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Stem cells in cutaneous wound healing
Clinics in Dermatology (2007) 25, 73 – 78
Stem cells in cutaneous wound healingB Jisun Cha, MDc, Vincent Falanga, MDa,b,c,* a
Department of Dermatology, Boston University School of Medicine Boston, MA, USA Department of Biochemistry. Boston University School of Medicine Boston, MA, USA c Department of Dermatology, Roger Williams Medical Center Providence, RI, USA b
Abstract Treatment of chronic wounds remains difficult, in spite of better understanding of pathophysiologic principles and greater adherence to recognized standards of care. Even with recent advances stemming from breakthroughs in recombinant growth factors and bioengineered skin, up to almost 50% of chronic wounds that have been present for more than a year remain resistant to treatment. Because of these realities, there is excitement in the use of stem cells to offset impaired healing. Early data appear encouraging, but much work remains to be done. Although pilot studies suggest that multipotent adult stem cells can accelerate wound repair or even reconstitute the wound bed, the answers will need to come from randomized clinical trials. Thus far, considerable focus has been placed on bone marrow–derived mesenchymal stem cells, and there are now promising approaches for introducing them into the wound. It might turn out, however, that other types of stem cells will be more effective, including those derived from hair follicles or, perhaps, subsets of bone marrow–derived cultured cells. Still, proper wound care and adherence to basic principles cannot be bypassed, even by the most sophisticated approaches. D 2007 Elsevier Inc. All rights reserved.
Introduction The overall therapy for nonhealing wounds has largely focused on the identification and correction of the precipitating and perpetuating factors. This approach includes antibiotic use for accompanying cellulitis, revascularization of ischemic limbs, rigorous off-loading for decubitus (pressure) ulcers, and compression devices for venous ulcers. Appropriate wound care is critical, and various treatment modalities are used to improve the wound bed. Debridement of the necrotic tissue, removal of edema fluid, B This study was supported by NIH, grants AR46557 and DK067836, and the Wound Biotechnology Foundation * Corresponding author. Department of Dermatology and Skin Surgery, Roger Williams Medical Center, Providence, RI 02908, USA. Tel.: +1 401 456 2521; fax: +1 401 456 6449. E-mail address: [email protected] (V. Falanga).
0738-081X/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.clindermatol.2006.10.002
decreasing the bacterial burden, and providing the right balance of moisture to the wound bed are all required components of wound care. Many ulcers heal when one is able to implement these steps, although there are still chronic wounds that either do not heal at all or do so very slowly. In addition, there is also a need to accelerate the healing of acute wounds, which would generally heal within a defined time frame. Therefore, in recent years, there have been efforts to develop more advanced treatment modalities. Topically applied growth factors, in particular, plateletderived growth factor and cell-based therapies have come of age. Some of these treatment modalities are either in the development phase, available as off-label use or actually approved by the Food and Drug Administration for specific indications. Some of these therapies are outlined in Table 1. Platelet-derived growth factor–BB is approved for use in the treatment of diabetic neuropathic ulcers of the foot. As for
74 Table 1
J. Cha, V. Falanga Representative skin substitutes
Biobrane [Bertek Pharmaceuticals; Morgantown, WV]: acellular dressing, collagen bound to nylon fabric Epicel [Genzyme; Cambridge, MA]: autologous epidermal graft AlloDerm [LifeCell Corporation in Palo Alto, CA]: accellular, allogeneic dermal graft Integra Artificial Skin [Integra Lifesciences; Plainsboro, NJ]: bovine collagen and chondroitin-6-sulfate Oasis [Cook Biotech, Inc. West Lafayette, IN]: pig intestinal mucosa TranCyte [Advanced Tissue Sciences; La Jolla, CA]: devitalized fibroblasts on nylon mesh Dermagraft [Smith & Nephew, La Jolla, CA]: human fibroblasts in an absorbable matrix (approved in United States for diabetic ulcers) Orcel [Ortec International, Inc. New York, NY]: human fibroblasts and keratinocytes in a bovine collagen sponge Apligraf [Novartis; East Hanover, NJ]: human fibroblasts and keratinocytes in a bovine collagen matrix (approved in United States for venous and diabetic ulcers)
bioengineered skin, a bilayered living skin construct has been approved for venous and diabetic ulcers. A living dermal skin equivalent was also approved for diabetic neuropathic ulcers. Taken together, one can state that in the last 10 years or so, tremendous progress has been made in chronic wounds, where the mainstay of treatment has largely relied on improvements in dressings and compression bandages. In addition to standard and more advanced treatments, other less commonly used and unusual therapeutic products, not necessarily new, are being used by some clinicians.1 For example, cadaver skin is such an alternative; it is a true allograft and is always eventually rejected by the recipient. Skin that lacks a dermis is less able to resist trauma and is prone to contraction, resulting in a poor functional and cosmetic outcome. All currently available examples of artificial dermis lack a vascular plexus for the nourishment of the epidermis and require host vasculogenesis into the dermis graft to supply nourishment to the grafted epidermis.1 Other strategies for treatment of chronic non–healing cutaneous wounds include temporary substitutes such as porcine xenografts, synthetic membranes, and autologous and allogeneic epidermal substitutes.2 Recent artificial dermal substitutes are structurally optimized to incorporate the surrounding tissue and to allow cell invasion by fibroblasts and capillaries for subsequent dermal remodeling.3 Although topically applied platelet-derived growth factor–BB remains the only recombinant growth factor approved for wound healing in the United States, there are several reports that other cytokines or growth factors are critical to wound healing.4 These growth factors play major roles in local inflammation, reepithelialization, granulation tissue formation, neovascularization, and extracellular matrix production from various cell sources and through diverse mechanisms.5 There have been extensive investigations into wound healing by the exogenous application
