Preparation of chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and its effects on wound healing of full-thickness skin defects in rats

Liu Ying; Cheng Feng; Wang Zewei; Jin Hongxu; Cao Binyan; You Pingfei; Hu An; Shi Xiuyun; Du Juan; Yuan Zhixin

doi:10.3760/cma.j.cn501225-20230928-00101

Volume 40 Issue 1

Jan. 2024

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Article Navigation > CHINESE JOURNAL OF BURNS AND WOUNDS > 2024 > 40(1): 50-56

Shi JJ,Sun Y,Peng ZJ.Effects of wedge-shaped heel pad in the treatment of extremely severe burn patients with foot drop deformity[J].Chin J Burns,2021,37(6):562-567.DOI: 10.3760/cma.j.cn501120-20200319-00181.

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Liu Y,Cheng F,Wang ZW,et al.Preparation of chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and its effects on wound healing of full-thickness skin defects in rats[J].Chin J Burns Wounds,2024,40(1):50-56.DOI: 10.3760/cma.j.cn501225-20230928-00101.

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Preparation of chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and its effects on wound healing of full-thickness skin defects in rats

doi: 10.3760/cma.j.cn501225-20230928-00101

1.
Department of Emergency Medicine, Northern Theater Command General Hospital, Shenyang 110016, China
2.
Laboratory of Acute and Critical Care Research and Transformation, Jilin Provincial People's Hospital, Changchun 130021, China
3.
Department of Emergency Medicine, Jilin Provincial People's Hospital, Changchun 130021, China

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Doctoral Research Foundation of Liaoning Province of China 2023-BS-032

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Corresponding author: Jin Hongxu, Email: cszx_jhx@163.com; Du Juan, Email: dujuan0512@126.com
Received Date: 2023-09-28

Abstract

Abstract

Objective To prepare the chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and to explore its effects on wound healing of full-thickness skin defects in rats. Methods The research was an experimental research. Chitin nanofibers were prepared by acid hydrolysis and alkaline extraction method, and then mixed with hyaluronic acid and collagen to prepare chitin/hyaluronic acid/collagen hydrogels (hereinafter referred to as hydrogels). Besides, the hydrogels loaded with mouse adipose-derived stem cells were prepared. Thirty male 12-week-old guinea pigs were divided into negative control group, positive control group, and hydrogel group according to the random number table, with 10 guinea pigs in each group. Ethanol, 4-aminobenzoic acid ethyl ester, or the aforementioned prepared hydrogels without cells were topically applied on both sides of back of guinea pigs respectively for induced contact and stimulated contact, and skin edema and erythema formation were observed at 24 and 48 h after stimulated contact. Adipose-derived stem cells from mice were divided into normal control group cultured routinely and hydrogel group cultured with the aforementioned prepared hydrogels without cells. After 3 d of culture, protein expressions of platelet-derived growth factor-D (PDGF-D), insulin-like growth factor-Ⅰ (IGF-Ⅰ), and transforming growth factor β ₁ (TGF-β ₁) were detected by Western blotting ( n=3). Eight male 8-week-old Sprague-Dawley rats were taken and a circular full-thickness skin defect wound was created on each side of the back. The wounds were divided into blank control group without any treatment and hydrogel group with the aforementioned prepared hydrogels loaded with adipose-derived stem cells applied. Wound healing was observed at 0 (immediately), 2, 4, 8, and 10 d after injury, and the wound healing rate was calculated at 2, 4, 8, and 10 d after injury. Wound tissue samples at 10 d after injury were collected, the new tissue formation was observed by hematoxylin-eosin staining; the concentrations of interleukin-1α (IL-1α), IL-6, IL-4, and IL-10 were detected by enzyme-linked immunosorbent assay method; the expressions of CD16 and CD206 positive cells were observed by immunohistochemical staining and the percentages of positive cells were calculated. The sample numbers in animal experiment were all 8. Results At 24 h after stimulated contact, no skin edema was observed in the three groups of guinea pigs, and only mild skin erythema was observed in 7 guinea pigs in positive control group. At 48 h after stimulated contact, skin erythema was observed in 8 guinea pigs and skin edema was observed in 4 guinea pigs in positive control group, while no obvious skin erythema or edema was observed in guinea pigs in the other two groups. After 3 d of culture, the protein expression levels of PDGF-D, IGF-I, and TGF-β ₁ in adipose-derived stem cells in hydrogel group were significantly higher than those in normal control group (with t values of 12.91, 11.83, and 7.92, respectively, P<0.05). From 0 to 10 d after injury, the wound areas in both groups gradually decreased, and the wounds in hydrogel group were almost completely healed at 10 d after injury. At 4, 8, and 10 d after injury, the wound healing rates in hydrogel group were (38±4)%, (54±5)%, and (69±6)%, respectively, which were significantly higher than (21±6)%, (29±7)%, and (31±7)% in blank control group (with t values of 3.82, 3.97, and 4.05, respectively, Pvalues all <0.05). At 10 d after injury, compared with those in blank control group, the epidermis in wound in hydrogel group was more intact, and there were increases in hair follicles, blood vessels, and other skin appendages. At 10 d after injury, the concentrations of IL-1α and IL-6 in wound tissue in hydrogel group were significantly lower than those in blank control group (with tvalues of 8.21 and 7.99, respectively, P<0.05), while the concentrations of IL-4 and IL-10 were significantly higher than those in blank control group (with tvalues of 6.57 and 9.03, respectively, P<0.05). The percentage of CD16 positive cells in wound tissue in hydrogel group was significantly lower than that in blank control group ( t=8.02, P<0.05), while the percentage of CD206 positive cells was significantly higher than that in blank control group ( t=7.21, P<0.05). Conclusions The hydrogel loaded with mouse adipose-derived stem cells is non-allergenic, can promote the secretion of growth factors in adipose-derived stem cells, promote the polarization of macrophages to M2 phenotype in wound tissue in rats with full-thickness skin defects, and alleviate inflammatory reaction, thereby promoting wound healing.
- Biocompatible materials,
- Hydrogel,
- Inflammation,
- Macrophages,
- Adipose-derived stem cells,
- Wound repair

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References

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Preparation of chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and its effects on wound healing of full-thickness skin defects in rats

doi: 10.3760/cma.j.cn501225-20230928-00101

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Preparation of chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and its effects on wound healing of full-thickness skin defects in rats

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