Ononin-modified Chitosan/β-glycerophosphate hydrogel-loaded adipose-derived stem cells promote the repair of diabetic wounds in rats.

Abstract

OBJECTIVE: To evaluate the mechanistic synergy of an ononin-modified chitosan/β-glycerophosphate (CS/β-GP) hydrogel loaded with adipose-derived stem cells (ADSCs) in repairing diabetic wounds (DWs). We specifically aimed to determine if ononin could biochemically remodel the hostile diabetic microenvironment to enhance ADSCs survival and potency. METHODS: Primary rat ADSCs were characterized by flow cytometry. A thermosensitive CS/β-GP hydrogel was fabricated, and its microstructure was characterized by scanning electron microscopy (SEM). he biological response of ADSCs to ononin was assessed via CCK-8, scratch assays and Live/Dead staining to identify the optimal concentration. A diabetic wound model in rats was established, with all rats randomly assigned into five groups: Control, model, ononin (On), ADSCs, and ononin+ADSCs (On+ADSCs). Wound healing rates were recorded at days 0, 4, 8, and 12 post-treatment. At 12th day, the synergistic impact of ononin and ADSCs on the wound microenvironment was evaluated by measuring healing rates, inflammatory cytokines (TNF-α, IL-1β, IL-6). Additionally, the cumulative release profiles of IGF-1, VEGF, and TGF-β3 from the hydrogel-loaded ADSCs were monitored over 96 h, fitting a Korsmeyer-Peppas kinetic model to characterize the sustained release behavior. Histopathological evaluation, immunohistochemistry for CD31, collagen I, and α-SMA, and Western blot analysis for VEGF, VEGFR2, and CD31 were performed for mechanistic exploration. RESULTS: SEM confirmed a three-dimensional interconnected porous microstructure of the CS/β-GP hydrogel, providing a biomimetic scaffold for cell retention. 20µM ononin significantly promoted ADSCs proliferation and migration without observable cytotoxicity (P < 0.05). The Ononin release profile from the hydrogel followed a sustained release pattern, in accordance with the Korsmeyer-Peppas model. The hydrogel demonstrated sustained release of both Ononin and ADSCs-derived growth factors. Notably, ononin acted as a biochemical modifier that enhanced the paracrine activity of ADSCs. Compared with the model group, the On+ADSCs group exhibited the most significant acceleration in wound closure, re-epithelialization, and collagen deposition. This was accompanied by a robust suppression of inflammatory cytokines and a marked upregulation of CD31, collagen I, and α-SMA. Mechanistically, the combination therapy potently activated the VEGF/VEGFR2 signaling axis, significantly outperforming monotherapies (P < 0.05). CONCLUSION: Ononin-modified CS/β-GP hydrogel effectively promotes diabetic wound healing by optimizing the inflammatory microenvironment and amplifying ADSCs-mediated angiogenesis through the VEGF/VEGFR2 axis. This composite biomaterial represents a promising strategy for chronic wound management.