The Secrets of Healing Without Scars Skin Regeneration Is an Imperfect Process That Is Impeded by a Host of Factors

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REGENERATION The secrets of healing without scars Skin regeneration is an imperfect process that is impeded by a host of factors. Working out the part played by each could lead to fresh approaches to treating burns and scars.

BY CASSANDRA WILLYARD When Michael Harrison, a paediatric surgeon deciphering what drives this in utero healing, at the University of California, San Francisco, he might discover ways to prompt scar-free he body’s largest organ might seem began to perform the first ever surgeries on healing outside the womb. “My reluctant one barely more than cellular wrapping fetuses, he noticed something curious about year in the lab became four,” Longaker says. “I paper, but skin has roles that range the babies who survived. Incisions he had became obsessed with scarring.” MARCO MELGRATI Tfrom fending off microorganisms to regulating made in them in the womb seemed to heal Longaker, now a plastic surgeon with a body temperature. It also has a considerable without scarring. focus on regenerative medicine at Stanford flaw: severely damaged skin can heal, but it Harrison asked Michael Longaker, a University in California, has not yet unrav- can’t regenerate. Instead, it forms scars. These postdoctoral researcher in his laboratory, to elled the mystery completely. Nor have other marks are not just cosmetic defects. Scar tissue investigate the phenomenon. Longaker was researchers. Although many studies have can inhibit a person’s movement and, because it sceptical. Because his boss was the only phy- provided valuable insight into how scarring lacks sweat glands, prevent the body from cool- sician who was performing fetal surgeries, occurs, they have yielded few clinically useful ing off. Although scars seem to be thicker than he says, “My first reaction was, ‘Gosh, that treatments. “There’s been some improvement,” normal skin, the tissue is actually weaker. doesn’t seem like a big health-care problem says Stephen Badylak, deputy director of the Scarring seems to be an inevitable part of because you’re the only one making [fetal] McGowan Institute for Regenerative Medicine being human. But three decades ago, it became wounds.’” But it didn’t take long for Longaker at the University of Pittsburgh in Pennsylvania. clear that the youngest patients don’t scar. to understand the potential implications: by But it’s still far from the expectations raised by

