In this review, the authors discuss the stages of skin wound healing, the role of stem cells in accelerating skin wound healing, and the mechanism by which these stem cells may reconstitute the skin in the context of tissue engineering.
Key points
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The current gold standard of care for full-thickness burn injuries is to debride and use skin grafts to cover the wounds.
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These approaches are limited by the amount of skin available for grafting, particularly when the burns cover a large total body surface area.
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Stem cells are a unique cell population characterized by self-renewal and differentiation capabilities, which may allow for regeneration of the skin and its contents.
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Advances in tissue engineering have increased the interest in applying stem cells onto tissue engineered scaffolds to further reconstitute the damaged tissue and develop skin regenerative therapies.
Introduction
Skin functions to preserve processes such as hydration, to protect against the external environment, and to regulate temperature. Severe damage to the skin as in burn injuries may interfere with these processes and, in severe cases, may be life threatening. Although the current gold standard of care for full-thickness burn injuries is to debride and use skin grafts to cover the wounds, these approaches are limited by the amount of skin available for grafting, particularly when the burns cover a large total body surface area. Recently, there has been significant interest in the use of stem cells for skin regeneration. Stem cells are a unique cell population characterized by self-renewal and differentiation capabilities. Several populations of stem cells have been identified that have shown to assist in wound healing through immunomodulation, reepithelialization, revascularization, and collagen deposition. Furthermore, the advances in tissue engineering have increased the interest in applying stem cells onto tissue engineered scaffolds to further reconstitute the damaged tissue. Although these applications of stem cells in wound regenerative therapies have provided encouraging results, long-term clinical studies are necessary to determine whether the observed effects are clinically significant and whether regeneration can be achieved. In this review, we discuss the stages of skin wound healing, the role of stem cells in accelerating skin wound healing, and the mechanism by which these stem cells may reconstitute the skin in the context of tissue engineering.
Introduction
Skin functions to preserve processes such as hydration, to protect against the external environment, and to regulate temperature. Severe damage to the skin as in burn injuries may interfere with these processes and, in severe cases, may be life threatening. Although the current gold standard of care for full-thickness burn injuries is to debride and use skin grafts to cover the wounds, these approaches are limited by the amount of skin available for grafting, particularly when the burns cover a large total body surface area. Recently, there has been significant interest in the use of stem cells for skin regeneration. Stem cells are a unique cell population characterized by self-renewal and differentiation capabilities. Several populations of stem cells have been identified that have shown to assist in wound healing through immunomodulation, reepithelialization, revascularization, and collagen deposition. Furthermore, the advances in tissue engineering have increased the interest in applying stem cells onto tissue engineered scaffolds to further reconstitute the damaged tissue. Although these applications of stem cells in wound regenerative therapies have provided encouraging results, long-term clinical studies are necessary to determine whether the observed effects are clinically significant and whether regeneration can be achieved. In this review, we discuss the stages of skin wound healing, the role of stem cells in accelerating skin wound healing, and the mechanism by which these stem cells may reconstitute the skin in the context of tissue engineering.
Background
Skin is a soft tissue that forms roughly 8% of the total body mass and covers the entire surface area of the body. It is a self-repairing, self-renewing organ that forms an important barrier that separates the outer environment from the internal organs. The skin is composed of the epidermis, dermis, and hypodermis ( Fig. 1 ). The epidermis is the outermost layer of the skin and is characterized by a stratified structure and acts as a protective barrier. The layers of the epidermis include the stratum corneum, stratum granulosum, stratum lucidum, stratum spinosum, and stratum basale (see Fig. 1 ). Underneath the epidermis is the dermis, which is enriched for dermal fibroblasts that produce collagens and elastin fibers and provides the skin with structural support and elasticity. The dermis is divided into 2 layers—the more superficial papillary layer and deeper reticular layer. Within the dermis are also epidermal appendages that give rise to hair follicles, sebaceous glands, and sweat glands that provide sensation and support to the skin. Important differences exist between the papillary and reticular layers of the dermis that play a role in scarring depending on the depth of burn injury. The dermal fibroblasts located in the papillary dermis are heterogeneous in terms of morphology and proliferation kinetics, whereas dermal fibroblasts located in the reticular dermis have myofibroblast-like characteristics. Thus, with progressively deeper wounds, the fibroblasts in the reticular dermis are activated producing more alpha-smooth muscle protein and higher collagen lattice contraction, leading to scarring. In contrast, burns affecting the papillary dermis are less likely to result in hypertrophic scar formation. Below the dermis lies the hypodermis, which consists mainly of adipose tissue and blood vessels. The hypodermis not only insulate the skin and provide an energy reservoir, but it also ensures the life-saving mechanical and thermoregulatory characteristics of the skin. Deep to the hypodermis is fascia, muscle, and bone, which provide mechanical and structural support for all the layers more superficial to it. Together, these components form a highly organized and complex environment for which different cell types must communicate to continuously repair and regenerate the skin during normal homeostasis and during injury.
