Concentrated Growth Factor Enhances the Effect of Hypoxia-Pretreated Adipose-Derived Stem Cell Sheets on Skin Wound Healing

Abstract

Background: Most significant wounds often exhibit poor healing. Hence, the exploration of novel approaches to enhance skin wound healing (SWH) and further investigate potential underlying mechanisms remains crucial. Our aim was to delineate the collective impact of concentrated growth factor (CGF) and hypoxia-pretreated adipose-derived stem cell (ADSC) sheets on SWH and investigate the underlying mechanism. Methods: The optimal concentration of CoCl₂, a hypoxia-inducing reagent, and CGF extract for cell proliferation and ADSC growth factor production in rats was determined using dose gradient experiments. Mice were categorized into six groups according to ADSC treatment: (A) control, (B) ADSC sheets, (C) ADSC sheets treated with CoCl₂, (D) CGF particles, (E) CGF and ADSC sheets, and (F) CGF and ADSC sheet combination treated with CoCl₂. The impact of CGF and hypoxia on ADSC proliferation was investigated through western blotting and Cell Counting Kit-8 (CCK-8) assay. Immunohistochemistry, Masson staining, and hematoxylin and eosin staining were conducted ten days after the surgical procedure to assess the effects. Key markers for skin wound healing were evaluated, and the underlying mechanism in human ADSCs was examined. Results: In vitro studies demonstrated synergistic promotion of proliferation, transwell migration, and growth factor production through the combined application of 10% CGF extract and 50 μM CoCl₂. This combination notably activated the Integrin-linked kinase (ILK)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR)/hypoxia-inducible factor 1-alpha (HIF-1α) pathway in ADSCs derived from both rats and humans, leading to a significant acceleration of skin wound healing (SWH) in vivo. Conclusion: The acceleration of skin wound healing (SWH) in nude mice was observed when using rat-derived ADSC sheets treated with the combination of CGF and CoCl₂, which was achieved through the activation of the ILK/AKT/mTOR/HIF-1α signaling pathway.