1454 Investigating the utility and exploring biological targets of micro-wound healing compounds in 2D and 3D skin models

An injury to the epidermis can result in a micro-wound, characterized by its localization above the dermal-epidermal junction and an absence of vessel damage. Re-epithelialization is driven by keratinocytes which organize into overlapping migrating and proliferating zones to ensure faithful micro-would closure. Although still relatively unexplored, keratinocyte-mediated cytokine secretion is thought to be a feature of the micro-wound re-epithelialization response. A failure to re-epithelize, also characteristic of chronic wounds, can lead to reoccurring infections and hyperpigmentation. Although wound healing has been extensively researched, there are gaps in our understanding of the cellular mechanisms involved in the onset and completion of re-epithelization, leading to a scarcity of available enhanced micro-wound healing compounds (MHCs). To investigate the utility of a panel of putative MHCs, a new high-throughput scratch assay method was devised. A guide for multichannel pipette scratches was produced by 3D-printing an autoclavable 96-well plate lid with custom-made lengthwise slits. Using an immortalized keratinocyte cell line (N/TERT), the rate of scratch healing was characterized. Critical re-epithelization time points have been determined, and markers of migration are being explored within the identified periods. Moreover, known keratinocyte-migration-inducing compounds are being tested to serve as a positive control for enhanced MHC screening. Any putative MHC hits will be further explored using proteomic approaches to elucidate the signaling pathways involved in the onset and completion of re-epithelization. This analysis will assist in the identification of novel enhanced MHC targets and further our understanding of keratinocyte re-epithelization.