Any disruption in the normal anatomic structure and functional integrity of the skin ultimately leads to a wound. Wound healing relies on a complex dynamic process that involves the interaction of many cell types, growth factors, cytokines, and chemokines. Dysfunction of this mechanism can result in chronic, nonhealing wounds that lead to infection, sepsis, insensible fluid loss, impaired temperature regulation, and, in severe cases, death. Disruption in the wound-healing process can also lead to the formation of excess granulation tissue, which can ultimately lead to hypertrophic scar formation.
Particularly challenging wounds are large burn wounds. In addition to the acute systemic responses that occur immediately after the injury, these large burn wounds are also more likely to enter a prolonged period of hypermetabolism, chronic inflammation, and lean body mass wasting, all of which may impair wound healing. Furthermore, an increased susceptibility to infection owing to altered immune status may lead to sepsis, which can be lethal if not treated appropriately. With respect to sustained hypermetabolism and inflammation, these processes impair wound healing through delayed reepithelialization. Furthermore, limited availability of donor skin grafts becomes a significant challenge in large burn wounds. Split thickness skin grafts, often meshed to maximize the harvested skin, can cause secondary contracture during the healing process owing to variable thickness of the dermis. In addition to the thickness of the skin graft, the depth of the underlying wound bed also affects the extent and type of scarring. Autologous keratinocytes cultured in vitro have been used as an alternative to treat serious burns; however, this approach requires weeks to generate enough material to cover large burn wounds. Current treatment strategies require 2 biopsies followed by shipping of the samples, expansion, and return of the cultured tissue. These cultured tissues represent the only option for large burn injury patients; however, when used alone, they have increased risk of infection and take longer to incorporate. Epicel (Genzyme Biosurgery, Cambridge, MA) is the most commonly used cultured epithelial autograft. To help mitigate the high risk of infection and graft failure, special antimicrobial soaks such as sulfamylon and silver nitrate can be used. Additionally, the cultured epithelial autograft can be used on top of a widely meshed autograft (4:1, 6:1, or MEEK) to help autograft incorporation. A cultured epithelial autograft is not without risks as squamous cell carcinoma has been reported in grafted regions.
Others have focused their efforts on regenerating the dermal component of the skin with tissue engineered skin substitutes or acellular dermal substitutes, and although these efforts have been promising, disadvantages of these substitutes include slow vascularization, poor integration, and rejection. Acellular dermal substitutes are also often applied in a 2-step procedure that still requires skin grafting. Thus, there is an increased interest to identify an alternative treatment plan to augment the wound healing process to accelerate wound closure, minimize scar formation, and minimize additional operations.
Interest in the use of stem cells to regenerate different aspects of the skin has increased in the last decade. The skin harbors several distinct populations of stem cells and a rich array of cell types that have the potential to repair the skin. Studies have focused recently on characterizing the capacity of these cells for wound healing and tissue engineering purposes. In this review, we discuss the various stem cell populations found within the different layers of the skin and their potential application for skin regeneration. Enriching these stem cell populations and cultivating a microenvironment that facilitates the survival of these stem cells may assist in the regeneration of the skin and its contents.
