Disrupting Mechanotransduction Decreases Fibrosis And Contracture In Split Thickness Skin Grafting
Dominic Henn, MD1,2, Kellen Chen, PhD1, Michael Januszyk, MD PhD1, Janos Barrera, MD1, Chikage Noichiki, MD1, Michelle Griffin, MD1, Artem Trotsyuk, BS1, Ruth Tevlin, MD1, Jagannath Padmanabhan, PhD3, Sun Hyung Kwon, PhD1, Dharshan Sivaraj, BS1, Nestor Diaz, BS1, Christopher Lavin, MD1, Andreas Keller, PhD4, Michael T. Longaker, MD1, Geoffrey C. Gurter, MD1.
1Hagey Laboratory for Pediatric Regenerative Medicine, Div. of Plastic Surgery, Stanford University, Stanford, CA, USA, 2Dept. of Plastic Surgery, UT Southwestern Medical Center, Dallas, TX, USA, 3Hagey Laboratory for Pediatric Regenerative Medicine, Stanford, CA, USA, 4Chair for Clinical Bioinformatics, Saarland University, Saarbruecken, Germany.
PURPOSE: Humans and other large organisms heal wounds by fibrosis, with the formation of hypertrophic scars (HTS) that severely compromise physiologic skin function. The current standard-of-care for partial- and full- thickness soft tissue defects without exposed bone or neurovascular structures is split-thickness skin grafting (STSG); however, skin grafts often result in HTS formation and have other disadvantages including fragility, abnormal pigmentation, and altered sensation. Currently, there are no effective therapeutics on the market that can prevent HTS formation resulting from STSG.
METHODS: We have developed the first large animal model of STSG using clinically relevant surgical techniques and established treatment protocols for postoperative wound care. Full-thickness excisional wounds were created on the back of red Duroc pigs. STSG were harvested in a thickness of 0.01 inches using an electric dermatome and meshed in a 1:1.5 ratio. Grafted wounds were either treated with focal adhesion kinase inhibitor (FAKI) releasing hydrogels, blank hydrogels (placebo), or standard dressings as controls. Wound healing and scar quality were assessed over time by serial quantification of scar area in photographic images as well as cutometer measurements of the scars over time. After 90 days, animals were euthanized, and explanted scar tissue as well as unwounded pig skin were processed for single-cell RNA sequencing (scRNA-seq). In addition, we performed immunofluorescent protein staining of tissue sections and investigated the effect of FAKI on cultured porcine fibroblasts in a 3D cell culture model.
RESULTS: Wounds treated with STSG developed substantial contracture as well as hypertrophic scars at 90 days. From scRNA-seq, we identified pathways critical to driving scar formation, such as aberrant mechanotransduction signaling. FAK inhibition significantly reduced contracture, fibrotic collagen deposition, and improved biomechanical properties. Using scRNA-seq, we observed an increase in myeloid and lymphoid cells in untreated scars compared to scars treated with FAKI and unwounded skin. Moreover, we identified that fibroblast sub-populations in untreated scars showed an enrichment of mechanotransduction pathways as well as osteogenic and chondrogenic differentiation fates, whereas FAKI drove fibroblasts toward pro-angiogenic and adipogenic differentiation states characteristic of unwounded skin. We were able to recapitulate this transcriptomic shift in fibroblast differentiation using 3D-cultured fibroblasts in vitro, and we also confirmed these genes on the protein level using immunofluorescent staining.
CONCLUSION: Our findings indicate that FAKI-releasing patches provide and effective therapeutic strategy to mitigate HTS formation resulting from STSG by restoring the transcriptional differentiation fates of unwounded skin within scar fibroblasts. Our surgically relevant STSG model, translationally applicable therapy, and clinically relevant endpoint measurements demonstrate the potential of our therapeutic approach for future human clinical trials.
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