Light-Intensity-Dependent Control of Collagen Hydrogel Properties via Riboflavin Phosphate-Mediated Photocrosslinking

While photocrosslinked collagen hydrogels show promise in tissue engineering, conventional approaches for property control often require complex chemical modifications or concentration changes that alter their biochemical composition. Here, we present the first systematic investigation of light-intensity-dependent control in riboflavin phosphate (RFP)-mediated photocrosslinking as a novel, single-parameter approach to modulate hydrogel properties while preserving native biochemical environments. We systematically investigated the effects of varying light intensities (100 K, 50 K, and 10 K lux) during hydrogel fabrication through comprehensive structural, mechanical, and biological characterization. Scanning electron microscopy revealed unprecedented control over network architecture, where higher light intensities produced more uniform and compact networks, while swelling ratio analysis showed significant differences between 100 K lux (246 ± 2-fold) and 10 K lux (265 ± 4-fold) conditions. Most significantly, we discovered that intermediate intensity (50 K lux) uniquely optimized mechanical performance in physiological conditions, achieving storage modulus of about 220 Pa after 24 h swelling, compared to about 160 and 109 Pa for 100 K and 10 K lux conditions, respectively. Remarkably, cellular studies using NIH/3T3 fibroblasts demonstrated that lower light intensity (10 K lux) enhanced cell proliferation by 2.8-fold compared to 100 K lux conditions after 7 days of culture, with superior cell network formation in both 2D and 3D environments. This groundbreaking approach establishes light intensity as a powerful single parameter for precise control of both mechanical and biological properties, offering a transformative tool for tailoring collagen-based biomaterials in tissue engineering applications.