of various growth factors.6 There have been reports that cultured cells such as dermal fibroblasts from chronic cutaneous wounds do not respond to the certain cytokinelike transforming growth factor–b, which is critical in the healing process.7 Presently, several technologies are under active development to aid cutaneous wound repair.8 It is becoming increasingly clear, however, that more radical steps need to be taken to propel the treatment of chronic wounds in a direction that allows greater effectiveness. For example, in spite of advances in the treatment of chronic wounds with bioengineered skin, there remain almost 50% of patients who do not heal when their ulcers have been previously resistant to conventional therapy.9
Stem cells Stem cells are characterized by their prolonged selfrenewal capacity and by their asymmetric replication. Asymmetric replication describes a special property of stem cells: with every cell division, one of the cells retains its self-renewing capacity, whereas the other enters a differentiation pathway and joins a mature nondividing population.10 Stem cells were first identified as pluripotent cells in embryos, and these were called embryonic stem (ES) cells. It is now clear that stem cells are also present in many, if not all, tissues in adult animals and contribute to the maintenance of tissue renewal and homeostasis.
Embryonic stem cells The early embryo contains totipotent ES cells, which can give rise to all the tissues of the human body. Such cells can be isolated from normal blastocysts—the structures formed at about the 32-cell stage during embryonic development.11 Embryonic stem cells can be maintained in culture as undifferentiated cell lines or induced to differentiate into many different lineages.12,13 Embryonic stem cells have had an enormous impact on biology and medicine. They have been used to study the specific signals and differentiation steps required for the development of many tissues. They have made possible the production of knockout mice. To produce knockout mice, a specific gene is inactivated or deleted from cultured ES cells. These cells are then injected into blastocysts, which are implanted into the uterus of a surrogate mother. The genetically modified implanted blastocysts develop into full embryos. In spite of their tremendous potential, however, ES cells are the subject of considerable controversy. For one, there are ethical concerns and issues regarding the possibility that the use of human ES cells could lead to the almost industrial production of human embryos for the sole purpose of obtaining their cells. There are new data suggesting that ES cells can be obtaining without destroying the embryo.14 It is unclear whether this approach would definitely remove the ethical issues and whether enough ES cells could be generated this way. Concerns about human cloning have also played a major role in this controversy.
Stem cells in cutaneous wound healing Finally, ES cells may not be safe, and neoplasia could be stimulated by the introduction of these totipotent cells.
Adult stem cells It is the ethical concerns, however, that have played the major role in the inability of obtain fresh supplies of ES cells. This problem has stimulated investigators to explore the capabilities of adult stem cells to contribute to remodeling of diverse tissues and organs. Bone marrow stem cells have been a favored target of investigation. The ability of bone marrow–derived stem cells to contribute to a number of different tissues has been demonstrated in vitro and in vivo in animal models and, more recently, in human transplantation chimeras.15 Tissue damage caused by different mechanisms is believed to be responsible for the homing of stem cells and their differentiation into specific tissue-related cells.16 Many tissues in adult animals have been shown to contain reservoirs of stem cells, which are called badult stem cells.Q Compared to ES cells, adult stem cells have a more restricted differentiation capacity and are usually lineage-specific. The adult bone marrow harbors a heterogeneous population of stem cells, which appear to have very broad developmental capabilities.17 These cells, called bmultipotent adult progenitor cells,Q have been isolated from postnatal human and rodent bone marrow. They proliferate in culture without senescence and can differentiate into mesodermal, endodermal, and neuroectodermal cell types. Interestingly, multipotent adult progenitor cells are not confined to the bone marrow. They have been isolated from muscle, brain, and skin and can be made to differentiate into endothelium, neurons, hepatocytes, and other cell types.17,18 It is not known whether a single type of adult bone marrow stem cell is capable of generating all tissue lineages or if, alternatively, there are multiple types of bone marrow stem cells, each committed to differentiate into a specific tissue or a group of related tissues.19 In addition to bone marrow cells that may migrate to various tissues after injury, adult stem cells reside permanently in most organs. These cells (known as tissue stem cells) can generate the mature cells of the organs in which they reside. Their differentiation commitment, however, can change when they are transplanted into a different tissue. Stem cells located outside of the bone marrow are generally referred to as btissue stem cells.Q Tissue stem cells are located in sites called niches, which differ among various tissues. For instance, in the gastrointestinal tract, they are located at the isthmus of stomach glands and at the base of the crypts of the colon. Niches have been identified in the bulge area of hair follicles and the limbus of the cornea. A change in stem cell differentiation from one cell type to another is called transdifferentiation, and the multiplicity of stem cell differentiation options is known as developmental plasticity.19,20 The relative contribution of true transdifferentiation or cell fusion to the development of various mature cell types from hematopoi-
