Dynamic Perfusion and Cell Seeding Density Govern Remodeling and Mechanical Maturation of Bioprinted Collagen Constructs

Hydrogel-based three-dimensional culture systems are widely used in tissue engineering; however, their maturation is often limited under static conditions. This study investigates the combined effects of dynamic perfusion and initial cell seeding density on remodeling behavior and mechanical properties of bioprinted collagen hydrogel constructs, with additional assessment of smooth muscle cell-like (SMC) differentiation. Rectangular constructs (30 × 15 × 1.5 mm) were fabricated using high-concentration collagen (30 mg/mL) with cell densities of 10 and 20 million cells/mL. MCDB- and DMEM-based media were first compared using growth curves, leading to the selection of MCDB differentiation medium for subsequent experiments. Constructs were then cultured statically or under dynamic perfusion (20 mL/min) for up to 7 days. Remodeling was evaluated by monitoring changes in construct dimensions over time. Static constructs exhibited non-uniform deformation and rolling, whereas dynamically perfused samples retained their geometry and underwent homogeneous contraction. Mechanical testing revealed a transition from stiff and brittle to more compliant and ductile behavior, with preserved load-bearing capacity at large strains. Remodeling and mechanical outcomes were strongly influenced by cell density and culture medium, with differentiation conditions promoting more stable constructs. These changes were accompanied by increased expression of smooth muscle–related markers under dynamic culture. Overall, dynamic perfusion and cell seeding density jointly govern remodeling and mechanical maturation of bioprinted collagen constructs, highlighting their importance for functional hydrogel-based tissue development.