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Hypertrophic scars (HTSs) result from excessive collagen accumulation and impaired wound remodelling, leading to considerable aesthetic and functional concerns. Despite the availability of various treatment strategies, their clinical success remains limited, emphasising the need for alternative approaches. Human amniotic fluid (hAF), naturally enriched with cytokines and growth factors, has emerged as a promising biological material for tissue regeneration. This study investigated the therapeutic potential of two forms of hAF—pooled-frozen and pooled-frozen gamma-irradiated—in a rat model of hypertrophic scarring. Fifteen adult male Sprague–Dawley rats were randomly assigned to receive subcutaneous injections of either saline, pooled-frozen hAF, or pooled-frozen gamma-irradiated hAF at the wound margins on days 1, 3 and 5 following the induction of hypertrophic scars via talc powder application. After 21 days, wound healing was evaluated through histological and immunohistochemical analyses. Both treatment groups demonstrated significantly improved wound healing compared to the control group. Granulation tissue formation was enhanced in the treated groups, particularly in animals receiving gamma-irradiated fluid, which also showed superior collagen remodelling characterised by aligned and mature collagen bundles. Both treatment groups demonstrated an increase in M2 macrophage density, as evidenced by elevated Arg+/CD68+ cell ratios; however, this effect was more pronounced in the gamma-irradiated group, indicating a stronger shift towards a regenerative immune profile. Enhanced reepithelialisation, increased hair follicle density and reduced scar thickness were also observed. These findings suggest that gamma-irradiated hAF provides a more effective and minimally invasive therapeutic option for modulating scar formation and improving wound healing outcomes, supporting its potential translation into clinical applications for the management of hypertrophic scars.
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