75 etic stem cells is unclear at present. Human umbilical cord blood has also been explored as an alternative source of stem cells to repopulate the bone marrow in the treatment of diseases in children and adults.21-23 Because of the ease with which they are obtained, human mesenchymal stem cells (hMSCs) derived from bone marrow have received considerable attention. These cells are self-renewing and are capable of differentiating into multiple cells and tissues.24 A single bone marrow–derived stem cell is able to differentiate into epithelial cells of the liver, lung, gastrointestinal tract, and skin.25 It has been shown that long-term repopulation by green fluorescent protein–labeled bone marrow–derived cell transplantation in wounded skin results in differentiation into nonhematopoietic skin structures.26 That type of analysis, however, needs confirmation and further studies, as the amount of newly developed green fluorescent protein+ structures was certainly not dramatic. Mixture of green fluorescent protein–labeled bone marrow cells and embryonic skin cells successfully differentiated into epidermal keratinocytes, sebaceous glands, follicular epithelial cells, and dendritic cells.27 Human stem cells may offer considerable opportunities for providing differentiated cells for gene therapy, drug discovery, and regenerative medicine.28 Stem cells could be harvested and transduced ex vivo and the corrected cells reintroduced into the host. Growth advantages of the corrected stem cells could offer new therapeutic approaches for genetic diseases. In essence, there are at least 4 different ways of using mesenchymal stem cells (MSCs). These include local implantation of MSCs for localized diseases, systemic transplantation, combining stem cell therapy with gene therapy, and the use of MSCs in tissue engineering protocols. Clinical trials have shown promising results with the administration of MSCs for osteogenesis imperfecta,29,30 HurlerTs syndrome, and methachromatic leukodystrophy31 and to enhance engraftment of heterologous bone marrow transplantation.32
Possible mechanisms of action Immediately after tissue injury and formation of a fibrin clot, the bone marrow is known to contribute inflammatory cells, such as granulocytes and monocytes, to initiate the process of wound repair. Monocytes, later macrophages, are particularly important.33 Most inflammatory cells in the wound are presumed to either undergo apoptosis or migrate back into the circulation once the wound-derived chemotactic signal has subsided. Subpopulation of bone marrow– derived cells integrate into the healed wound as antigenpresenting dendritic cells, often termed skin-associated lymphoid tissue and fibrocytes.34-36 These cells were defined as bone–marrow derived cells because they expressed the cell-surface marker CD45.37 It has been known for several decades that the epidermis of the skin contains a subpopulation of basal cells that
76 exhibit the properties expected of somatic stem cells: slow cell cycle, high proliferative potential, location in a protected niche, capacity to maintain and repair the tissue in which they reside, and long life span.38-45 Slowly, cycling epidermal stem cells have been identified by long-term nuclear retention of tritiated thymidine or bromodeoxyuridine label.39,40,46 These undifferentiated label-retaining stem cells have been shown to reside in the bulge area of the hair follicle,43,47,48 and in the interfollicular basal layer of the epidermis.39,40,49 They are self-renewing and able to produce daughter transient amplifying cells that undergo a finite number of cell divisions before they differentiate and leave the proliferative basal compartment, a property similar to stem cells in other continuously renewing tissues.42,50 The label-retaining stem cells from the skin express K14,51 a marker for basal epidermal cells, and do not express K1,52 a marker for differentiated epidermal cells. This putative stem cell population does not express CD34 or Sca-1 and typical hematopoietic markers and shows slow cell proliferation, long-term in vitro growth, long-term expression of recombinant genes, and complete recapitulation of the epidermis with long-term maintenance of the engineered tissue, whereas the transient amplifying population does not exhibit any of these characteristics.53,54 In normal skin, bone marrow–derived cells achieve steady-state presence in the dermis and are responsible for the production of collagen type III in skin. Collagen type I is the predominant collagen in normal human skin and exceeds collagen type III by a ratio of 4 to 1. During wound healing, this ratio decreases to 2:1 because of an early increase in the deposition of collagen type III.55 Fathke et al37 showed that the bone marrow–derived cells were able to contract a collagen matrix and transcribe collagen types I and III, whereas the skin-resident cells transcribed only collagen type I. A wound stimulus prompts upward migration of marked bulge cells, whereas mesenchymal epithelial interactions at the start of the hair cycle stimulate downward movement.56 - 58 When cultured in vitro, bulge keratinocytes yield larger colonies than those from other skin sites.57-60 Like all organs, the skin has a complex tissue architecture and is composed of a large variety of cell types of ectodermal and mesodermal origins. Most of these cells are produced from corresponding progenitor/stem cells by tightly regulated mechanisms. A subpopulation of cells located in the bulge regions of hair follicles has been shown to give rise to epidermal keratinocytes as well as hair follicles.56,60 - 62 Adult epithelial stem cells have a number of distinguishing features, and hair follicle stem cells have additional properties specific for this unique organ. As mentioned earlier, all stem cells undergo asymmetric cell division to generate daughter cells that retain the stem cell phenotype, as well as daughter transient amplifying cells that proliferate and differentiate to replenish cells lost to the environment after terminal differentiation. Stem cells are generally slow-cycling in vivo, but they have a high proliferative potential during tissue expansion such as fetal
