Effects and mechanisms of allogeneic epidermal stem cells on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds

Huang Shaobin; Hu Zhicheng; Zhang Yi; Tang Bing; Wang Peng; Xu Hailin; Wang Zhiyong; Dong Yunxian; Cheng Pu; Rong Yanchao; Wu Jun; Zhu Jiayuan

doi:10.3760/cma.j.cn501120-20200704-00339

Volume 37 Issue 11

Nov. 2021

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Article Navigation > CHINESE JOURNAL OF BURNS AND WOUNDS > 2021 > 37(11): 1061-1069

Huang SB,Hu ZC,Zhang Y,et al.Effects and mechanisms of allogeneic epidermal stem cells on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds[J].Chin J Burns,2021,37(11):1061-1069.DOI: 10.3760/cma.j.cn501120-20200704-00339.

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Effects and mechanisms of allogeneic epidermal stem cells on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds

doi: 10.3760/cma.j.cn501120-20200704-00339

1.
Department of Burns, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China
2.
Department of Burns and Plastic Surgery, Affiliated Hospital of Nantong University, Nantong 226001, China
3.
Department of Burns and Plastic Surgery, the First Affiliated Hospital of Shenzhen University, Shenzhen 518037, China

Funds:

General Program of National Natural Science Foundation of China 81871565

Science and Technology Planning Project of Guangdong Province of China 2016B090916001

Foundation for Basic and Applied Basic Research of Guangdong Province of China 2019A1515012208

Sun Yat-sen University Clinical Research 5010 Program 2013001, 2018003

Fundamental Scientific Research of Sun Yat-sen University 19ykpy66

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Corresponding author: Zhu Jiayuan, Email: zhujiay@mail.sysu.edu.cn
Received Date: 2020-07-04

Abstract

Abstract

Objective To investigate the effects and mechanisms of allogeneic epidermal stem cells (ESCs) on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds. Methods Experimental research methods were applied. Primary ESCs that appeared paving stone-like after being cultured for 7 d were obtained by enzymatic digestion method from one 4-week-old male BALB/c-NU nude mouse (the same strain, age, and sex below). The cells of third passage were identified by flow cytometry to positively express ESC marker CD44 and negatively express CD45, meanwhile, the positive expression of ESC markers of p63 and integrin 6α, and negative expression of CD71 were identified by immunofluorescence method. The ESCs of third passage in the logarithmic growth phase were used for the following experiments. Twenty-six nude mice were equally divided into phosphate buffered saline (PBS) group and ESCs group according to the random number table. A full-thickness skin defect wound was made on the back of each nude mouse, and then the wounds of the two groups were sprayed with equal volumes of PBS and ESCs, respectively. The wounds were transplanted with full-thickness skin grafts cut from the backs of 4 other nude mice. Each ten nude mice from the two groups were selected, the wound healing and skin survival on post surgery day (PSD) 0 (immediately), 3, 7, 14, and 21 were observed, and the survival ratio and shrinkage rate of skin grafts on PSD 3, 7, 14, and 21 were calculated (the number of sample was the number of surviving skin grafts at each time point); the blood perfusion in the skin grafts on PSD 3, 7, and 14 was detected by the laser speckle blood flow imager, and the blood flow ratio of nude mice skin grafts in ESCs group to PBS group at each time point was calculated (the number of sample was the pair number of surviving skin grafts in group pairing at each time point). The skin graft tissue of each 3 nude mice remained in the two groups were collected on PSD 7, and the mRNA expressions and protein expressions of tumor necrosis factor α (TNF-α), interleukin 8 (IL-8), IL-10, type Ⅰ collagen, type Ⅲ collagen, and matrix metalloproteinase 9 (MMP-9) in the tissue were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Data were statistically analyzed with Log-rank test, analysis of variance for repeated measurement, one-way analysis of variance, independent sample t test, and Bonferroni correction. Results Taking the condition on PSD 0 as a reference, the wounds of nude mice in the two groups healed gradually on PSD 3, 7, 14, and 21, and the shrinkage of skin grafts was gradually obvious. Among them, the shrinkage healing of wound of nude mice in PBS group was more significant than that in ESCs group. On PSD 3, the skin graft of 1 nude mouse failed in ESCs group, while the skin graft of 3 nude mice failed in PBS group. On PSD 7, the skin graft of another nude mouse failed in PBS group. The survival ratio of skin grafts of nude mice in the two groups was similar on PSD 3, 7, 14, and 21 (P>0.05). On PSD 3, 7, 14, and 21, the shrinkage rates of skin grafts of nude mice in ESCs group were (9.2±0.4)%, (19.7±1.2)%, (53.6±3.5)%, and (62.2±5.1)%, respectively, which was significantly lower than (11.0±0.9)%, (47.8±2.8)%, (86.1±7.1)%, and (89.7±9.0)% in PBS group (t=5.719, 26.650, 11.940, 7.617, P<0.01). On PSD 3, 7, and 14, blood perfusion signals were observed in the skin grafts of nude mice in the two groups. The average blood perfusion ratios of the skin grafts of nude mice in ESCs group to PBS group were greater than 1, and there was no statistically significant difference in the overall comparison of 3 time points (P>0.05). On PSD 7, compared with those of PBS group, the mRNA and protein expressions of TNF-α, IL-8, type Ⅰ collagen, and type Ⅲ collagen in the skin graft tissue of nude mice in ESCs group were significantly reduced, while the mRNA and protein expressions of IL-10 and MMP-9 in the skin graft tissue of nude mice in ESCs group were significantly increased (in mRNA comparison, t=2.823, 2.934, 2.845, 2.860, 3.877, 2.916, P<0.05). Conclusions Allogeneic ESCs can reduce the shrinkage of allogeneic full-thickness skin grafts transplanted on full-thickness skin defect wounds in nude mice, promote the formation of new blood vessels between the skin graft and the wound, reduce inflammation and collagen protein expression, and promote the expression of MMP-9, thus improving the survival quality of skin grafts.
- Stem cell transplantation,
- Skin transplantation,
- Wound healing,
- Epidermal stem cells

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References

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Effects and mechanisms of allogeneic epidermal stem cells on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds

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Effects and mechanisms of allogeneic epidermal stem cells on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds

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