See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/244480572 Tissue Engineering of Skin Article in Journal of the American College of Surgeons · June 2013 DOI: 10.1016/j.jamcollsurg.2013.03.027 · Source: PubMed CITATIONS READS 28 525 5 authors, including: Rami A Kamel Joon Faii Ong 4 PUBLICATIONS 49 CITATIONS 8 PUBLICATIONS 172 CITATIONS University of Toronto SEE PROFILE Harvard Medical School SEE PROFILE Elof Eriksson Edward Caterson 105 PUBLICATIONS 1,781 CITATIONS 113 PUBLICATIONS 2,427 CITATIONS Massachusetts General Hospital SEE PROFILE Brigham and Women's Hospital SEE PROFILE All content following this page was uploaded by Joon Faii Ong on 30 November 2016. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are linked to publications on ResearchGate, letting you access and read them immediately. Tissue Engineering of Skin Rami A Kamel, MD, Joon Faii Ong, Edward J Caterson, MD, PhD BSc, Elof Eriksson, Johan PE Junker, PhD, elastin, and reticular fibers. The main cellular component of the dermis is the fibroblasts, which provide constant secretion of the collagen and proteoglycan matrix. From a developmental perspective, fetal wound repair displays an absence of scarring and fibrosis. This regenerative process is characterized by minimal inflammation and restoration of normal collagen deposition and skin adnexae.6 The growth factor profile in fetal healing skin is substantially different from that of the adult one, being characterized by higher levels of transforming growth factor (TGF)-b3 and lower levels of platelet-derived growth factor (PDGF), TGF-b1, and TGF-b2.7 Tissue engineering (TE) and regenerative medicine are a blend of developmental biology, life sciences, and engineering efforts that attempts to address clinical problems. Tissue engineering was defined in 1988 as the application of principles and methods of engineering and life sciences toward fundamental understanding of structure function relationships in normal and pathological mammalian tissues and the development of biologic substitutes to restore, maintain, or improve tissue function1 (Fig. 1). It has been studied and applied to various organs2 with the potential for decreasing the need for organ transplantation.3 The themes of current and future molecular efforts involve coalescing approaches to recapitulate normal development in clinical scenarios where skin reconstruction is needed. An increased understanding of stem cells, scaffolding, and signaling with extracellular matrix (ECM) interactions will make this tissueengineered future possible.4 Tissue engineering of the skin has offered major advances in burn treatment and wound care, especially considering the limited availability of autologous donor skin for coverage of large defects. GOALS OF SKIN TISSUE ENGINEERING The essential goals of skin TE are the effective healing and complete simulation of physiological skin, with close to native mechanical qualities, and lack of host toxicity or immune rejection.8 In addition, the restoration of skin anatomy needs to go beyond rehabilitation of structural architecture to include reinstitution of skin pigmentation, nerve, vascular plexus, and adnexa. The design of skin substitutes also needs to consider the genotype of the transplanted skin cells, the biocompatibility of materials used, the complexity of fabrication, and storage issues.9 The restoration of an intact barrier and prevention of sepsis are crucial in skin TE, particularly in the treatment of large burns. Another important goal is the initiation of wound healing in the case of chronic wounds. Yannas and Burke outlined the basic foundations for the design of artificial skin by describing physicochemical, biochemical, and mechanical considerations.10 The skin substitute should control fluid loss, infection, contracture, and scarring. ANATOMICAL AND DEVELOPMENTAL CONSIDERATIONS Human skin consists of epidermal and dermal layers permeated by a complex vascular and nervous network. Beneath this is the hypodermis, composed primarily of loose connective tissue and fat (Fig. 2). Epidermal basal cells and stem cells residing in the basal layer and hair follicles are responsible for a continuous process of renewal of the epidermis.5 Other cell types present in the epidermis include melanocytes, Langerhans cells, and Merkel cells. The dermis consists of 2 layers: an upper papillary layer composed of a thin arrangement of collagen fibers and a thick lower reticular layer consisting of thick collagen fibers parallel to the surface of the skin. The