Skin regeneration and hair follicle development
Wound healing is a complex and dynamic process that begins immediately after injury. Normal wound healing is divided into overlapping phases: hemostasis, inflammation, proliferation, and remodeling. Hemostasis is the first phase and starts immediately after the initial injury to prevent exsanguination and initiate clot formation. In the second phase, inflammatory cells are recruited to the site of injury to remove cellular debris and initiate cellular signaling cascades for further wound healing. During the proliferative phase, keratinocytes proliferate to close the wound while the myofibroblasts contract to decrease the wound size. Endothelial cells are simultaneously proliferating throughout all phases of wound healing to revascularize the damaged tissue. Last, over weeks to years, the extracellular matrix (ECM) remodels and forms a scar with a tensile strength close to 80% compared with normal uninjured skin.
Most wounds to the skin cause leakage of blood from damaged blood vessels and a clot forms to serve as a temporary shield to protect the denuded wound tissue and provide a provisional matrix through which cells can migrate during the reparative process. The clot consists of platelets embedded in a mesh of cross-linked fibrin fibers together with small amounts of plasma fibronectin, vitronectin, and thrombospondin. It also serves as a reservoir of cytokines and growth factors, such as platelet-derived growth factor, transforming growth factor-β (TGF-β), epidermal growth factor, and insulin-like growth factor-1, which are released by degranulating platelets. Owing to the high concentration of cytokines and growth factors, the clot also provides chemotactic cues to recruit circulating inflammatory cells to the wound site and initiates and stimulates angiogenesis. After hemostasis is achieved (within minutes), chemotactic signals released by degranulating platelets as well as byproducts of proteolysis of fibrin and other matrix components recruit neutrophils and monocytes to the wound site. Neutrophils arrive within minutes of injury at the wound site and serve to clear the initial rush of contaminating bacteria as well as provide a source of proinflammatory cytokines that serve as the earliest signals to activate local fibroblast and keratinocytes. In contrast, macrophages accumulate at the wound site several days after the initial injury and are essential for effective wound healing. The macrophages phagocytose any remaining pathogenic organisms and cellular debris. Once activated, macrophages also release a battery of growth factors and cytokines at the wound site, thus amplifying the earlier wound signals released by degranulating platelets and neutrophils. Therefore, these crucial M2 macrophages secrete growth factors and antiinflammatory cytokines to mediate the reparative process.
When ongoing injury has ceased, hemostasis has been achieved, and an immune response successfully set in place, the acute wound shifts toward tissue repair. The proliferative phase starts on the third day after wounding and lasts for approximately 2 weeks thereafter. This phase is characterized by fibroblast migration and deposition of newly synthesized ECM to replace the provisional fibrin network. Fibroblasts and myofibroblasts in the surrounding tissue are stimulated to proliferate for the first 3 days, after which these cells migrate into the wound. Once in the wound, these cells proliferate profusely and produce the matrix proteins fibronectin, hyaluronan, and proteoglycans. By the end of the first week, abundant ECM accumulates, which provides support for the wound and further supports cell migration. The fibroblasts then convert into myofibroblasts, which initiates wound contraction in the reparative process that helps to approximate the wound edges.
In the proliferative phase, reepithelialization through migration of keratinocytes also occurs and starts from the wound edges within a few hours of wounding. In the unwounded skin, the basal keratinocyte layer attaches to the basement membrane. These keratinocytes are anchored primarily to hemidesmosomes, which bind to laminin in the basement membrane by way of integrins and have intracellular links to the keratin–cytoskeletal network. During wound healing, the hemidesmosomes dissolve and the leading edge keratinocytes express new integrins that allow for the cells to migrate over the provisional wound matrix. In addition, the leading-edge keratinocytes also have the ability to dissolve the fibrin barrier ahead of them to allow for the migration. The chief fibrinolytic enzyme is plasmin, which is derived from plasminogen within the clot itself, and can be activated by either tissue-type plasminogen activator or urokinase-type plasminogen activator (uPA). Both of these activators and the receptor for uPA are upregulated in the migrating keratinocyte. Furthermore, various members of the matrix metalloproteinase (MMP) family are also upregulated by the wound edge keratinocytes. More specifically, MMP-9 can cut collagen type IV and VII and is thought to be responsible for releasing keratinocytes from the basement membrane. Additional MMPs have been identified in the wound that degrades native collagens and presumably aids keratinocyte crawling by cutting collagen type I and III at the sites of focal adhesion attachment to the dermis. Once the denuded wound surface has been covered by a monolayer of keratinocytes, epithelial migration ceases and a new stratified epidermis with underlying basal lamina is reestablished from the margins of the wound inward.