J. Cha, V. Falanga development and wound healing.47,56,63,64 The location of stem cells is in specific niches and discrete locations, generally tightly adherent to the basement membrane in well-protected areas.45,65 Hair follicle stem cells also have the capacity to respond to stress such as wounding or phorbol esters.66 Finally, they appear to be multipotent, giving rise to lower follicle components as well as sebaceous gland and epidermis.58,60 Two of the most important features of epithelial stem cells are their seemingly unlimited capacity for selfrenewal and their slow-cycling nature. In the epidermis, self-renewal has been characterized by in vitro clonogenicity or colony-forming efficiency.67,68 Although stem cells are considered to progress through the cell cycle at a slower rate than transient amplifying basal cells, they have a higher proliferative potential, and it is thought that they increase their proliferative rate at times of tissue regeneration, such as during fetal development and wound healing.69
Stem cells and wound healing studies A recent review of this subject has been published with respect to cutaneous wound healing.70 Enriched populations of hematopoietic stem cells homing to bone marrow within 48 hours could incorporate and function in a variety of tissues, including stomach, kidney, lung, and skin.25 These cells persisted for 11 months after grafting; they were identified as male cells in a female host. Such long-term persistence of grafted male cells in females has been previously reported,71 as well as the persistence for 27 years of male cells in the blood of females who had given birth to male offspring.72 Such data suggest that stem cells may exist in all tissues, that these stem cells may have the capacity to differentiate into a variety of tissues,73-75 and have a very long life. It has been proposed that individual stem cells might persist throughout the lifetime of an organism.42,76 Borue et al77 showed that lethally irradiated female mice that have been reconstituted with male bone marrow cells and subsequently wounded express a significant number of Y chromosome–positive epithelial cells in healing wounds. This expression is apparent within days after wounding and increases over the next 21 days, with the greatest increase seen between days 3 and 5. This increase occurs just after a peak of mitotic activity of the bone marrow–derived epithelial cells immediately adjacent to the wound.77 In another study, Han et al78 showed the potential of human bone marrow stromal cells to accelerate wound healing in vitro by measuring the amount of collagen synthesis and the levels of basic fibroblast growth factor and vascular endothelial growth factor. The levels of these growth factors were much higher in tissue culture of the bone marrow stromal cells group compared to the fibroblast group. This interaction of stem cells with growth factors, or their
Stem cells in cutaneous wound healing possible interdependence, is interesting and may require further work. Nakagawa et al24 suggested that hMSCs, together with basic fibroblast growth factor in a skin defect model, accelerates wound healing and showed that the hMSCs transdifferentiated into the epithelium in rats. Ichioka et al79 showed that the addition of bone marrow significantly increases the collagen matrix to induce wound healing angiogenesis especially in the early stage of the repair process. In a human study of chronic nonhealing wounds, our group showed that directly applied bone marrow–derived cells can lead to wound closure and possible rebuilding of tissues.80 That particular study, although interesting, was hampered by the lack of an efficient delivery system for the autologous bone marrow– derived cultured cells. More recently, Falanga et al81 have fully characterized the human autologous bone marrow– derived cultured cells as being MSCs (hMSCs) and have applied it to nonhealing and acute wounds, using a specialized fibrin spray system. This approach appears to be safe and may represent a rather ideal way of introducing cells, and not just stem cells (perhaps even soluble mediators), into injury sites. Of particular interest is that the wounds treated with the hMSCs developed new elastic fibers. Thus, there are indications that some degree of dermal reconstitution is being achieved.
Conclusions Chronic wounds remain a formidable challenge. In spite of recent advances from breakthroughs in recombinant growth factors and bioengineered skin, up to 50% of chronic wounds that have been present for more than a year remain resistant to treatment. Stem cells offer the possibility that some structures within the wound may be reconstituted. To what extent this will be possible remains uncertain, and randomized clinical studies will be needed to determine the effectiveness of the approach. Because of the relative ease with which they are cultured, bone marrow–derived mesenchymal stem cells have been tested in pilot studies, and there are now promising approaches for introducing them into the wound. It might turn out, however, that other types of stem cells will be more effective, including those derived from hair follicles, or perhaps subsets of bone marrow–derived cultured cells. Still, proper wound care and adherence to basic principles cannot be bypassed, even by the most sophisticated approaches.
