culture, the skin organoids expressed CK14, CD31, and vimentin. SOEVs extracted from the culture supernatant of skin organoids on day 7 of culture exhibited a typical disc-shaped vesicular structure, with an average particle size of 70.1 nm. At 24 h post-scratching, the migration rates of HaCaT cells and HUVECs in SOEV group were significantly higher than those in corresponding control group (with t values of 16.73 and 7.71, respectively, P<0.05). From PID 0 to 14, the wound area of mice gradually decreased in all three groups. At PID 3, 7, 10, and 14, the wound healing rates of mice in SOEV@GelMA group were (56.47±9.26)%, (73.87±6.02)%, (92.62±3.92)%, and (98.92±0.26)%,respectively, which were significantly higher than (28.18±15.63)%, (49.21±11.96)%, (72.53±7.93)%, and (86.73±2.34)% in control group (P<0.05); at PID 3, 7, and 10, the wound healing rates of mice in SOEV@GelMA group were significantly higher than those in GelMA group ((34.51±14.43)%, (58.30±8.00)%, and (79.16±4.15)%, respectively, P<0.05). At PID 14, the area of neo-epithelial coverage in the wounds of mice in GelMA group and SOEV@GelMA group was greater than that in control group; separation of the epidermis and dermis was still observed in partial wounds of mice in GelMA group and control group, whereas the wounds of mice in SOEV@GelMA group achieved almost complete re-epithelialization. At PID 14, collagen fiber deposition was observed in the wound tissue of mice in all three groups. Among them, the collagen fibers in the dermal layer of the wound tissue in SOEV@GelMA group were arranged in a relatively orderly manner. The percentage of collagen fiber positive area in the wound tissue of mice in GelMA group and SOEV@GelMA group was significantly higher than that in control group (P<0.05), and the percentage of collagen fiber positive area in the wound tissue of mice in SOEV@GelMA group was significantly higher than that in GelMA group (P<0.05). Conclusions The SOEV composite hydrogel can promote collagen fiber deposition in full-thickness skin defect wounds of mice, accelerate the wound repair process, significantly enhance the healing efficacy, and improve the quality of wound healing. -
Key words:
- Skin /
- Organoids /
- Extracellular vesicles /
- Hydrogel /
- Wound healing
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参考文献
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图 1 将HaCaT细胞、HSF及HUVEC混合培养后各时间点形成的皮肤类器官形态 倒置相差显微镜 ×100。1A.培养1 d,皮肤类器官呈现为规则的圆球形,形态初步形成；1B.培养3 d,皮肤类器官内部结构进一步紧缩,球体边界更加清晰；1C.培养7 d,皮肤类器官持续生长,体积略有增加,仍保持球形；1D.培养14 d,皮肤类器官结构变得更加致密
注：将HaCaT细胞、人皮肤成纤维细胞（HSF）、人脐静脉血管内皮细胞（HUVEC）按2∶1∶1的数量比混合
图 2 将HaCaT细胞、HSF及HUVEC混合培养7 d后形成的皮肤类器官中细胞角蛋白14、CD31和波形蛋白的表达情况。2A.细胞角蛋白14主要表达于皮肤类器官的外层 Alexa Fluor 488-4',6-二脒基-2-苯基吲哚 ×100；2B.CD31主要表达于皮肤类器官的核心 Alexa Fluor 647-4',6-二脒基-2-苯基吲哚 ×100；2C.波形蛋白主要表达于皮肤类器官的核心 Alexa Fluor 488-4',6-二脒基-2-苯基吲哚 ×100
注：将HaCaT细胞、人皮肤成纤维细胞（HSF）、人脐静脉血管内皮细胞（HUVEC）按2∶1∶1的数量比混合；细胞角蛋白14和波形蛋白阳性染色为绿色,CD31阳性染色为紫红色,细胞核阳性染色为蓝色
图 3 SOEV的表征。3A.SOEV呈现盘状囊泡样结构 透射电子显微镜 ×100 000；3B.SOEV的平均粒径为70.1 nm
注：将HaCaT细胞、人皮肤成纤维细胞（HSF）、人脐静脉血管内皮细胞（HUVEC）按2∶1∶1的数量比混合培养7 d得到皮肤类器官；SOEV为皮肤类器官来源细胞外囊泡
图 4 2组HUVEC和2组HaCaT细胞划痕后各时间点迁移情况 倒置相差显微镜 ×100。4A、4B.分别为对照组、SOEV组HUVEC划痕后0 h（即刻）的划痕情况；4C、4D.分别为对照组、SOEV组HaCaT细胞划痕后0 h（即刻）的划痕情况；4E、4F.分别为对照组、SOEV组HUVEC划痕后24 h的划痕情况,图4E划痕面积略小于图4A,图4F划痕面积明显小于图4B、4E；4G、4H.分别为对照组、SOEV组HaCaT细胞划痕后24 h的划痕情况,图4G划痕面积与图4C相近,图4H划痕面积明显小于图4D、4G
注：皮肤类器官来源细胞外囊泡（SOEV）组细胞培养时加入30 μg/mL SOEV,对照组细胞常规培养；HUVEC为人脐静脉血管内皮细胞
图 6 3组全层皮肤缺损小鼠伤后各时间点创面愈合情况。6A、6B、6C.分别为对照组伤后0（即刻）、7、14 d创面,逐渐愈合；6D、6E、6F.分别为GelMA组伤后0、7、14 d创面,逐渐愈合；6G、6H、6I.分别为SOEV@GelMA组伤后0、7、14 d创面,图6G创面面积与图6A、6D相近,图6H创面面积明显小于图6B,图6I创面面积明显小于图6C
注：分别于对照组、甲基丙烯酸酐化明胶（GelMA）组、皮肤类器官来源细胞外囊泡（SOEV）@GelMA组小鼠创面施加磷酸盐缓冲液、GelMA水凝胶、含30 μg/mL SOEV的GelMA水凝胶即SOEV复合水凝胶
Table 1. 3组全层皮肤缺损小鼠伤后各时间点创面愈合率比较（%,
组别 样本数 3 d 7 d 10 d 14 d 对照组 6 28.18±15.63 49.21±11.96 72.53±7.93 86.73±2.34 GelMA组 6 34.51±14.43 58.30±8.00 79.16±4.15 90.16±0.80 SOEV@GelMA组 6 56.47±9.26 73.87±6.02 92.62±3.92 98.92±0.26 F值 7.37 11.51 19.75 115.62 P值 0.002 0.008 0.002 <0.001 P1值 0.413 0.167 0.380 0.769 P2值 <0.001 <0.001 <0.001 0.044 P3值 <0.001 0.007 0.023 0.189 注：分别于对照组、甲基丙烯酸酐化明胶（GelMA）组、皮肤类器官来源细胞外囊泡（SOEV）@GelMA组小鼠创面施加磷酸盐缓冲液、GelMA水凝胶、含30 μg/mL SOEV的GelMA水凝胶即SOEV复合水凝胶；时间因素主效应,F=216.09,P<0.001；处理因素主效应,F=17.90,P<0.001；两者交互作用,F=1.87,P=0.156；F值、P值为3组间各时间点总体比较所得,P1值、P2值、P3值分别为对照组与GelMA组、对照组与SOEV@GelMA组、GelMA组和SOEV@GelMA组各时间点比较所得 
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