“After injury, a scar is what makes you whole.”
—China Miéville, The Scar
Forward-thinking surgeons are looking to regenerative medicine to engineer a living, growing tissue that can replace or repair tissue damaged by trauma and do so while leaving little or no scar. Such tissue engineering could revolutionize trauma care, affording surgeons the opportunity to “replace tissue, repair tissue, cover defects, and do certain operations we couldn’t otherwise do,” according to Samir Fakhry, M.D., Chief of the Division of General Surgery, who leads the Level I MUSC Trauma Center as the Medical Director for Surgical and Acute Critical Care.
Seeing the promise that tissue engineering holds for improving surgical care, the Division of General Surgery at MUSC established a regenerative medicine laboratory in 2012 with the recruitment of Michael J. Yost, PhD, Associate Professor in the Department of Surgery and a bioengineer whose research focuses on regenerating skeletal muscle. Dr. Fakhry is convinced that the revolutionary treatments of tomorrow will spring from close collaboration between surgeons on the frontlines of treating trauma and scientists who are trying to equip them with better technology for doing so: “It is important to have a laboratory where we can take questions and begin to develop solutions working with scientist colleagues.”
Why New Treatment Options Are Needed
Trauma can be caused by a violent assault (eg, gunshot or stabbing), a car crash, or by surgery. In each case, the body is penetrated and tissue is damaged or lost. Trauma surgeons, who witness the effects of injury every day, currently have too few options for addressing such tissue damage, and none of them is without drawback. They can harvest skin or muscle flaps from elsewhere on the patient’s body for grafting to the site of traumatic injury, but sufficient tissue can sometimes be lacking for major injuries and the donor sites can often become infected. They can graft tissue donated from human or animal donors, but those grafts can be rejected by the patient’s immune system. Finally, they can use commercially available synthetic (eg, polymers, plastic) materials to close the wound, but these materials are expensive, easily become infected, and are inert, ie, they do not adapt to the body’s needs, limiting their functionality.
The body’s own response to trauma is scar. “The body will eventually replace most defects—fill in holes and bridge gaps—the body has only one means of doing that and it’s very undifferentiated and it’s called scar—a very clever solution to a complex set of problems but not the ideal solution,” explains Dr. Fakhry.
Scar tissue covers over the wound and allows for healing, but it can also disfigure, lower self-esteem, and limit function by causing contractures that restrict range of motion. Scar tissue is also as much as 30% weaker than healthy tissue.
Scar may be the body’s natural response to trauma, but it is in some ways an outmoded, even primitive one. “If in prehistoric times I were bitten by a saber-toothed tiger, I’d want the inflammatory response calling in neutrophils that would blast everything so I could live. Today, I don’t want the scar,” explains Dr. Yost.
Dr. Yost is working to offer a more sophisticated response to trauma, one whose resolution is regeneration and not scar, by taking a two-fold approach. First, he is working to engineer tissue that can live and grow and function as normal tissue in the body. Second, he is trying to learn to mute the body’s own inflammatory response to such tissue so that it remains viable and so that scarring can be minimized.
Regenerating Skeletal Muscle
FIGURE. Illustration showing the steps of muscle repair and healing. Top circle: Macrophages move to the injured area, cleaning up and removing the damaged muscle fibers. Middle circle: Blood vessels deliver oxygen and other nutrients necessary for muscle repair. Bottom circle: Specialized cells, called satellite cells, are activated and fuse together to regenerate a new muscle fiber. Illustration by Elise Walmsley, Bsc, AAM, used with permission.
Simple administration of satellite cells (the stem cells for skeletal muscle) will not ensure the regeneration of skeletal tissue. A scaffold of some sort must be provided for the satellite cells to improve their viability and preserve their orientation and architecture. Dr. Yost’s laboratory has successfully regenerated skeletal muscle fibers by administering satellite cells with an extracellular matrix (ie, collagen).¹ The co-administration of satellite cells and matrix to the trauma site prompts the body to recognize the need to generate skeletal muscle in that location and to initiate the events necessary to do so, including the influx of additional satellite cells and the creation of vasculature. Satellite cells grow together to form myocytes, the building blocks of muscle (Figure).
Although Dr. Yost and his team are able to obtain muscle fibers with this method, they are not able to produce “nice, organized, fascicular bundles of skeletal muscle” with good contractility, and there is still some interstitial scarring, likely due to an initial inflammatory response. “We have not seen a lot of good voluntary contraction of the muscle—nobody has—but we are getting muscle in there and that is an important step.” In Dr. Yost’s opinion, achieving organized bundles of muscle tissue will require better “communication” between the regenerating muscle and neurons. The regenerating skeletal muscle must provide cues to recruit motor neurons, but innervation by those recruited motor neurons is in turn necessary to cue the regenerating muscle to develop into organized bundles. Dr. Yost has established the presence of activated motor neurons in the skeletal muscle he has regenerated; however, that muscle does not yet seem to be able to take the cues from the motor neurons. Further studies are needed to understand how to make the regenerating muscle more receptive to those cues.
The advent of bioprinter technology will help realize the promise of such regenerated tissues by allowing medical centers to produce patient-specific tissue on site for surgical applications.