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Stem cells in cutaneous wound healingB Jisun Cha, MDc, Vincent Falanga, MDa,b,c,* a
Department of Dermatology, Boston University School of Medicine Boston, MA, USA Department of Biochemistry. Boston University School of Medicine Boston, MA, USA c Department of Dermatology, Roger Williams Medical Center Providence, RI, USA b
Abstract Treatment of chronic wounds remains difficult, in spite of better understanding of pathophysiologic principles and greater adherence to recognized standards of care. Even with recent advances stemming from breakthroughs in recombinant growth factors and bioengineered skin, up to almost 50% of chronic wounds that have been present for more than a year remain resistant to treatment. Because of these realities, there is excitement in the use of stem cells to offset impaired healing. Early data appear encouraging, but much work remains to be done. Although pilot studies suggest that multipotent adult stem cells can accelerate wound repair or even reconstitute the wound bed, the answers will need to come from randomized clinical trials. Thus far, considerable focus has been placed on bone marrow–derived mesenchymal stem cells, and there are now promising approaches for introducing them into the wound. It might turn out, however, that other types of stem cells will be more effective, including those derived from hair follicles or, perhaps, subsets of bone marrow–derived cultured cells. Still, proper wound care and adherence to basic principles cannot be bypassed, even by the most sophisticated approaches. D 2007 Elsevier Inc. All rights reserved.
Introduction The overall therapy for nonhealing wounds has largely focused on the identification and correction of the precipitating and perpetuating factors. This approach includes antibiotic use for accompanying cellulitis, revascularization of ischemic limbs, rigorous off-loading for decubitus (pressure) ulcers, and compression devices for venous ulcers. Appropriate wound care is critical, and various treatment modalities are used to improve the wound bed. Debridement of the necrotic tissue, removal of edema fluid, B This study was supported by NIH, grants AR46557 and DK067836, and the Wound Biotechnology Foundation * Corresponding author. Department of Dermatology and Skin Surgery, Roger Williams Medical Center, Providence, RI 02908, USA. Tel.: +1 401 456 2521; fax: +1 401 456 6449. E-mail address: [email protected] (V. Falanga).
0738-081X/$ – see front matter D 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.clindermatol.2006.10.002
decreasing the bacterial burden, and providing the right balance of moisture to the wound bed are all required components of wound care. Many ulcers heal when one is able to implement these steps, although there are still chronic wounds that either do not heal at all or do so very slowly. In addition, there is also a need to accelerate the healing of acute wounds, which would generally heal within a defined time frame. Therefore, in recent years, there have been efforts to develop more advanced treatment modalities. Topically applied growth factors, in particular, plateletderived growth factor and cell-based therapies have come of age. Some of these treatment modalities are either in the development phase, available as off-label use or actually approved by the Food and Drug Administration for specific indications. Some of these therapies are outlined in Table 1. Platelet-derived growth factor–BB is approved for use in the treatment of diabetic neuropathic ulcers of the foot. As for
74 Table 1
J. Cha, V. Falanga Representative skin substitutes
Biobrane [Bertek Pharmaceuticals; Morgantown, WV]: acellular dressing, collagen bound to nylon fabric Epicel [Genzyme; Cambridge, MA]: autologous epidermal graft AlloDerm [LifeCell Corporation in Palo Alto, CA]: accellular, allogeneic dermal graft Integra Artificial Skin [Integra Lifesciences; Plainsboro, NJ]: bovine collagen and chondroitin-6-sulfate Oasis [Cook Biotech, Inc. West Lafayette, IN]: pig intestinal mucosa TranCyte [Advanced Tissue Sciences; La Jolla, CA]: devitalized fibroblasts on nylon mesh Dermagraft [Smith & Nephew, La Jolla, CA]: human fibroblasts in an absorbable matrix (approved in United States for diabetic ulcers) Orcel [Ortec International, Inc. New York, NY]: human fibroblasts and keratinocytes in a bovine collagen sponge Apligraf [Novartis; East Hanover, NJ]: human fibroblasts and keratinocytes in a bovine collagen matrix (approved in United States for venous and diabetic ulcers)
bioengineered skin, a bilayered living skin construct has been approved for venous and diabetic ulcers. A living dermal skin equivalent was also approved for diabetic neuropathic ulcers. Taken together, one can state that in the last 10 years or so, tremendous progress has been made in chronic wounds, where the mainstay of treatment has largely relied on improvements in dressings and compression bandages. In addition to standard and more advanced treatments, other less commonly used and unusual therapeutic products, not necessarily new, are being used by some clinicians.1 For example, cadaver skin is such an alternative; it is a true allograft and is always eventually rejected by the recipient. Skin that lacks a dermis is less able to resist trauma and is prone to contraction, resulting in a poor functional and cosmetic outcome. All currently available examples of artificial dermis lack a vascular plexus for the nourishment of the epidermis and require host vasculogenesis into the dermis graft to supply nourishment to the grafted epidermis.1 Other strategies for treatment of chronic non–healing cutaneous wounds include temporary substitutes such as porcine xenografts, synthetic membranes, and autologous and allogeneic epidermal substitutes.2 Recent artificial dermal substitutes are structurally optimized to incorporate the surrounding tissue and to allow cell invasion by fibroblasts and capillaries for subsequent dermal remodeling.3 Although topically applied platelet-derived growth factor–BB remains the only recombinant growth factor approved for wound healing in the United States, there are several reports that other cytokines or growth factors are critical to wound healing.4 These growth factors play major roles in local inflammation, reepithelialization, granulation tissue formation, neovascularization, and extracellular matrix production from various cell sources and through diverse mechanisms.5 There have been extensive investigations into wound healing by the exogenous application
