The Role of Collagen Rheology in Human Keratinocyte Differentiation: Implications for Skin Substitute Development

Type I collagen hydrogels are widely employed as scaffolds in tissue engineering due to their biocompatibility and ability to mimic the extracellular matrix (ECM). ECM viscoelasticity plays a critical role in regulating key cellular functions such as adhesion, proliferation, and differentiation. This study evaluates how collagen source and quality influence hydrogel architecture, mechanical properties, and keratinocyte behavior. Hydrogels were prepared at a concentration of 2.3 mg/mL using collagen from Advanced Biomatrix (AB, GLP grade) and Collagen Solutions (CS, GMP grade), and assessed for fibrillogenesis, rheological performance, and their ability to support stratified HaCaT keratinocyte cultures. AB-derived hydrogels exhibited higher porosity but lower mechanical resilience, characterized by a linear viscoelastic region (LVER) of 2.54%. In contrast, CS-derived hydrogels showed reduced porosity, denser fiber networks, and a higher LVER of 9.96%, indicating enhanced strain tolerance. HaCaT cells cultured on AB hydrogels showed diminished proliferation, metabolic activity, stratification, and expression of differentiation markers compared to those on CS hydrogels, which supported a more robust epidermal architecture. These findings highlight the critical role of collagen quality and mechanical characteristics on scaffold performance and epidermal tissue formation, emphasizing the need to optimize biomaterial properties for effective regenerative outcomes.