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

Abstract

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.

Metal–Electrolyte Solution Dual‐Mode Electrospinning Process for In Situ Fabrication of Electrospun Bilayer Membrane

“…After 10 h, for the stable adhesion and spreading of the cells, the PDMS block was carefully removed from the membrane, which generated a wound gap with 6 mm diameter at the center region of the membrane, and the cells were additionally cultured for 6 d to investigate the wound coverage by the cells on the membrane. [ 31 ] After the cell culture, the cells were investigated by staining with the Live/Dead cell viability kit and TRITC‐conjugated phalloidin and DAPI. For the Live/Dead staining, the cells were washed with PBS and treated with 4 × 10 −3 m calcein acetoxymethyl ester (green) and 2 × 10 −3 m ethidium homodimer‐1 (red) at 37 °C for 30 min.…”

Collagen-Based Biomimetic Systems to Study the Biophysical Tumour Microenvironment

“…Collagen glycation also proved to be an effective stiffening method, although it is time consuming [ 81 , 82 ]. Finally, the use of ultraviolet crosslinking in the presence of riboflavin possesses the advantage of being a simple and effective method that may also act on a local scale: by using light masks that spatially filter light transmission, it is possible to create patterns or gradients of crosslinked fibrils in the gel [ 83 , 84 ]. Light irradiation, especially in the UV range, may induce cytotoxic effects.…”

Mechanical Properties of Bioengineered Corneal Stroma

“…[43][44][45][46][47] Keratocytes also exhibit durotaxis and mechanosensing, i.e., the migration to stiffer regions of materials. [48,49] Alteration in ECM stiffness can change, for instance, actin stress fiber production and focal adhesions, as well as alter keratocytes' phenotype. [50,60] Significant differences in keratocyte alignment, morphology and matrix reorganization are observed between anisotropic and isotropic environments.…”