Reprogramming Wound Healing: GAG ‐Based Bioactive Scaffold Drives Pro‐Regenerative Cellular Cross‐Talk

ABSTRACT Effective wound healing depends on coordinated fibroblast‐macrophage cross‐talk that governs the transition from inflammation to regeneration. Here, we evaluated whether a glycosaainoglycan (GAG)‐based viscoelastic scaffold composed of chitosan, chondroitin sulfate, and hyaluronic acid (CH‐(CS‐HA)) can modulate this cross‐talk and promote regenerative repair. In vitro coculture studies demonstrated excellent cytocompatibility and significant phenotypic modulation. In fibroblasts, α‐SMA ( acta2 ) expression increased ~4.4‐fold, and fibronectin ( fn1 ) was markedly upregulated at 96 h in the CH‐(CS‐HA) + conditioned media (CM) group. Importantly, the Col‐I/Col‐III ( col1a1 / col1a3 ) ratio decreased from ~2.1 to ~1.1 over time, while the TGF‐β1/TGF‐β3 ( tgfb1 / tgfb3 ) ratio shifted from ~2.0 to ~0.7, indicating antifibrotic ECM remodeling. In macrophages, CD86 ( Cd86 ) expression decreased from ~2.8‐fold to ~1.5‐fold, whereas CD206 ( Mrc1 ) increased from ~3.4‐fold to ~6.7‐fold between 48 and 96 h, confirming M2 polarization. The TGF‐β1/TGF‐β3 ( tgfb1 / tgfb3 ) ratio similarly declined from ~1.0 to ~0.3, reinforcing an anti‐inflammatory phenotype. In a full‐thickness rat wound model, CH‐(CS‐HA) treatment achieved ~91% wound closure by day 17 compared to ~63% in controls. Gene expression in healed tissue showed elevated tgfb3 (~6.4‐fold) relative to tgfb1 (~4.3‐fold), increased col1a1 (~5.3‐fold) with reduced col1a3 (~2.7‐fold), and enhanced acta2 and fn1 expression, consistent with organized matrix maturation. Histology confirmed complete re‐epithelialization and early hair follicle regeneration. Collectively, these findings demonstrate that CH‐(CS‐HA) orchestrates immune‐stromal cross‐talk to promote inflammation resolution, balanced ECM remodeling, and functional tissue regeneration.