of various growth factors.6 There have been reports that cultured cells such as dermal fibroblasts from chronic cutaneous wounds do not respond to the certain cytokinelike transforming growth factor–b, which is critical in the healing process.7 Presently, several technologies are under active development to aid cutaneous wound repair.8 It is becoming increasingly clear, however, that more radical steps need to be taken to propel the treatment of chronic wounds in a direction that allows greater effectiveness. For example, in spite of advances in the treatment of chronic wounds with bioengineered skin, there remain almost 50% of patients who do not heal when their ulcers have been previously resistant to conventional therapy.9
Stem cells Stem cells are characterized by their prolonged selfrenewal capacity and by their asymmetric replication. Asymmetric replication describes a special property of stem cells: with every cell division, one of the cells retains its self-renewing capacity, whereas the other enters a differentiation pathway and joins a mature nondividing population.10 Stem cells were first identified as pluripotent cells in embryos, and these were called embryonic stem (ES) cells. It is now clear that stem cells are also present in many, if not all, tissues in adult animals and contribute to the maintenance of tissue renewal and homeostasis.
Embryonic stem cells The early embryo contains totipotent ES cells, which can give rise to all the tissues of the human body. Such cells can be isolated from normal blastocysts—the structures formed at about the 32-cell stage during embryonic development.11 Embryonic stem cells can be maintained in culture as undifferentiated cell lines or induced to differentiate into many different lineages.12,13 Embryonic stem cells have had an enormous impact on biology and medicine. They have been used to study the specific signals and differentiation steps required for the development of many tissues. They have made possible the production of knockout mice. To produce knockout mice, a specific gene is inactivated or deleted from cultured ES cells. These cells are then injected into blastocysts, which are implanted into the uterus of a surrogate mother. The genetically modified implanted blastocysts develop into full embryos. In spite of their tremendous potential, however, ES cells are the subject of considerable controversy. For one, there are ethical concerns and issues regarding the possibility that the use of human ES cells could lead to the almost industrial production of human embryos for the sole purpose of obtaining their cells. There are new data suggesting that ES cells can be obtaining without destroying the embryo.14 It is unclear whether this approach would definitely remove the ethical issues and whether enough ES cells could be generated this way. Concerns about human cloning have also played a major role in this controversy.
Stem cells in cutaneous wound healing Finally, ES cells may not be safe, and neoplasia could be stimulated by the introduction of these totipotent cells.
Adult stem cells It is the ethical concerns, however, that have played the major role in the inability of obtain fresh supplies of ES cells. This problem has stimulated investigators to explore the capabilities of adult stem cells to contribute to remodeling of diverse tissues and organs. Bone marrow stem cells have been a favored target of investigation. The ability of bone marrow–derived stem cells to contribute to a number of different tissues has been demonstrated in vitro and in vivo in animal models and, more recently, in human transplantation chimeras.15 Tissue damage caused by different mechanisms is believed to be responsible for the homing of stem cells and their differentiation into specific tissue-related cells.16 Many tissues in adult animals have been shown to contain reservoirs of stem cells, which are called badult stem cells.Q Compared to ES cells, adult stem cells have a more restricted differentiation capacity and are usually lineage-specific. The adult bone marrow harbors a heterogeneous population of stem cells, which appear to have very broad developmental capabilities.17 These cells, called bmultipotent adult progenitor cells,Q have been isolated from postnatal human and rodent bone marrow. They proliferate in culture without senescence and can differentiate into mesodermal, endodermal, and neuroectodermal cell types. Interestingly, multipotent adult progenitor cells are not confined to the bone marrow. They have been isolated from muscle, brain, and skin and can be made to differentiate into endothelium, neurons, hepatocytes, and other cell types.17,18 It is not known whether a single type of adult bone marrow stem cell is capable of generating all tissue lineages or if, alternatively, there are multiple types of bone marrow stem cells, each committed to differentiate into a specific tissue or a group of related tissues.19 In addition to bone marrow cells that may migrate to various tissues after injury, adult stem cells reside permanently in most organs. These cells (known as tissue stem cells) can generate the mature cells of the organs in which they reside. Their differentiation commitment, however, can change when they are transplanted into a different tissue. Stem cells located outside of the bone marrow are generally referred to as btissue stem cells.Q Tissue stem cells are located in sites called niches, which differ among various tissues. For instance, in the gastrointestinal tract, they are located at the isthmus of stomach glands and at the base of the crypts of the colon. Niches have been identified in the bulge area of hair follicles and the limbus of the cornea. A change in stem cell differentiation from one cell type to another is called transdifferentiation, and the multiplicity of stem cell differentiation options is known as developmental plasticity.19,20 The relative contribution of true transdifferentiation or cell fusion to the development of various mature cell types from hematopoi-