dermal ECM is composed mainly of collagen, STRATEGIES AND APPROACHES Three different elements interplay substantially in TE of skin: the cells in consideration, the matrices as support materials, and the different growth factors governing the development of the bioengineered tissue. An in vitro manipulation of cells, matrices, or their combination can be resorted to in the production of skin substitutes (Fig. 3). Disclosure Information: Nothing to disclose. Received February 7, 2013; Revised March 15, 2013; Accepted March 18, 2013. From the Division of Plastic Surgery, Brigham and Women’s Surgery, Harvard Medical School, Boston, MA. Correspondence address: Edward J Caterson, MD, PhD, Division of Plastic Surgery, Tissue Repair and Gene Therapy Laboratory, 75 Francis St, Boston, MA 02115. email: ecaterson@partners.org ª 2013 by the American College of Surgeons Published by Elsevier Inc. MD, PhD, FACS, Cells Cells are central to TE of skin. It is the interplay between the cells, the molecular signals, and the microenviroment 533 ISSN 1072-7515/13/$36.00 http://dx.doi.org/10.1016/j.jamcollsurg.2013.03.027 534 Kamel et al Tissue Engineering of Skin J Am Coll Surg Abbreviations and Acronyms ASC ECM EGF ES HA PDGF TE TGF ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ adipose-derived stem cells extracellular matrix epidermal growth factor engineered skin hyaluronic acid platelet-derived growth factor tissue engineering transforming growth factor within the scaffold that permits TE as reality. A crucial consideration here is locating suitable sources of cells. These sources need to provide a high quantity of cells for sufficient regeneration to generate skin bioconstructs. Cells are derived from a variety of lineages, from stem cells to differentiated somatic cells. These populations can be classified as local, systemic, or progenitor. The cells are generally obtained from the host, close relatives, or other individuals. The different cells considered for skin substitutes include keratinocytes, fibroblasts, adipocytes, melanocytes, hair follicle associated cells, and various progenitor cells. Although most of the current commercially available skin substitutes rely on keratinocytes and fibroblasts, the other cell types are being investigated for future generations of bioengineered skin. Multiple cell types other than skin cells are involved in the extensive process of wound healing. Investigating the role of different cells, such as platelets, macrophages, neutrophils, and fibroblasts in wound healing will yield more information about cellular signaling that can be used to build smarter skin substitutes. Keratinocytes Keratinocytes constitute 95% to 97% of the epidermis and being on the surface of the skin makes them one of the most easily accessed skin cell types. In comparison with the other cells of the dermis and epidermis, keratinocytes are the only type present in uninjured skin, splitthickness autografts, and healed skin post grafting,11 making them very important in the development of engineered skin (ES) substitutes. In addition, the capacity of keratinocytes to renew and proliferate,12 and maintain their ability to reconstruct the epidermis after long-term expansion13 is useful in engineered constructs. Autologous or allogenic keratinocytes have been grown into sheets, made into suspensions, or delivered using various dressings ranging from xenogeneic collagen to synthetic polymers in the pursuit of a commercial skin substitute. Most of the cell-based skin substitutes, whether scaffold-free substitutes or cell-seeded scaffolds, rely mainly on keratinocytes for their manufacturing. In addition, Figure 1. Concept of tissue engineering. The figure shows the interdisciplinary nature of tissue engineering involving developmental and cellular biology that aims at understanding the growth and function of cells and tissues and the signals governing cellular processes and the principles and methods of engineering leading to the fabrication of matrices and development of bioreactors to generate tissue engineered products implicated in an array of laboratory and clinical uses. keratinocytes have been used as a source of induced pluripotent cells. Aasen and colleagues claim a 100-fold and 2-fold increase in efficiency and speed, respectively, of retroviral reprogramming of human primary keratinocytes over human fibroblasts14 Given the ready access to keratinocytes, they are potentially useful as a source of stem cells for both experimental models and engineered therapeutic