Why Some Wounds Don’t Leave Scars

An open wound is like a battlefield. On one side are pathogenic microbes, trying their best to sneak into the cozy confines of the body. Standing guard are immune and connective tissue cells, working hard to neutralize any invaders and close the skin barrier as quickly as possible, respectively. Typically for injuries to human skin, this healing process leaves behind a scar.

“Scarring is a process that has been selected throughout our evolutionary history as a way to effectively heal an injury, but it has consequences,” said Ophir Klein, a developmental biologist at Cedars-Sinai Medical Center who studies regeneration of the oral mucosa. “After a scar, the tissue is different than it was before and doesn't always function as well, even if the goal of healing is achieved.” But there are certain parts of the body, such as the uterine endometrium and the oral mucosa, that heal without scarring, all the while achieving speedy repairs.¹ However, the molecular mechanisms that regulate rapid regeneration of such tissues are relatively unexplored.

To understand why injuries in the oral mucosa leave no trace behind, Klein and his group teamed up with Michael Longaker, a plastic surgeon at Stanford University who studies wound repair and skin scarring. In a recent paper published in Science Translational Medicine, the researchers identified molecular mechanisms that facilitate regeneration over scarring.² Upon synthetically activating these pathways in facial skin, wounds healed quicker and with reduced scarring. These findings pave the way for advanced regenerative therapeutics in the future.

“For decades, there have been studies comparing oral wound healing to skin, but not in depth,” said Lari Hӓkkinen, a cell and molecular biologist studying wound healing at the University of British Columbia, who was not involved in the study. “This study takes it to another dimension with comprehensive analysis using bioinformatics approaches.”

Klein and his colleagues compared wound repair in the oral mucosa and facial skin since both tissues have the same embryonic origin. They removed tiny, circular patches of tissues from the two locations in mice and monitored the wounds over the next seven days. While the oral wounds regenerated scarlessly by day four, appearing the same as unwounded mucosa, facial wounds healed with a scar by day seven. The team took a closer look at the various cell types migrating to the wounds, such as neutrophils, fibroblasts, and vascular cells. The majority of cells in the oral mucosa were anti-inflammatory, which is thought to be a key factor in reducing scarring. Contrastingly, facial wounds contained mostly inflammatory immune cells and fibroblasts enriched in scar-inducing pathways.

How were all these different cell types communicating to drive tissue repair? To find out, the team performed a CellChat analysis, a computational tool which predicts cell interactions based on the receptors and ligands present on their surfaces.³ While some of the estimated links were similar between oral and facial wounds, three fibroblast types talked to each other more in the mucosa than in the skin. Using single-cell RNA sequencing, the team identified enrichment of several molecular pathways in the oral wounds, but one stood out for being highly expressed in these three chatty cell populations: signaling between the cell surface receptor-tyrosine kinase angiotoxin receptor-like (Axl) and its ligand growth arrest specific-6 (Gas6).

The AXL-GAS6 pathway is involved in various functions such as cell proliferation, migration, adhesion, and survival.⁴ However, its role in wound healing seems to be tissue specific. There’s evidence for the pathway’s scar-promoting role in lungs, kidneys, and liver, among other organs.^5,6 But scientists have also reported its contribution towards tissue regeneration.⁷ In this study, Klein and his team observed an increase in expression of AXL and GAS6 in oral wounds as compared to facial wounds, hinting at its regeneration-promoting function. Next, the team probed potential downstream targets of AXL signaling in facial and oral fibroblasts, focusing on a protein that helps cells respond to mechanical stimuli: focal adhesion kinase (FAK). They hypothesized that the pliable mechanical properties of oral mucosa, which is similar to non-scarring fetal skin, underlies its regenerative potential, as opposed to facial skin which has more tension.

Upon immunolabeling FAK, Klein and his colleagues observed lower expression of the protein in oral wound fibroblasts compared to facial wound cells. Facial wounds that had genetically reduced FAK levels healed like oral tissue—showing reduced scarring. To determine if tissue mechanical strain altered FAK expression, the researchers designed an in vitro experiment where they stretched cultured oral and facial fibroblasts. While skin cells increased their FAK levels and secreted scar-inducing molecules in response to stretching, oral mucosal cells did not; instead, they retained the high levels of AXL and GAS6 from before the intervention. Inhibiting FAK in facial fibroblasts or AXL in oral fibroblasts drove the cells to adopt the opposite phenotypes. These findings indicated that high levels of AXL reduced FAK expression in oral wound fibroblasts that experienced mechanical strain.

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To test whether these mechanisms were conserved in humans, Klein and his colleagues obtained five biopsies of repetitive oral wounds that healed with a scar and three facial wound scars, from five individuals. Both oral and facial scars had low levels of AXL and GAS6 and high levels of FAK, suggesting that scarring programs supersede regenerative processes in aberrant wound healing. The team could drive similar wounds in mice to heal without scarring by treating the injuries with GAS6.

Klein hopes these findings lead to therapies for better wound healing. But Hӓkkinen is cautious. “It has been shown that [AXL-GAS6] signaling is also involved in cancer. So, there's obviously these kinds of issues that need to be considered so that we don't do harm by affecting this pathway,” he said.

Now, the team wants to explore additional molecular candidates and pathways that differ between the tissues and how disrupting them affects other aspects of wound healing. Klein noted the unique connections needed to bring such a project to fruition. “[This study] was a conversation between the two labs and probably something that wouldn't have happened as well if one of the labs had tried to do this on their own,” he said.