Comprehensive analysis of in vitro wound healing potential of three-dimensionally macroporous cryogels

Assessment of wound healing capability is an important criterion for evaluating a biomaterial as a wound care material. The scratch assay has been widely used to assess the in vitro wound healing potential of biomaterials in various forms, including nanoparticles, extracts, and drugs. However, assessing the in vitro wound healing potential in the presence of solid three-dimensional (3D) biomaterials on scratch-based wounds is limited due to: (1) cell death because of the high density of 3D biomaterials, and (2) difficulty in following the wound healing patterns based on phase-contrast microscopic images, considering the hindrance in the presence of biomaterial. In the present work, we improved the evaluation of the wound healing potential of cryogels, as low-density 3D macroporous biomaterials, by combining phase-contrast microscopy with LIVE/DEAD cell imaging, proposing it as an enhanced method for scratch assay, using a previously developed biomaterial, i.e., hydroxyethyl methacrylate (HEMA)-chitosan (HC) cryogels. The healing of the scratch was assessed over 24 h through phase-contrast imaging at 0 h, 2 h, 4 h, 6 h, 8 h, and 24 h intervals. This was further coupled with LIVE/DEAD cell imaging at 0 h and 24 h intervals. LIVE/DEAD cell imaging of the scratch area in the presence of scaffold showed that it acted as bridge between the two ends of the scratch, with cells migrated not only at the scratch area but also within the scaffolds. Such a phenomenon is otherwise difficult to analyse solely from the phase-contrast images. Therefore, in the current study, a combination of phase-contrast and LIVE/DEAD cell imaging is proposed to assess wound healing in the presence of 3D macroporous cryogel, as an advancement to the conventional methods used for analysing cryogel-assisted in vitro wound healing.