Biomimetic Collagen Scaffolds Natural Cross-Linking Strategies via Transglutaminase and Methylglyoxal for Skin Repair

Collagen-based scaffolds are widely used in tissue engineering for skin repair due to their biocompatibility and structural resemblance to extracellular matrix. However, rapid degradation and insufficient mechanical stability limit their effectiveness. This study investigates natural cross-linking strategies utilizing Transglutaminase (TG) and Methylglyoxal (MGO) to enhance scaffold durability while maintaining biocompatibility. Monolayer and bilayer collagen scaffolds were fabricated and cross-linked using enzymatic (TG) and non-enzymatic (MGO) treatments. Cross-linking efficiency was assessed via TNBS/Hydroxyproline assays, while AFM and mechanical testing evaluated structural and mechanical properties. Cell viability (LIVE/DEAD), metabolic activity (MTS assay), and cell migration (in-growth and out-growth assays) were analyzed to assess biocompatibility and scaffold functionality. Wound healing was also assessed histologically. TG and MGO crosslinking significantly enhanced collagen fibril organization, scaffold stiffness, and enzymatic resistance. TG- and MGO-treated scaffolds exhibited a homogeneous fibrillar structure with improved mechanical integrity. Both cross-linking strategies supported high fibroblast viability and sustained metabolic activity over time. Migration assays confirmed that TG and MGO scaffolds facilitated fibroblast infiltration and wound closure. In vivo, the bilayer scaffolds with TG/MGO cross-linking showed full epithelialization, with the Tg/25 MGO group exhibiting reduced inflammation, organized collagen, and hair follicle formation, suggesting improved regeneration. These findings highlight the potential of biomimetic collagen scaffolds as wound dressings for dermal tissue regeneration and skin repair.