products. Fibroblasts and fibrocytes In response to injury, local fibroblasts migrate into and proliferate in injury sites in response to the release of Vol. 217, No. 3, September 2013 Kamel et al Tissue Engineering of Skin 535 Figure 2. Schematic presentation of skin. The diagram shows the epidermis, dermis, and underlying hypodermis. Blood vessels and nerves course through the dermis, which also contains specialized elements, such as sweat glands and hair follicles and their adnexae, such as the erector pili muscle and sebaceous glands. (Adapted from MacNeil,88 with permission.) growth factors and cytokines such as TGF-b1.15 To repair the wound, fibroblasts deposit collagen and other relevant ECM components.16 Many current ES products depend on cultured allogenic neonatal fibroblasts or cultured autologous fibroblasts for their production. Although keratinocytes and fibroblasts can be harvested from local skin tissue, fibrocytes are present systemically in the blood stream and bone marrow. Looking back, the concept of wound fibroblasts originating from peripheral blood cells was postulated almost a century ago.17 Since then, numerous studies have reported the differentiation of peripheral mononuclear cells into fibroblast-like cells. Fibrocytes constitute a novel cell population, constituting 0.1% to 0.5% of peripheral blood leukocytes, and are characterized by the expression of collagen type I, CD13, CD34, and CD45.18,19 Fibrocytes have also been shown to play an important role in wound healing by acting like myofibroblasts and producing numerous proinflammatory cytokines and growth factors, secreting collagen and other ECM proteins, and enhancing collagen production in response to TGF-b1.18,20 Therefore, future research can incorporate fibrocytes into new models of ES substitutes. These melanocytes communicate with overlying keratinocytes and the underlying fibroblasts via both secreted factors and intercellular junctions.22,23 As human pigmentation has substantial social, cosmetic, and photoprotective implications, there have been long-term efforts to regulate skin pigmentation. Melanocytes have been used in suspension with keratinocytes for the production of the epidermal replacement product “ReCell.”24 Skin substitutes have been used in the laboratory to study melanocytes behavior and its implications in melanoma. Melanocytes Melanocytes are derived from neural crest cells located in the epidermis and synthesize the pigment melanin. Melanin provides visible pigmentation to the skin, hair, and eyes, and also protect skin from ultraviolet radiation.21-23 Melanocytes are located at the border of the dermis and epidermis in a regularly dispersed pattern. Adipose-derived stem cells Although adipocytes from the hypodermis are usually not incorporated into current skin substitutes, there has been a recent push to develop adipose-derived stem cells (ASCs) as novel therapy, taking advantage of this readily available and abundant source of multipotent progenitor cells. Adipose-derived stem cells are sought as a viable Figure 3. Skin tissue engineering strategies and approaches. Diagram showing different elements contributing to the production of skin substitutes. Several tissue engineering strategies depend on either direct application of freshly isolated or cultured cells, in situ local regeneration of the defective tissue or the delivery of tissues assembled in vitro from cells and scaffolds. In vitro manipulation could involve cultivation, bioreactors or different ways of physical and chemical stimulation. Image of Apligraf as an example of skin substitutes is used with permission from Organogenesis Inc. EGF, epidermal growth factor; FGF, fibroblast growth factors; PDGF, platelet-derived growth factor; TGF-b, transforming growth factor b. 536 Kamel et al Tissue Engineering of Skin alternative to autologous tissue flaps, autologous fat transplantation, and alloplastic implants.25 In burn eschars, a population of multipotent stem cells might be an interesting resource for TE approaches to heal burn wounds. Although the origin of these cells remains unknown, their resemblance to ASCs could also be cause for speculation that in deep burns, the subcutaneous adipose tissue might be an important stem cell source for the healing wound.26 A study by Kim and colleagues demonstrated the beneficial effect of human ASCs on healing of ischemic wound in diabetic nude mice.27 In addition, it has been suggested that ASC transplantation could be associated with a beneficial effect on skin-graft