The Inflammatory Response and Scar Formation
Engineering living, growing, functional tissue is only half the battle. The implanted tissue must then be protected from the body’s innate defenses. The body assumes that all intrusions are hostile ones and reacts to all the same, making no distinction between stab wound and surgical incision, foreign toxin and bioengineered tissue. As Dr. Yost notes, “the body has no plan for sterile surgical trauma, and it has no plan for exogenous tissue to be implanted as a repair material.” As a result, those seeking regenerative medicine solutions to tissue damage find themselves battling the body’s own first-line of defense—the inflammatory response that is an integral part of innate immunity.²
Innate immunity is the body’s first line of defense, its emergency first responders. Inflammation helps to get the emergency first responders to the area of damaged tissue, where they can begin to clear up cellular debris and remove any noxious or foreign agents.
The initial step in inflammation, usually occurring within seconds to a few minutes of injury, is vascular: arterioles and venules near the wound site contract and then dilate, allowing increased blood flow to the site. At the same time, the walls of the vessels become more permeable, allowing fluid to enter the damaged tissue. This increased blood flow and leakage of fluid from the blood vessel into the surrounding tissue are responsible for the redness and swelling associated with inflammation.
The next step in inflammation, occurring in the course of several hours, is cellular. Damaged cells leak adenosine triphosphate (ATP) into the extracellular space, triggering migration of blood-borne leukocytes, especially neutrophils, to the site. The flow of blood through the dilated vessels becomes sluggish, providing the opportunity for neutrophils and macrophages to accumulate in the region of injury, move to the vessel walls, and eventually pass through the walls into the damaged tissue. There, they phagocytize any noxious or threatening agent as well as cellular debris and, on occasion, healthy cells as well.
This “cleaning away” of damaged tissue is necessary before healing can begin. If the wound has been minor, the normal architecture of the tissue can be fully restored. However, in many cases, a scar will form, as the body seeks to fill the gaps in tissue left by the removal of the damaged cells by depositing fibroblasts and collagen.
From Scar to Regeneration
The goal of Dr. Yost’s research is to move healing away from scar and toward regeneration. Although inflammation is essential to protect the body against assaults and to remove toxins, pathogens, and cellular debris, it can also damage healthy tissue (eg, through the release of free radicals, through the phagocytosis of healthy cells). How can the inflammatory response, necessary in order to trigger regeneration, be attenuated so as to minimize damage to healthy tissue and to lessen the likelihood that any engineered cells that are implanted into the wound site will be attacked by neutrophils and other first responders?
Dr. Yost’s laboratory is trying to modulate the inflammatory response by removing its trigger—the leakage of ATP from damaged cells into the extracellular space. ATP typically escapes from cells via gap junctions, openings between adjacent endothelial cells that are formed when the hemichannels of the two adjacent cells come together. Each hemichannel is composed of six connexin proteins. If the connexins can be prevented from forming hemichannels, then the ATP would have no means of escaping from the cell into the extracellular matrix to trigger inflammation. Two peptides capable of disrupting formation of the hemichannels by connexins have been identified by research done at MUSC.
The first, the α–connexin carboxyl-terminal (ACT1) peptide, which was developed in the laboratory of former MUSC researcher Robert Gourdie, PhD (who continues in an adjunct position at MUSC), was shown to reduce scarring and promote wound healing in animal models by reducing the inflammatory response and the area of scar progenitor tissue and to improve skin strength and extensibility.³ MUSC licensed the rights to the peptide to First String Research, which took a topical gel containing the peptide into clinical trials. In July 2013 at the International Gap Junction Conference in Charleston, SC, First String announced the promising results of phase 2 trials in three patient populations—those with diabetic foot ulcers, those with venous leg ulcers, and those who had undergone laparoscopic incisions. Significant increases were reported in the mean percent of wound closure at four and twelve weeks as well as the incidence of 100% wound closure. A phase 3 trial of the topical gel is in the planning stages.
The second peptide, the development and testing of which has been a collaborative effort between the laboratories of Dr. Yost and Dr. Gourdie, has shown impressive anti-fibrotic, anti-inflammatory, and pro-regenerative activities in animal models. Like ACT1, it disrupts formation of hemichannels by connexins, but via a different mechanism. Preliminary results suggest a strong efficacy in preventing scar formation and promoting regeneration of tissue with normal architecture.
More Than a Cosmetic Benefit
Because the same mechanisms are involved in scar formation throughout the body, therapies that prove effective in preventing or diminishing scarring could have broad applications. For instance, such therapies could prevent tissue scarring that follows myocardial infarction or results in macular degeneration. A standard therapy or set of therapies for reducing or preventing scar formation could spare patients some of the most devastating consequences of these diseases.
And, of course, for those who have undergone traumatic injury, regenerative therapies offer the hope of being made whole without having to bear the lifelong scars of a past tragedy.
¹ Logan MS, Propst JT, Nottingham JM, Goodwin RL, Pabon DG, Terracio L, Yost MJ, Fann SA. Human satellite progenitor cells for use in myofascial repair: isolation and characterization. Annals of Plastic Surgery. 2010;64(6):794-799.
² Rhett JM, Ghatnekar GS, Palatinus JA, Quinn M, Yost MJ, Gourdie RG. Novel therapies for scar reduction and regenerative healing of skin wounds. Trends Biotechnol. 2008 Apr;26(4):173-80.
³ Ghatnekar GS, O’Quinn MP, Jourdan JL, Gurjarpadhye AA, Draughn RL, Gourdie RG. Connexin43 carboxyl-terminal peptides reduce scar progenitor and promote regenerative healing following skin wounding. Regen Med. 2009;4(2): 205-223.