Scaffold-free three-dimensional human umbilical cord mesenchymal stem cell secretome repairs mouse skin injury

doi:10.12307/2025.982

Abstract

Abstract: BACKGROUND: The mesenchymal stem cell secretome contains bioactive substances, cytokines, and growth factors. Three-dimensional cell culture can regulate the secretion of these components, potentially enhancing the ability to promote injury repair.
OBJECTIVE: To investigate the repair effect of three-dimensional cultured human umbilical cord mesenchymal stem cell secretome on skin injuries in mice.
METHODS: Human umbilical cord mesenchymal stem cells were cultured in conventional two-dimensional culture dishes and 96-well U-bottom cell culture plates, from which their secretory components were subsequently collected. The expression of skin damage repair related secretory factors in umbilical cord mesenchymal stem cells was analyzed using RT-qPCR. The protein expression level of skin damage repair related factors in umbilical cord mesenchymal stem cell secretome was detected using enzyme-linked immunosorbent assay. The potential of human umbilical cord mesenchymal stem cell secretome to repair vascular injuries was evaluated using an immortalized human umbilical vein endothelial cell migration model. A mouse skin injury model was established, and the human umbilical cord mesenchymal stem cell secretome was injected subcutaneously. Repair effects on skin injury were assessed through wound healing rates and histopathological analysis.
RESULTS AND CONCLUSION: (1) After three days of cultivation, human umbilical cord mesenchymal stem cells cultured in two dimensions exhibited a fibroblast-like, swirling growth pattern, whereas three-dimensional culture led to the formation of uniform microspheres. (2) Compared with two-dimensional culture, three-dimensional culture significantly increased the mRNA expression of transforming growth factor β and basic fibroblast growth factor in human umbilical cord mesenchymal stem cells. (3) Compared with two-dimensional culture, three-dimensional cultured human umbilical cord mesenchymal stem cell secretome significantly enhanced the protein expression of vascular endothelial growth factor, interleukin-10, and granulocyte-macrophage colony-stimulating factor in the human umbilical cord mesenchymal stem cell secretome. (4) Compared with two-dimensional culture, three-dimensional cultured human umbilical cord mesenchymal stem cell secretome significantly promoted the migration of immortalized human umbilical cord mesenchymal stem cells. (5) Compared with the untreated control group and the two-dimensional cultured human umbilical cord mesenchymal stem cell secretome, the three-dimensional cultured human umbilical cord mesenchymal stem cell secretome can significantly accelerate the skin wound healing rate and wound skin structure remodeling in mice. These results indicate that three-dimensional culture can enhance the expression of paracrine factors of human umbilical cord mesenchymal stem cells, and their secretome can significantly promote the repair of mouse skin damage.

Key words: umbilical cord mesenchymal stem cell, three-dimensional cell culture, paracrine, secretome, cell migration, skin defect, wound healing, engineered stem cell

CLC Number:

Ma Wenjing, Zhang Jinyu, Jiang Mingxia, Xiu Bingshui, Bai Rui, Liu Yuhan, Chen Xuyi, Yuan Zengqiang, Liu Zhiqiang. Scaffold-free three-dimensional human umbilical cord mesenchymal stem cell secretome repairs mouse skin injury[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(1): 68-77.

Figures/Tables 7

2.6 二维和三维HUCMSCs-Sec对小鼠皮肤损伤组织病理学的影响 2.6.1 苏木精-伊红染色结果小鼠皮肤损伤后16 d，取伤口及其周围1.0-2.0 mm处的皮肤进行病理染色，如图6A显示对照组小鼠皮肤角化过度如红色箭头所示，伴有局部棘层增厚如白色箭头所示，胶原纤维缺失，未见皮肤附属器，同时可见少量炎症细胞浸润如黄色箭头所示。此外，局部区域还出现细胞坏死现象，表现为胞核碎裂、固缩及深染，如黑色箭头所示。二维HUCMSCs-Sec治疗组小鼠皮肤损伤程度较对照组稍有改善，肉芽组织区域有所减少，皮肤附属器官稍有增多，如黄色箭头所示毛囊；棘层较厚，未见明显变化，真皮层胶原纤维排列疏松，仍可见大量炎症细胞及成纤维细胞。与对照组相比，三维HUCMSCs-Sec治疗组小鼠皮肤表皮层结构保持相对完整，未见明显增厚，可观察到少量皮肤附属器，包括毛囊和皮脂腺，分别为蓝色和绿色箭头所示。更重要的是，三维HUCMSCs-Sec治疗组小鼠皮肤的炎症和坏死症状有所减轻，如黄色箭头所示，三维HUCMSCs-Sec治疗组小鼠皮肤中有较少的炎症细胞。进一步定量分析显示各小鼠在表皮、肉芽组织、真皮厚度方面有显著差异。如图6B-D所示，三维HUCMSCs-Sec治疗组小鼠的表皮厚度(38.1±15.3) μm显著低于对照组的(85.6±33.7) μm和二维HUCMSCs-Sec治疗组的(66.5±20.5) μm；三维HUCMSCs-Sec治疗组小鼠肉芽组织厚度(124.0±34.2) μm同样低于对照组的(302.3±95.0) μm和二维HUCMSCs-Sec治疗组的(275.3±54.6) μm，而三维HUCMSCs-Sec治疗组小鼠的真皮厚度(544.6±424.1) μm显著高于对照组的(246.6±117.5) μm和二维HUCMSCs-Sec治疗组的(360.6±121.6) μm，表明三维HUCMSCs-Sec组治疗后小鼠损伤皮肤结构恢复更为完整。"

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