Chitosan hydrogel loaded with human umbilical cord mesenchymal stem cell-derived exosomes promotes healing of chronic diabetic wounds in rats

doi:10.12122/j.issn.1673-4254.2025.10.04

Abstract

Abstract:

Objective To investigate the mechanism by which chitosan (CS) hydrogel loaded with human umbilical cord mesenchymal stem cell (HUVECs)-derived exosomes (hUCMSC-exos) (Exos@CS-Gel) improves diabetic wound healing. Methods hUCMSC-exos were extracted and Exos@CS-Gel was prepared. The effect of Exos@CS-Gel on proliferation and migration of HUVECs were evaluated using scratch wound assay and CCK-8 assay. Diabetic rat models with full-thickness skin wounds established by streptozotocin induction were randomized divided into 4 groups for treatment with Exos@CS-Gel (100 µg hUCMSC-exos dissolved in 100 µL 24% CS hydrogel), hUCMSC-exos (100 µg hUCMSC-exos dissolved in 100 µL PBS), CS hydrogel (100 µL 24% CS hydrogel), or PBS (control group). Wound healing and the therapeutic mechanisms were assessed using immunohistochemistry, HE staining, immunofluorescence, and qRT-PCR. Results In cultured HUVECs, Exos@CS-Gel treatment significantly promoted cell proliferation and migration. In the rat models of chronic diabetic wounds, the wound healing rate in Exos@CS-Gel group reached 92.7% on day 14, significantly higher than those in hUCMSC-exos group (9.12%), CS hydrogel group (16.28%), and control group (25.98%). Microvessel density and the expression levels of vascular endothelial growth factor and transforming growth factor β-1 were significantly increased in the Exos@CS-Gel group. Conclusion Exos@CS-Gel promotes survival capacity of hUCMSC-exos in vitro and accelerates diabetic wound healing in rats by promoting angiogenesis and cell proliferation.

Key words: angiogenesis, diabetic wound, exosomes, mesenchymal stem cells, chitosan, hydrogel

Xiaohui QIU, Meng WANG, Jiangjie TANG, Jianda ZHOU, Chen JIN. Chitosan hydrogel loaded with human umbilical cord mesenchymal stem cell-derived exosomes promotes healing of chronic diabetic wounds in rats[J]. Journal of Southern Medical University, 2025, 45(10): 2082-2091.

Figures/Tables 8

Fig.1 Isolation and characterization of human umbilical cord mesenchymal stem cells (HUVECs)-derived exosomes (hUCMSC-exos). A: Transmission electron microscopy (TEM) of hUCMSC-exos. B: Histogram showing the diameter and concentration distribution of hUCMSC-exos. C: Western blotting of exosomal markers (Alix, TSG101, CD63, and CD81) in hUCMSC-exos.

Tab.1 Initial gelation temperature （IGT） of different concentrations of Exos@CS-Gel and their gelation time at 37 ℃ (Mean±SD)

Concentration	IGT (℃)	Gelation time at 37 ℃ (s)
20%	17.2±1.4	92.7±6.3
22%	16.3±1.2	78.5±5.2
24%	14.2±1.5	66.3±3.4
26%	12.9±1.1	51.2±2.7
28%	11.8±0.9	46.9±2.9

Fig.2 Cellular localization of exosomes tracked by PKH67 staining (Original magnification: ×400). A: PBS group. B: CS group. C: hUCMSCs-exos group. D: Exos@CS-Gel group. Green indicates PKH67 (exosome staining dye), and blue indicates DAPI (nuclear staining dye).

Fig.3 Effect of Exos@CS-Gel on HUVEC migration and proliferation. A: Wound healing assay showing the effect of Exos@CS-Gel on HUVEC cell migration ability. B: Comparison of wound healing rates among different groups. C: CCK-8 assay evaluating the effect of Exos@CS-Gel on HUVEC proliferation ability. ***P<0.001.

Fig.4 Wound healing c in each group of rats. A: Representative wound images of each group on days 0, 3, 7, 14. B: Comparison of wound area changes over time among the groups. **P<0.01, ***P<0.001 vs PBS group.

Fig.5 HE staining of the wound tissues in each group on day 14 after treatments (×100).

Fig.6 Effect of Exos@CS-Gel on angiogenesis in rat wound tissues assess by immunohistochemical detection of CD31 expression (vascular endothelial cell marker) in the wound tissues from each group on day 7 and 14 after the treatments (immunohistochemical staining, ×100).

Fig.8 Effect of Exos@CS-Gel on VEGF and TGF-β1 expression levels in rat wound tissues, assessed by qRT-PCR of mRNA expression levels of VEGF (A, B) and TGF-β1 (C, D) in the wound granulation tissues on day 7 and day 14 after treatments. *P<0.05, **P<0.01, ***P<0.001 vs PBS group; #P<0.05, ##P<0.01 vs CS-Gel group.

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