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the hype of the work that began in the 1980s. a dermatologist at the Perelman School of Yet many researchers are cautiously Medicine at the University of Pennsylvania in optimistic that a better understanding of the Philadelphia, noticed that older people often mechanisms that lead to scarring will pave develop thinner scars than do younger adults. VSHYUKOVA/SPL the way for innovative strategies for reducing To understand why, Leung turned to mice. the formation of scar tissue. In September, the He and his colleagues compared wound heal- US Food and Drug Administration approved ing in young and old mice by punching holes the first treatment to involve a ‘spray-on’ skin, in the rodents’ ears1. In one-month-old ani- and numerous other skin-healing products are mals, such wounds healed with a thick scar in clinical trials. The field of skin regeneration and never closed fully — similar to earring is moving in a different direction, Badylak says. holes in people, Leung says. In 18-month-old Rather than growing skin in Petri dishes in mice, which are roughly equivalent to 65-year- the lab, and then transplanting it onto people, old people, healing took longer, but the holes researchers are using the body as a bioreactor closed completely, and with less scarring. The and encouraging skin to do what it did during same observations held for wounds on the backs fetal development — regenerate. They want to of the mice. find out more about how scarring occurs, as Leung and his colleagues wondered whether well as how it might be stopped. a component of the blood of young mice promotes scar formation. To test the idea, they EVOLUTIONARY ADVANTAGE joined together old and young mice, giving Cut the skin and it will bleed. And then it will them a shared circulatory system through a heal. Initially, a clot forms to staunch blood surgical technique called parabiosis. The team flow, which kicks off a massive inflamma- found that exposure to the blood of young ani- tory response. Immune cells flood the region mals caused wounds in elderly mice to scar1. to clear bacteria and debris, while cells called Further experiments revealed the probable keratinocytes in skin’s outer layer divide rap- culprit: Cxcl12, a gene that encodes a protein idly in a race to close the wound and prevent called stromal cell-derived factor 1 (SDF1). Fluorescence micrograph of human skin fibroblasts. infection. Next, the wound begins to fill. When the team knocked out SDF1, even Spindle-shaped cells known as fibroblasts wounds in young animals healed with mini- other researchers can find a way to identify and migrate to the damaged area and churn out mal scarring. This discovery suggests a route block the same fibroblasts in people, it might collagen and other proteins that provide tis- towards scar-free wound healing in people: be possible to prompt wound healing to follow sue with structure. Within three weeks of the suppressing the activity of CXCL12. a more regenerative pathway. “I would be disap- injury occurring, the wound has healed. In fact, there’s already a drug on the pointed if we’re not doing something like that in But such speedy healing has a major market that interferes with the SDF1 humans in the next five to seven years,” he says. downside. These quick repairs often result in pathway — plerixafor. The drug is used to Although some fibroblasts are clear drivers scars, particularly when the wound is deep. In mobilize stem cells from bone marrow in peo- of scar formation, other research suggests that healthy skin, collagen fibres form a lattice. But ple with certain types of cancer. Leung and his fibroblasts also contribute to regenerative heal- during wound healing, fibroblasts lay down colleagues hope to test whether plerixafor can ing. About a decade ago, George Cotsarelis, collagen fibres parallel to each other, which cre- minimize the recurrence of keloids — thick, a dermatologist at the Perelman School of ates tissue that is stiff and weak. That’s because raised scars that tend to keep growing — in a Medicine, and his colleagues were trying to evolution has selected speed over perfection: clinical trial. The team is also looking at how develop a mouse model to understand the role before the discovery of antibiotics, slow heal- SDF1 promotes initial scar formation. of stem cells in hair follicles. Scientists had long ing would probably have meant acquiring an Scarring is a complex process, and SDF1 is thought that when an adult hair follicle is lost, infection or experiencing prolonged bleeding. only part of the story. Fibroblasts are another it is gone for ever. But then the team noticed “It’s really a matter of survival versus aesthetics,” prominent player. something odd: when they made a large wound says Jeff Biernaskie, a stem-cell biologist at the These cells have long “The really cool on the back of a genetically normal mouse, hair University of Calgary in Alberta, Canada. been blamed for part is that once regrew in the middle of the wound3. When such repairs to skin are small, they scar tissue. “We’ve you get a hair Even more strangely, skin around hair don’t pose much of a problem. But large scars had this assump- follicle, it kind follicles seemed to be normal, and a layer of can be life-changing. Scar tissue “doesn’t have the tion that fibroblasts of normalizes fat formed beneath — something that doesn’t stretch and the mobility and the range of motion are all the same,” the skin.” usually occur under scar tissue. In 2017, a that normal skin does,” says Angela Gibson, a Biernaskie says. But team led by Cotsarelis showed in mice that burn surgeon who studies wound healing at the research in the past five years has revealed that new hair follicles secrete growth factors called University of Wisconsin School of Medicine and fibroblasts comprise a diverse group of cells, bone morphogenetic proteins (BMPs) that can Public Health in Madison. That can be especially and that some seem to have a larger role in scar transform fibroblasts into fat cells4. “The really problematic when scars cover joints. Imagine, formation than do others. cool part,” Costarelis says, is that “once you get Gibson says, not being able to hold a fork or to In 2015, Longaker and his colleagues a hair follicle, it kind of normalizes the skin”. raise your arms to wash your hair. conducted an inventory of the fibroblasts on Human fibroblasts also seem able to make But scarring might not be inevitable. Fetal the skin of a mouse’s back2. When they cre- the leap from fibroblast to fat. When the team skin begins to scar only late in gestation, which ated a wound on the back, they found that only took such cells from a keloid scar and exposed suggests that human skin possesses at least one of two lineages of fibroblast — expressing them to a BMP, or placed them near a BMP- some regenerative capabilities. All researchers homeobox protein engrailed-1 — was respon- secreting hair follicle, they too turned into fat have to do is to work out how to unlock them. sible for the formation of most scar tissue. And cells. These findings suggest that it might be when the team disabled those cells in mice, possible to prod injured skin towards regenera- FANTASTIC FIBROBLASTS wounds healed more slowly but also formed less tion rather than scar formation. But translating Fetal wounds are not the only wounds that scar tissue, similar to what happened in mice the work into a treatment protocol poses con- are resistant to scarring. Thomas Leung, that lack SDF1. Longaker thinks that if he and siderable difficulties, Cotsarelis says. Skin

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mouse, which regenerates, and another in the spray-on skin system approved by the Food ear of a conventional lab mouse, which does and Drug Administration earlier this year, and not, and then evaluate how the healing process marketed as ReCell by biotechnology company differs. His team is now beginning to define Avita Medical in Valencia, California, is an those differences. example of an early success.