In the final phase of wound healing, the remodeling phase is responsible for the development of new epithelium and scar tissue formation, which can last for up to 1 to 2 years. In this phase, the ECM begins to mature. The collagen bundles increase in diameter and the tensile strength of the wound increases progressively to approximately 80% of the original strength compared with unwounded tissue. In addition to collagen deposition, the collagen fibers become more organized, which leads to increased contraction that initially began in the inflammatory phase. As the wound matures, the density of fibroblasts and macrophages is reduced by apoptosis. With time, the growth of the capillaries stops, blood flow to the area declines, and metabolic activity at the wound site decreases. The end result is a fully mature scar with a reduced number of cells, decreased blood vessels, and high tensile strength.
Epithelial stem cells: interfollicular epidermal stem cells and hair follicle stem cells
Epithelial stem cells, in general, fit a broader definition of adult tissue resident stem cells. Scores of epithelial stem cell populations have been identified based on both in vitro and in vivo methods and are believed to interact to accelerate skin repair and regeneration during injury. Epithelial stem cells are divided into 2 broad categories: interfollicular epidermal stem cells and hair follicle stem cells, each of which have their own distinct characteristics.
Interfollicular epidermal stem cells are located in the basal layer of the epidermis and are believed to have self-renewal capacity. Because epidermal renewal is continuous throughout life, it has been postulated that at least a portion of the epidermal basal cells behave like stem cells. After injury, these epidermal stem cells undergo a limited number of replications before undergoing differentiation. Thus, these cells are unipotent and differentiate to form the mature epidermis in adult skin.
In contrast, hair follicle stem cells isolated from the bulge region have been shown to have the potential to regenerate hair follicles as well as contribute to epidermal regeneration in response to wounding. These bulge stem cells are located in the deepest portion of the permanent hair follicle and represent a promising source of multipotent stem cells. Human stem cells extracted from the bulge region of the hair follicle have been induced to form epidermal and sebaceous cells and have the potential to differentiate toward other hair follicle components in vitro. In response to injury, these bulge stem cells can be rapidly mobilized and migrate to repair damaged epidermis in vivo. Studies have also shown that defects in the hair follicle bulge lead to an acute delay in reepithelialization of cutaneous wounds.
Additional epidermal stem cells have been identified in the isthmus of the hair follicle, which is the region between the bulge and the sebaceous gland. These isthmus stem cells have been shown to have the potential to differentiate into all epidermal cell lineages after transplantation. Recently, an additional population of hair follicle stem cells has been isolated from the isthmus that expressed Lgr6. These Lgr6-expressing stem cells have been shown to give rise to all epidermal lineages after transplantation and contribute to the homeostasis of the isthmus region and sebaceous gland.
Clinically, transplantation of cultured epithelial cells containing epidermal stem cells and keratinocytes, either in cell suspension or as cell sheets, has been well-established for the treatment of extensive and deep wounds, particularly when using fibrin matrices as vehicles to facilitate the transplantation of epidermal stem cells. The use of cultured epithelial allografts to treat chronic skin ulcers or deep dermal burns has also been reported. Meanwhile, there is increasing evidence to suggest immune privilege of skin–dermal stem cells. For instance, it was reported that hair follicles were produced surprisingly without inducing immunogenic rejection after transplantation of allogenic follicle dermal sheath cells. Considering their capacity to regenerate the epidermis in a relatively simple manner, the epidermal stem cells may become an important source for novel therapeutic approaches in the management of wounds. In the future, clinical trials will be necessary to determine the safety and efficacy of these cells for skin regeneration and repair.
Mesenchymal stem cells
In addition to epidermal stem cells, our current understanding and development of stem cell therapy for the treatment of wounds has been through the study of mesenchymal stem cells (MSCs). MSCs have been isolated not only from bone marrow aspirates but also from adipose tissue and umbilical cord blood. Their role in wound closure has been well-documented through the years and their potential use in tissue engineering and skin regeneration has gained significant interest in recent years. MSCs have been shown to traffic to areas of injury and secrete cytokines, chemokines, and growth factors to stimulate repair in a paracrine fashion ( Fig. 2 ). These factors have been shown to immunomodulate the inflammatory process, recruit various cell types for neovascularization, and stimulate proliferation during this regenerative process (see Fig. 2 ). Furthermore, studies have demonstrated that MSCs are capable of self-renewal and differentiation into various lineages, including osteogenic, adipogenic, and chrondrogenic lineages, to replace the damaged tissue.
