Chitosan-based hydrogel, along with citrus-derived extracellular vesicles and clove extract: advanced skin wound...

Plant-derived Extracellular vesicles (EVs), particularly those from Citrus limettioides Tan, represent a promising strategy for wound-healing applications. This study investigated the antibacterial and wound-healing efficacy of a chitosan-based hydrogel containing clove extract (CTS/Clo) and EVs through both in vitro and in vivo analyses. Chitosan hydrogels (1.5% and 2%) were formulated with clove extract at concentrations of 0.0156% and 0.0625%. Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and drug release profiles showed that the porous structure, combined with a uniform mixture of CTS and Clo, helped the gradual release of Clo. Antibacterial assays against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa confirmed the effectiveness of the hydrogel formulation. Isolated EVs displayed a consistent size distribution and expressed key EV markers. Treatment of human mesenchymal stem cells (hMSCs) and human dermal fibroblasts (HDFs) with the hydrogel and EVs significantly enhanced cell viability (p<0.0001). In an animal model, the combined use of hydrogels with EVs significantly sped up wound closure (up to 95%), increased gene expression (Col1a1, Col3a1, MMP9, VEGFA, TGF-β) related to tissue regeneration, and improved collagen production through collagen deposition (p<0.001) and Col1a1 protein expression.