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 ~ 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 ~ 1–10 kPa 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.