survival in rats and might represent a novel therapeutic approach to preventing skin-graft ischemic necrosis in humans.28 A recent study concludes that ASCs can serve as an alternative for enhancing wound healing through differentiation and angiogenesis. Administration of allogeneic ASCs using an acellular dermal matrix as a scaffold can have a promising therapeutic effect in the treatment of cutaneous wounds.29 Hair follicle cells Progenitor cells are found in stem cell niches such as hair follicles. Because of their potency and relative abundance they, together with ASCs, might be the most promising cell candidates studied to date.30 Through lineagetracing studies, hair follicle stem cells have been shown to mobilize to the wound and contribute to its repair.31-33 A recent study by Mascré and colleagues confirms the existence of 2 distinct progenitor populations, namely slow-cycling stem cells and committed progenitor cells. Together, these 2 populations are responsible for the repair and maintenance of homeostasis of the epidermis.34 Materials for matrices The ECM is in a state of continuous reciprocity with its resident cellular population35 and also serves as a scaffold post injury, which enhances remodeling of tissue. Although the cells secrete ECM components and communicate with each other through it, the ECM modulates the phenotype, genetic expression, development, proteome, and function of these cells. This interaction is influenced by the microenvironment, which provides a niche for homeostatic modulation of the ECM. When considering skin substitutes, bioengineered matrices should aim to mimic the ECM by incorporating appropriate cytokines, enzymes, pharmacological agents, and other signaling peptides at physiological quantities and durations. Matrices range from naturally occurring (eg, collagen, hydroxyapatite, alginate, polypeptides, glycosaminoglycans, hyaluronan, fibronectin) to synthetic (eg, polyvinyl J Am Coll Surg chloride, polylactide) and differ in their properties. Both matrix types need to be compatible with the biologic needs of the cells. Natural matrices are characterized by interactive properties, such as cell adhesiveness with low toxicity and low chronic inflammatory response. Synthetic matrices, on the other hand, are manufactured to allow better handling of material properties, such as geometrics, strength, degradation, and permeability. The advantages of synthetic scaffolds over their biological counterparts include the elimination of contamination and disease transmission, increased manufacturing uniformity, and better tailoring to clinical needs. The degradable polymers first used in TE were adapted from other surgical applications and had shortcomings concerning their mechanical and degradation properties.36 Because many TE strategies depend primarily on biodegradable polymer materials, a need for a new generation of materials emerged. Another major demand was for the fabrication of scaffold polymers with complex internal structures to better direct tissue growth.37 The introduction and application of nanotechnology aims to generate increasingly complex scaffolds that more closely mimic the ECM.38 Naturally occurring matrices Collagen Collagen has been widely used as a scaffold and carrier for cells in TE, particularly in skin.39,40 The principle for its use is to create reconstructed dermis that promotes the spontaneous formation of a human capillary-like network.41 A highly porous matrix composed of acellular collagen, glycosaminoglycans, and disaggregated autologous keratinocytes has been shown to regenerate dermis and epidermis in vivo in pigs.42 Fibronectin and fibrin Fibronectin is used as a cell adhesive layer due to the presence of a peptide sequence (arginine-serine-aspartate) involved in integrin-mediated cell adhesion.43 In dermal fibroblasts, interactions are mediated by b1-type integrins.44 Fibronectin has been implicated in the design of biomaterials for bioengineered constructs.45 Fibronectin has also been involved in the differentiation of stem cells.46 Fibrin, associated with fibronectin, enhances cellular proliferation and migration.47 Because fibrin can act as a glue, the use of fibronectin matrix as a delivery vehicle for cultured cells, such as keratinocytes and fibroblasts, might be similar to conventional skin grafts.48 Human plasma, a rich source of fibrin, has been investigated as a dermal scaffold in the hope of producing a completely autologous skin substitute.49 A study by Grant and colleagues suggests that the