Grayscale mask-assisted photochemical crosslinking for a dense collagen construct with stiffness gradient

Despite the potential of a collagen construct with a stiffness gradient for investigating cell-extracellular matrix (ECM) stiffness interaction or recapitulating an in vivo tissue interface, it has been developed in a limited way due to the low and poorly controllable mechanical properties of the collagen. This study proposes a novel fabrication process to achieve a compressed collagen construct with a stiffness gradient, named COSDIENT, at a level of similar to 1 MPa while maintaining in vivo ECM-like dense collagen fibrillar structures. The COSDIENT was fabricated by collagen compression followed by grayscale mask-assisted UV-riboflavin crosslinking. The collagen compression process enabled the remarkable increase in the stiffness of the collagen gel from similar to 1-10 kPa to similar to 1 MPa by physical compaction. The subsequent UV-riboflavin crosslinking with a continuous-tone grayscale mask could simply generate a gradual change of UV irradiation followed by modulating riboflavin-mediated crosslinking, thereby resulting in a continuous stiffness gradient with a range of 1.16-4.38 MPa in the single compressed collagen construct. The suggested grayscale mask-assisted photochemical crosslinking had no effect on the physical and optical properties of the original compressed collagen construct, while inducing gradual changes of chemical bonds among collagen fibrils. A skin wound healing assay with epidermal keratinocytes was finally applied as an application example of the COSDIENT to examine the effect of stiffness on the skin keratinocyte behavior.