Tri-Layer Amniotic Membrane Allografts promotes re-epithelialization in vitro via cJUN mediated mechanism

Introduction: Wound re-epithelialization is marked by the formation of a new epithelial layer that re-establishes the barrier between a wound and the environment allowing for successful wound closure. The cellular and molecular processes involved in re-epithelialization are driven by various regulatory proteins that coordinate the migration and proliferation of keratinocytes [1]. cJUN signaling plays a critical role in regulating keratinocyte activity [2]. In this in vitro study, the mechanism by which a tri-layer lyophilized human amnion chorion membrane (LHACM*) facilitates re-epithelialization is investigated.Methods:LHACM was prepared using the PURION® process, followed by lyophilization and terminal sterilization. Effects on cellular migration were assessed in HaCaT cells, utilizing an in vitro scratch wound assay. Using the same scratch wound assay the protein expression levels of phospho-cJUN, a key signaling molecule in the re-epithelialization process, were evaluated by immunofluorescence. Gene expression of cJUN target genes were determined utilizing qPCR. Multiplex Luminex assays were used to identify secreted matrix metalloproteinases (MMPs) responsible for promoting keratinocyte migration in conditioned media of cells treated with LHACM. Activity of secreted MMP1 was assessed using an in vitro activity assay.Results:Treatment with LHACM extract resulted in significant enhancement of HaCaT cell migration in an in vitro scratch wound assay. Immunofluorescence analysis of LHACM treated cells revealed increased expression of phospho-cJUN. Gene expression of cJUN target genes, MMP1, MMP9 and vimentin, was increased after LHACM treatment. Increased secreted MMP1 was detected in analyzed conditioned media of LHACM treated cells and was able to be activated and process a substrate in vitro.Discussion: The findings from this study suggest that LHACM modulates in vitro keratinocyte migration in part through the regulation of the cJUN signaling. This study provides insight into the mechanism by which LHACM modulates the re-epithelialization process and supports the clinical use of LHACM in wound healing applications to re-establish that critical epidermal barrier.References:1. Pastar, I., et al., Epithelialization in Wound Healing: A Comprehensive Review. Advances in Wound Care, 2014. 3(7): p. 445-464. 2. Li, G., et al., c-Jun Is Essential for Organization of the Epidermal Leading Edge. Developmental Cell, 2003. 4(6): p. 865-877.