application of Vol. 217, No. 3, September 2013 autologous keratinocytes, suspended in autologous fibrin sealant, to full-thickness wounds results in good reepithelialization.50 On a commercial level, some skin constructs, such as BioSeed-S, and some prospective products still under investigation, such as AcuDress, Allox, and Cyzact, depend on fibrin as a scaffold material.51 Hyaluronic acid Hyaluronic acid (HA) is used clinically in humans for knee pain and surgical adhesions.52,53 Hyaluronic acid was proven to have a proliferative effect on keratinocytes and fibroblasts and was reported to participate in scarless fetal wound healing.54 Also, a conjugation of HA with human growth factor secreted by fibroblasts was shown to enhance the transdermal delivery of protein therapeutics. This opens the door to additional uses for HA in various cosmetic and TE applications.55 Recombinant HA or its derivatives are currently used in skin substitutes such as LaserSkin, Hyalomatrix, and Hyalograft. Matrigel Matrigel is based on a mixture of proteins found normally in ECM, such as collagen, fibrin, laminin, and elastin.56 Matrigel resembles naturally occurring ECM with its complex signaling capacity and is used in cell cultures for skin-engineering purposes.57 Synthetic matrices Although naturally derived ECM components regulate and support the behavior, modification, and processing of native ECM molecules, photopolymerization, ionic cross linking, and enzymatic reactions can disrupt or compromise their natural physical and biochemical properties. Synthetic scaffolds have been investigated for the purpose of enhancing those properties. Self-assembly or enzyme-catalyzed approaches are used in creating 3-dimensional matrices from biopolymers. Advances in polymerization chemistry have allowed increasingly granular control of the production of multifunctional nanoparticles, with better control of the individual polymer components. This is seen especially in the assembly and conversion of these nanoparticles into structures of specific properties, with an ability to modulate response to specific stimuli through remote activation.58 The most widely used synthetic material in skin TE is silicone. However, many other materials have been investigated for the purpose of skin engineering, such as a copolymer of polyethyleneglycol-terephthalate and polybutylene terephthalate.59,60 Also, the incorporation of polyurethane foam into dressings or scaffolds has shown promising results in porcine wound-healing models.61 Kamel et al Tissue Engineering of Skin 537 Molecular signaling Complex and highly varied molecular cues are constantly changing during the process of regeneration. They alter the microenvironment in a manner consistent with the appropriate development of cells. These cues initiate a series of events that together are responsible for the entire system of proliferation and differentiation. With a better understanding of these molecular signals, especially in embryological development, we can enhance the adaptation of these signaling pathways to the fabrication of next-generation bioengineered skin products. Transforming growth factor b family Transforming growth factor b is a superfamily of multifunctional regulators of cellular growth, differentiation, and ECM production62 and plays a pivotal role in wound healing regulation and scarring. It is the growth factor most commonly involved in wound healing throughout the body.6 Three isoforms exist, namely b1, b2, and b3, all of which act on the cells through binding to dimeric receptor complexes. On activation, this receptor complex phosphorylates SMAD2 and SMAD3 proteins, which subsequently form dimers with SMAD4. Only then is the dimer able to migrate into the nucleus to act as a transcription factor. Transforming growth factor b1 and b2 activate the receptor complex and all subsequent signaling pathways, and TGF-b3 is a receptor antagonist functioning as an anti-scarring factor.63,64 In a previous study, it has been shown that the delivery of TGF-b1 in a collagen scaffold to a wound results in faster rates of epithelialization and contraction in rabbits.65 Platelet-derived growth factor Platelet-derived growth factor has many isoforms and mediates its actions through the activation of dimeric transmembrane tyrosine kinase receptors. It is a potent activator of cells of mesynchymal origin and has been implicated in chemotaxis, cellular proliferation, and induction of gene expression.66 It induces collagen deposition in cutaneous wounds through activation of fibroblasts. High levels of PDGF and its receptor have been confirmed in keloids and hypertrophic scars.67 In addition, it has been shown to induce fibroblasts to produce osteopontin, which enhances cellular adhesion to ECM and cellular migration, and provides an anti-apoptotic signal.68,69 A recent study suggests that the knockdown of osteopontin can result in more rapid healing with reduced scarring.70 In the context of skin TE, the control of PDGF production engenders the design of better skin substitutes, resulting in less scarring. 