75 etic stem cells is unclear at present. Human umbilical cord blood has also been explored as an alternative source of stem cells to repopulate the bone marrow in the treatment of diseases in children and adults.21-23 Because of the ease with which they are obtained, human mesenchymal stem cells (hMSCs) derived from bone marrow have received considerable attention. These cells are self-renewing and are capable of differentiating into multiple cells and tissues.24 A single bone marrow–derived stem cell is able to differentiate into epithelial cells of the liver, lung, gastrointestinal tract, and skin.25 It has been shown that long-term repopulation by green fluorescent protein–labeled bone marrow–derived cell transplantation in wounded skin results in differentiation into nonhematopoietic skin structures.26 That type of analysis, however, needs confirmation and further studies, as the amount of newly developed green fluorescent protein+ structures was certainly not dramatic. Mixture of green fluorescent protein–labeled bone marrow cells and embryonic skin cells successfully differentiated into epidermal keratinocytes, sebaceous glands, follicular epithelial cells, and dendritic cells.27 Human stem cells may offer considerable opportunities for providing differentiated cells for gene therapy, drug discovery, and regenerative medicine.28 Stem cells could be harvested and transduced ex vivo and the corrected cells reintroduced into the host. Growth advantages of the corrected stem cells could offer new therapeutic approaches for genetic diseases. In essence, there are at least 4 different ways of using mesenchymal stem cells (MSCs). These include local implantation of MSCs for localized diseases, systemic transplantation, combining stem cell therapy with gene therapy, and the use of MSCs in tissue engineering protocols. Clinical trials have shown promising results with the administration of MSCs for osteogenesis imperfecta,29,30 HurlerTs syndrome, and methachromatic leukodystrophy31 and to enhance engraftment of heterologous bone marrow transplantation.32
Possible mechanisms of action Immediately after tissue injury and formation of a fibrin clot, the bone marrow is known to contribute inflammatory cells, such as granulocytes and monocytes, to initiate the process of wound repair. Monocytes, later macrophages, are particularly important.33 Most inflammatory cells in the wound are presumed to either undergo apoptosis or migrate back into the circulation once the wound-derived chemotactic signal has subsided. Subpopulation of bone marrow– derived cells integrate into the healed wound as antigenpresenting dendritic cells, often termed skin-associated lymphoid tissue and fibrocytes.34-36 These cells were defined as bone–marrow derived cells because they expressed the cell-surface marker CD45.37 It has been known for several decades that the epidermis of the skin contains a subpopulation of basal cells that
76 exhibit the properties expected of somatic stem cells: slow cell cycle, high proliferative potential, location in a protected niche, capacity to maintain and repair the tissue in which they reside, and long life span.38-45 Slowly, cycling epidermal stem cells have been identified by long-term nuclear retention of tritiated thymidine or bromodeoxyuridine label.39,40,46 These undifferentiated label-retaining stem cells have been shown to reside in the bulge area of the hair follicle,43,47,48 and in the interfollicular basal layer of the epidermis.39,40,49 They are self-renewing and able to produce daughter transient amplifying cells that undergo a finite number of cell divisions before they differentiate and leave the proliferative basal compartment, a property similar to stem cells in other continuously renewing tissues.42,50 The label-retaining stem cells from the skin express K14,51 a marker for basal epidermal cells, and do not express K1,52 a marker for differentiated epidermal cells. This putative stem cell population does not express CD34 or Sca-1 and typical hematopoietic markers and shows slow cell proliferation, long-term in vitro growth, long-term expression of recombinant genes, and complete recapitulation of the epidermis with long-term maintenance of the engineered tissue, whereas the transient amplifying population does not exhibit any of these characteristics.53,54 In normal skin, bone marrow–derived cells achieve steady-state presence in the dermis and are responsible for the production of collagen type III in skin. Collagen type I is the predominant collagen in normal human skin and exceeds collagen type III by a ratio of 4 to 1. During wound healing, this ratio decreases to 2:1 because of an early increase in the deposition of collagen type III.55 Fathke et al37 showed that the bone marrow–derived cells were able to contract a collagen matrix and transcribe collagen types I and III, whereas the skin-resident cells transcribed only collagen type I. A wound stimulus prompts upward migration of marked bulge cells, whereas mesenchymal epithelial interactions at the start of the hair cycle stimulate downward movement.56 - 58 When cultured in vitro, bulge keratinocytes yield larger colonies than those from other skin sites.57-60 Like all organs, the skin has a complex tissue architecture and is composed of a large variety of cell types of ectodermal and mesodermal origins. Most of these cells are produced from corresponding progenitor/stem cells by tightly regulated mechanisms. A subpopulation of cells located in the bulge regions of hair follicles has been shown to give rise to epidermal keratinocytes as well as hair follicles.56,60 - 62 Adult epithelial stem cells have a number of distinguishing features, and hair follicle stem cells have additional properties specific for this unique organ. As mentioned earlier, all stem cells undergo asymmetric cell division to generate daughter cells that retain the stem cell phenotype, as well as daughter transient amplifying cells that proliferate and differentiate to replenish cells lost to the environment after terminal differentiation. Stem cells are generally slow-cycling in vivo, but they have a high proliferative potential during tissue expansion such as fetal