Bone Marrow
Bone marrow-derived MSCs (BMSCs) are isolated from bone marrow aspirates and are an attractive source of stem cells owing to their easy accessibility, expandable capacity in vitro, and low immunogenic potential. Little to no rejection has been observed after transplantation, when allogenic MSCs were administered systematically. Therefore, the application of BMSCs to facilitate wound repair has been of particular interest. Studies have shown that distant BMSCs contribute to the reconstruction of the dermal fibroblast population in cutaneous wounds in a chimeric mouse model. Moreover, BMSCs have been shown to express higher amounts of collagen, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF), compared with native dermal fibroblasts, indicating their potential use in accelerating the healing process. Furthermore, BMSCs were found to improve cutaneous healing by accelerating reepithelialization, increasing angiogenesis, as well as directly differentiating into epithelial cells expressing keratinocyte specific marker. These findings support the use of BMSCs to assist in the reparative process.
Emerging preclinical evidence also supports the therapeutic application of BMSCs. Injection of human BMSCs into the incisional wound led to improved wound closure with better tensile strength and significant reduction in scar formation in rabbits. Murine BMSCs applied to full-thickness excisional wounds exhibited an equivalent ability as control fibroblast to migrate through the tissue and to attenuate the local inflammatory response, thereby facilitating cutaneous healing in mice. Application of a fibrin polymer spray with cultured autologous BMSCs aided in the healing of acute and nonhealing wounds in both human and mice. This approach might represent a practical method to introduce cells to the wound and might eventually facilitate the clinical use of MSC-based therapies.
The application of BMSCs in cutaneous healing has been explored in the management of various clinical conditions as well. Studies have shown that the application of BMSCs impregnated in collagen matrices onto chronic leg ulcers that resisted conventional treatment for more than 1 year responded to BMSC treatment with reduced wound size, increased vascularity of the dermis, and increased dermal thickness of the wound bed. Moreover, application of autologous BMSCs together with skin fibroblast on biodegradable collagen on the wound edges of patients with diabetic foot ulcers improved healing. Promising results were reported in a case series that demonstrated the efficacy of transplanting BMSCs into lower extremity wounds. Together, the data demonstrated that topical application and peripheral injection of BMSCs into the wound were both safe and efficacious in healing wounds.
Adipose Tissue
Adipose tissue has been identified as another source for multipotent stem cells, with characteristics resembling those of BMSCs. The adipose-derived stem cells (ASCs) are capable of differentiating into cells of adipogenic, chondrogenic, myogenic, and osteogenic lineages in response to specific stimuli. ASCs can be isolated from liposuction aspirates or excised fat, which are obtained more easily in adequate quantities with minimal patient discomfort and negligible donor site morbidity as compared with bone marrow procurement. The application of ASCs in wound repair and tissue regeneration has been shown in a number of experimental models both in vitro and in vivo. Application of ASCs significantly accelerated the reepithelialization of cutaneous wounds by promoting human dermal fibroblast proliferation through direct cell–cell contact or through paracrine secretion of a variety of growth factors, including bFGF and TGF-β. Furthermore, these ASCs differentiate into adipocytes to provide a supportive architecture for dermal regeneration and reepithelialization.
In addition to the direct effects of administering ASCs directly into the wounds, the application of ASCs in tissue engineered constructs for the production of a new skin substitute has also been proposed. The application of autologous ASCs onto an atelocollagen matrix accelerated wound healing of diabetic ulcers in impaired diabetic mice. Topical application of human ASCs seeded on a silk fibrin–chitosan scaffold also showed improvement in wound repair, and these cells were shown to differentiate and contribute to fibrovascular, endothelial, and epithelial components of the reconstituted tissue. In addition to the promising results obtained in preclinical studies, ASCs were reported to improve tissue hydration and new vessel formation during the treatment of radiation-induced tissue damage in clinical studies. Although the mechanism of healing has not been understood completely, ASCs have been speculated to contribute to wound repair and tissue regeneration by actively secreting growth factors, such as VEGF and hepatocyte growth factor, and thus promote subsequent angiogenesis and proliferation of keratinocytes or dermal fibroblasts. These results together also suggest that ASCs are a viable option for wound repair and tissue engineering.