Some seem to involve the immune system. To prepare the treatment, surgeons remove a RON NIEBRUGGE/ALAMY Researchers tend to view inflammation as an piece of skin about the size of a postage stamp impediment to regenerative healing. Accord- from the patient and douse it with an enzyme ingly, the difference between scar formation in that liberates skin’s component cells: fibroblasts, adults and the fetus might be that adults mount keratinocytes and pigment-producing melano- a strong inflammatory response after injury cytes. These cells are then loaded into a nozzled whereas a fetus does not. But a connection syringe and sprayed onto the patient’s wound. between inflammation and regeneration has People with burns who require skin grafts typi- been difficult to establish. Efforts to prevent cally receive pieces of skin that are harvested scar formation by suppressing inflammation from unaffected parts of their bodies. Surgeons haven’t panned out, Seifert says. And he and take only the top layers of skin to create these his colleagues have found, at least in spiny grafts, which are known as split-thickness mice, that inflammation does not preclude grafts. One clinical trial showed that in people regenerative healing. In the wild, these mice with second-degree burns, which affect both mount a strong inflammatory response yet still skin’s epidermal and dermal layers, the ReCell manage to regenerate skin. therapy works as well as do conventional grafts, “We know that too much inflammation is but requires much less donor skin7. Although bad. And we know that no inflammation isn’t split-thickness grafts can be cut into a mesh helpful either,” Seifert says. In 2017, he and his that covers an area about three times their size, colleagues showed that macrophages, immune ReCell can treat skin wounds that are 80 times cells that are a key orchestrator of inflamma- larger than the donor piece of skin. ReCell can tion that is typically associated with scarring, also be combined with meshed grafts to treat are also required for regenerative healing in deeper burns. spiny mice6. Now, the team is trying to deter- Gibson is testing an alternative treatment mine which factors might tip macrophages for burns, a skin substitute called StrataGraft. Reindeer antler velvet has regenerative properties. and other immune cells away from scarring It comprises two layers of collagen: a bottom pathways and towards regeneration. layer that is seeded with human fibroblasts and regeneration will require the right signals to be A much larger mammal — reindeer a top layer that is seeded with cells that give delivered at the right time, and at the right dose. (Rangifer tarandus) — is also providing insight rise to keratinocytes. The therapy originated at For example, “When hair follicles form, their into the regenerative potential of skin. Both the University of Wisconsin, but is now being spacing is determined by gradients of growth male and female animals sprout new antlers developed by Mallinckrodt Pharmaceuticals factors,” he says. Altering those gradients, even each year. The downy velvet that covers the in Staines-upon-Thames, UK. One of the slightly, might alter the follicle pattern or even antlers as they grow is remarkably similar to first clinical trials of StrataGraft, published in function. “Precision is really required,” he says. human skin — thick with blood vessels, hair 2011, showed that it did not induce an acute follicles and sebaceous glands. But it differs in immune response8, and the substitute is now A MORE PERFECT MODEL one important way. “If we wound the velvet, being tested in a phase III trial. The mice in which most research on wound it regenerates perfectly,” Biernaskie says. “It’s Such therapies could be a boon for people healing is performed differ from people in really a beautiful and powerful model for skin with burns. Other companies are working on important ways. Their skin is loose, whereas healing.” treatments for tricky-to-heal wounds, such as that of humans is tight. Furthermore, mouse That capacity for regeneration seems to ulcers in people with diabetes or bedsores. “The wounds heal by contraction: such wounds pull be inherent to the velvet. Biernaskie and his market size is just gigantic,” Badylak says. But together rather than filling in. “I don’t know colleagues are now the main goal of these treatments is to promote how you can even begin to think you could “We know comparing changes in better healing, rather than to prompt skin to test something there and then translate it to that too much gene expression dur- regenerate. Achieving that next step — scar- humans,” Gibson says. inflammation ing wound healing in free healing — is “a tall order to fill”, Gibson In search of a better model, in 2009, Ashley is bad. And we two anatomical areas of says. However, she is optimistic that if clinicians Seifert, a developmental and regenerative know that no reindeer — skin on their who treat skin wounds collaborate closely with biologist at the University of Kentucky in inflammation backs, which doesn’t researchers who are working to understand Lexington, travelled to Kenya and began to isn’t helpful regenerate, and ant- scarring, the problem can be solved. “That’s study African spiny mice (Acomys kempi and either.” ler velvet, which does. when the science will move forward,” she says. ■ Acomys percivali) — species with a unique They hope that the defence mechanism. Because their skin tears comparison will help them to better understand Cassandra Willyard is a science journalist in easily, these mice can escape the jaws of pred- the signals that prompt velvet to regenerate, and Madison, Wisconsin. ators. Seifert expected to find that such mice perhaps lead them to treatments that promote 1. Nishiguchi, M. A., Spencer, C. A., Leung, D. H. & had speedy wound-repair processes or ways regeneration and prevent scarring. “We could Leung, T. H. Cell Rep. 24, 3383–3392 (2018). of preventing infection. But what he and his start to develop cocktails of drugs where we 2. Rinkevich, Y. et al. Science 348, aaa2151 (2015). colleagues found was much more intriguing: could mimic those signals,” Biernaskie says. 3. Ito, M. et al. Nature 447, 316–320 (2007). 5 4. Plikus, M. V. et al. Science 355, 748–752 (2017). spiny mouse wounds heal relatively scar free . 5. Seifert, A. W. et al. Nature 489, 561–565 (2012). The spiny mouse is one of only a few BENCH TO BEDSIDE 6. Simkin, J., Gawriluk, T. R., Gensel, J. C. & mammalian models of skin regeneration. But Skin regeneration is still a distant goal, but Seifert, A. W. eLife 6, e24623 (2017). 7. Holmes, J. H. IV et al. J. Burn Care Res. 39, 694–702 such mice provide a comparative framework. several companies are working to bring (2018). Seifert can punch a hole in the ear of a spiny wound-healing therapies to market. The 8. Centanni, J. M. et al. Ann. Surg. 253, 672–683 (2011).