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

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.