538 Kamel et al Tissue Engineering of Skin Fibroblast growth factors Fibroblast growth factors are a family of small polypeptide growth factors occurring in 21 isoforms that are engaged in many cellular activities from the differentiation to the control of proliferation and migration.71 Fibroblast growth factor 2 (basic fibroblast growth factor) has been reported to play a role in wound healing, as it is released by endothelial cells and macrophages at the wound site to enhance angiogenesis.72 It has been implicated in scar-free wound healing.73 Fibroblast growth factors 7 and 10 are secreted by fibroblasts to act on keratinocytes enhancing their proliferation and migration71 and were shown to be down-regulated in fetal scarless wounds.74 A balance between different isoforms must be achieved in skin substitutes to ensure adequate vascularization with minimal scarring. Recently, an in vitro skin TE model based on alginate microcapsules loaded with basic fibroblast growth factor and cultured dermal fibroblasts in autologous fibrin scaffolds has demonstrated increased cellular proliferation and viability.75 However, the authors believe that ascertaining clinical efficacy would require in vivo validation. Epidermal growth factor Epidermal growth factor (EGF) is involved in wound healing and homeostasis of various tissues.76 It also stimulates keratinocytes to produce HA.77 Epidermal growth factor signaling has been implicated in cellular motility and migration during wound repair.78,79 Recently, a wound dressing combining HA, EGF, and collagen studied in rats was suggested to promote wound healing by stimulating fibroblast function.80 A cell line, genetically modified to produce human EGF abundantly, was used to produce a skin equivalent that was shown to treat burns efficiently in a rat model.81 Bone marrow mesenchymal stem cells that were seeded on EGF microspheres for delivery to the wound site through biomimetic scaffolds not only improved wound healing, but also reconstituted sweat glands in the new skin.82 This offers a potential benefit in the administration of stem cells clinically and the development of complex skin adnexae for future skin constructs. Bioreactors Certain types of skin bioconstructs, such as Dermagraft and TransCyte, use bioreactors as modified culture systems to grow cells. Dermagraft uses roller bottle technology, promoting the cells to adhere to the bottle during the initial phase of cell expansion. A study by Sun and colleagues investigated the use of a peristaltic pump in the sterilization of scaffolds, cell seeding, and medium perfusion in a closed bioreactor system. This system has J Am Coll Surg been developed for the creation of TE skin at an air liquid interface and could be used either clinically or experimentally.83 More recently, a NASA-approved rotary bioreactor was used to investigate the proliferation and differentiation of human epidermal stem cells. Cells cultured on the rotary bioreactors aggregated on microcarriers, forming 3-dimensional epidermal structures. This can have positive implications on the development of future models of epidermal constructs.84 SKIN SUBSTITUTES A number of different strategies have been explored to integrate the cells and matrices mentioned here. The current trend is to deliver subconfluent cultured cells on a chemically inert carrier dressing to the wound bed in burns, chronic wounds, and vitiligo.85-87 Patient safety, clinical efficiency, and convenience are primary factors in designing skin bioconstructs.88 An ideal skin substitute would be an epidermal/dermal bilayer fully integrated into the wound bed: the epidermal layer engineered to regain the protective barrier function, with the dermal construct enabling rapid physiological repair of the skin lesion. This would provide rapid revascularization and re-innervation of the graft. Since the establishment of keratinocyte culture,89 many attempts at the ideal bioengineered skin have been made, beginning