J. Cha, V. Falanga development and wound healing.47,56,63,64 The location of stem cells is in specific niches and discrete locations, generally tightly adherent to the basement membrane in well-protected areas.45,65 Hair follicle stem cells also have the capacity to respond to stress such as wounding or phorbol esters.66 Finally, they appear to be multipotent, giving rise to lower follicle components as well as sebaceous gland and epidermis.58,60 Two of the most important features of epithelial stem cells are their seemingly unlimited capacity for selfrenewal and their slow-cycling nature. In the epidermis, self-renewal has been characterized by in vitro clonogenicity or colony-forming efficiency.67,68 Although stem cells are considered to progress through the cell cycle at a slower rate than transient amplifying basal cells, they have a higher proliferative potential, and it is thought that they increase their proliferative rate at times of tissue regeneration, such as during fetal development and wound healing.69
Stem cells and wound healing studies A recent review of this subject has been published with respect to cutaneous wound healing.70 Enriched populations of hematopoietic stem cells homing to bone marrow within 48 hours could incorporate and function in a variety of tissues, including stomach, kidney, lung, and skin.25 These cells persisted for 11 months after grafting; they were identified as male cells in a female host. Such long-term persistence of grafted male cells in females has been previously reported,71 as well as the persistence for 27 years of male cells in the blood of females who had given birth to male offspring.72 Such data suggest that stem cells may exist in all tissues, that these stem cells may have the capacity to differentiate into a variety of tissues,73-75 and have a very long life. It has been proposed that individual stem cells might persist throughout the lifetime of an organism.42,76 Borue et al77 showed that lethally irradiated female mice that have been reconstituted with male bone marrow cells and subsequently wounded express a significant number of Y chromosome–positive epithelial cells in healing wounds. This expression is apparent within days after wounding and increases over the next 21 days, with the greatest increase seen between days 3 and 5. This increase occurs just after a peak of mitotic activity of the bone marrow–derived epithelial cells immediately adjacent to the wound.77 In another study, Han et al78 showed the potential of human bone marrow stromal cells to accelerate wound healing in vitro by measuring the amount of collagen synthesis and the levels of basic fibroblast growth factor and vascular endothelial growth factor. The levels of these growth factors were much higher in tissue culture of the bone marrow stromal cells group compared to the fibroblast group. This interaction of stem cells with growth factors, or their
Stem cells in cutaneous wound healing possible interdependence, is interesting and may require further work. Nakagawa et al24 suggested that hMSCs, together with basic fibroblast growth factor in a skin defect model, accelerates wound healing and showed that the hMSCs transdifferentiated into the epithelium in rats. Ichioka et al79 showed that the addition of bone marrow significantly increases the collagen matrix to induce wound healing angiogenesis especially in the early stage of the repair process. In a human study of chronic nonhealing wounds, our group showed that directly applied bone marrow–derived cells can lead to wound closure and possible rebuilding of tissues.80 That particular study, although interesting, was hampered by the lack of an efficient delivery system for the autologous bone marrow– derived cultured cells. More recently, Falanga et al81 have fully characterized the human autologous bone marrow– derived cultured cells as being MSCs (hMSCs) and have applied it to nonhealing and acute wounds, using a specialized fibrin spray system. This approach appears to be safe and may represent a rather ideal way of introducing cells, and not just stem cells (perhaps even soluble mediators), into injury sites. Of particular interest is that the wounds treated with the hMSCs developed new elastic fibers. Thus, there are indications that some degree of dermal reconstitution is being achieved.
Conclusions Chronic wounds remain a formidable challenge. In spite of recent advances from breakthroughs in recombinant growth factors and bioengineered skin, up to 50% of chronic wounds that have been present for more than a year remain resistant to treatment. Stem cells offer the possibility that some structures within the wound may be reconstituted. To what extent this will be possible remains uncertain, and randomized clinical studies will be needed to determine the effectiveness of the approach. Because of the relative ease with which they are cultured, bone marrow–derived mesenchymal stem cells have been tested in pilot studies, and there are now promising approaches for introducing them into the wound. It might turn out, however, that other types of stem cells will be more effective, including those derived from hair follicles, or perhaps subsets of bone marrow–derived cultured cells. Still, proper wound care and adherence to basic principles cannot be bypassed, even by the most sophisticated approaches.
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