Umbilical Cord Derived
Umbilical cord blood has been proposed to be the largest untouched source of stem cells with naïve immune status. Stem cells isolated from umbilical cord blood have been shown to differentiate into epithelial cells in vitro. A recent in vivo study further confirmed that umbilical cord blood could improve obstinate skin wound healing by accelerating wound closure. The authors showed that these MSCs derived from umbilical cord blood were able to differentiate into keratinocytes in the wound bed. Thus, these stem cells isolated from umbilical cord blood could be used as a starting material for the expansion of cells needed for the treatment of large skin defects. However, although the studies have been promising, it should be noted that transplantation or transfusion of umbilical cord blood–derived stem cells may lead to immunologic rejection owing to their allogenic nature and thus their immunogenicity must be evaluated carefully before their use in clinical applications.
In addition to umbilical cord blood being used as a source of stem cells, different layers of the umbilical cord tissue could serve as a source of stem cells. The stem cells isolated from the umbilical cord have been termed umbilical cord MSCs (UC-MSCs). In general, UC-MSCs exhibited similar immunophenotypic and functional characteristics of BMSCs. Therefore, UC-MSCs are considered as a promising alternative to BMSCs in tissue repair and regeneration. Both in vitro and in vivo studies have demonstrated their potential to differentiate into epidermal tissue, and these studies suggest the potential clinical application of UC-MSCs for epithelial reconstitution in wound healing. Several ongoing clinical trials are underway currently to evaluate the application of these cells in the treatment of partial and full thickness burns, and chronic diabetic wounds. Immunologic rejection has not been observed in these trials and confirms the safety of amniotic stem cells and attenuates the concerns regarding their neoplastic potential in clinical applications. However, there remain questions regarding the effect of donor selection and culture condition on clinical outcomes, long-term therapeutic effect, and product consistency that require additional investigation before their use in the clinic.
Muscle Satellite Cells
Severe burns have the ability to damage structures that extend deep to the adipose tissue into the skeletal muscle. Interestingly, the damaged skeletal muscles have been shown to have the capacity to regenerate. Adult skeletal muscle is a stable tissue under normal circumstances, and has remarkable ability to repair itself after injury. Skeletal muscle regeneration is a highly orchestrated process involving the activation of various cellular and molecular responses. As a skeletal muscle stem cell, muscle satellite cells play an indispensable role in in the regeneration of skeletal muscle through the activation of various cellular and molecular responses. These satellite stem cells not only maintain the stem cell population through self-renewal, but also provide numerous myogenic cells that reconstitute a functional contractile apparatus. Studies demonstrate that these satellite cells express high levels of Pax3 and Pax7, and mutations in the Pax3 and Pax7 genes impair muscle regeneration. Animal studies have supported the importance of Pax7 in normal muscle development, as a mutation in Pax7 results in the loss of satellite cells in the postnatal period.
Interestingly, studies have revealed recently that satellite cells exhibit heterogeneity with respect to their cell fate potential and have the intrinsic potential to differentiate into multiple mesenchymal lineages. When cultured on solubilized basement membrane matrix (Matrigel), satellite cells from single myofibers spontaneously differentiated into myocytes, adipocytes, and osteocytes. These findings indicate that satellite cells may functionally resemble BMSCs. This notion is substantiated by other studies wherein satellite cells cultured in oxygen-rich culture conditions were found to assume mesenchymal lineage cells. Clonal analysis revealed that myogenic and adipogenic satellite cells have 2 separate populations in the satellite cell compartment. Both populations express the myogenic marker Pax7 and adipogenic markers, including peroxisome proliferator-activated receptors gamma and CCAAT/enhancer binding proteins. These results indicate that muscle satellite cells may represent a heterogeneous population of stem cells that have the potential to not only regenerate damaged skeletal muscle, but it highlights the possibility of these cells to develop into other mesenchymal lineage cells. Future advances in systems biology and bioengineering will be necessary to develop therapeutic approaches to regenerate loss skeletal muscle and other deeper structures.
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