Application of Gelatin/Vanillin/Fe³⁺/AGP-AgNPs Hydrogels Promotes Wound Contraction, Enhances Dermal Growth Factor Expression, and Minimizes Skin Irritation

Abstract

This study further investigates the potential of gelatin-based hydrogel cross-linked with vanillin and ferric ion (GVF), combined with andrographolide (AGP) and silver nanoparticles (AgNPs), as an anti-infection biomaterial for wound dressing, aimed at exploring the mechanisms that attenuate inflammation, enhance wound healing rates, and address allergic skin irritation. AGP-AgNPs were evaluated for cytotoxicity in human adult epidermal keratinocytes (HEKa) and the murine macrophage cell line (RAW 264.7), as well as for nitric oxide (NO) production in response to lipopolysaccharide-induced inflammation in RAW 264.7 macrophage cells. Skin-wound specimens from male Wistar rats were histologically analyzed for epidermal thickness and inflammatory changes. The mRNA expression profiling of dermal growth factors was assessed using RT-qPCR, and skin irritation tests were conducted in female New Zealand rabbits. These AGP-AgNPs exhibited significantly lower toxicity in HEKa and no toxicity in RAW 264.7. Interestingly, AGP-AgNPs at specific concentrations produced NO in RAW 264.7 control cells but were more effective in reducing inflammatory NO levels in RAW 264.7 cells pretreated with lipopolysaccharides, suggesting that AGP-AgNP composites are safe and effectively diminish inflammation. Furthermore, a marked increase in epidermal thickness and a reduction in histological inflammatory cells at wound sites were observed in rats treated with AGP-AgNPs/GVF hydrogels over 21 days. Upregulation of dermal genes promoting wound healing, including collagen types I and III, epidermal growth factor, transforming growth factor-beta, fibronectin, and vascular endothelial growth factor, but not fibroblast growth factor, was observed in a time-dependent manner. These results suggest that the anti-inflammatory properties of GVF/AGP-AgNP hydrogels could promote epithelialization, enhance cellular proliferation, support extracellular matrix synthesis, and facilitate angiogenesis. Additionally, rabbit skin in contact with GVF/AGP-AgNP hydrogels consistently displayed reduced levels of erythema and edema, with no swelling, and a standardized scoring system yielded low primary dermal irritation indices for this hydrogel. These findings suggest that the novel GVF/AGP-AgNP hydrogels possess anti-inflammatory-like activity and can modulate dermal growth factors for wound healing. This leads to reduced dermal irritation, making the formulation potentially suitable for safe topical applications in skin and wound care. However, comprehensive human studies and clinical trials should be required in the future.