with cultured epithelial autografts in patients90,91 to synthetic dermal alternatives,92 and sidestepping bovine collagen with autologous keratinocytes and fibroblasts as an alternative.93 Different examples of commercially available products are outlined in Tables 1 to 3. APPLICATIONS OF ENGINEERED SKIN Clinical applications Skin TE can be applied clinically in a variety of different scenarios, including disease, acute trauma, chronic wounds, or congenital abnormalities. The key to successfully adopting a therapeutic approach for the variety of these clinical conditions is to ensure maximum number of skin cells and the maintenance of their function. Extensive full-thickness burns In the case of thermal insult, the extent and depth of the wound, along with the age of the patient, is the main determinant of mortality.191 In addition, with fullthickness burns, regenerative elements are completely lost without the possibility of skin regeneration.51,192 Even with a mesh skin autograft to maximize the amount of skin available for grafting,193 donor sites are insufficient in providing complete coverage in extensive burns.194 Brand name CellSpray Uses/advantages Limitations Epicel Uses/advantages Limitations EpiDex Uses/advantages Limitations EpiBase Uses/advantages Uses/advantages Limitations LaserSkin Uses/advantages Biomaterial Scaffold source Cellular loading Lifespan References 539 Limitations Cell source 94,95 d d d Permanent Cultured or noncultured autologous keratinocytes Early wound coverage by activated proliferating keratinocytes Limited to partial-thickness and graft donor-site wounds 96 98 Petroleum gauze Synthetic In vitro Permanent Genzyme Biosurgery Cell sheet Confluent cultured backing autologous keratinocytes Little risk of rejection and relatively large area of application, high incidence of permanent take Time consuming to produce, susceptible to blistering, variable take rate, poor long-term outcomes, and necessity for dermal support, high cost, short shelf life Modex Cell sheet Confluent cultured Silicone membrane Synthetic In vitro Permanent 99 101 Therapeutiques autologous outer root sheath hair follicle cells Successful in treatment of chronic ulcers; cells have a good proliferative capacity Fragile product that takes almost 6 weeks to produce, difficulty in handling and application 102,103 d d In vitro Permanent Cell sheet Confluent cultured Laboratoires autologous Genevrier, Sophia keratinocytes Antipolis Has been effectively used in a case of cutaneous calciphylaxis Not totally satisfactory in cases of very deep dermal injuries. Synthetic In vitro Permanent 85,104 CellTran Ltd Cell-seeded scaffold Subconfluent cultured Synthetic silicone autologous support sheet with keratinocytes a specially formulated coating The coating enhances keratinocyte attachment and proliferation and provides a stable platform, used in neuropathic, pressure and diabetic foot ulcers, superficial burns and skin-graft donor sites Cellular growth requires almost 2 weeks and repeated application is needed, cannot be used alone for deep wound treatment Cell-seeded scaffold Subconfluent cultured Fibrin sealant BioTissue 105,106 Allogenic In vitro Permanent autologous Technologies keratinocytes on GmbH a fibrin matrix Proved efficient in treatment of recalcitrant venous ulcers, almost 50% increase in wound-healing efficiency compared with standard treatment No significant effect on ulcers >12 mos of duration, adverse effects should be investigated, additional information on use in burns is needed Matrix of hyaluronic Recombinent In vitro Permanent 107 110 Fidia Advanced Cell-seeded scaffold Confluent cultured acid ester Biopolymers autologous keratinocytes Interaction between cells and hyaluronic acid improve mechanical properties and allow better migration of keratinocytes to wound bed, good graft take rate, low infection rates Expansion of cells requires 3 wks, needs large-scale clinical trials (Continued) Tissue Engineering of Skin Limitations BioSeed-S Graft type Cell based Kamel et al Uses/advantages Limitations MySkin Manufacturer Clinical Cell Culture (C3) Vol. 217, No. 3, September 2013 Table 1. Examples of Commercially Available and Prospective Epidermal Skin Substitutes for Clinical and Laboratory Applications Continued Brand name CryoSkin Uses/advantages Limitations Suprathel Uses/advantages Limitations EpiSkin Uses/advantages Limitations Cell source Biomaterial Scaffold source Cellular loading Lifespan References Permanent 111 Temporary 112 Permanent 24 Temporary 113 115 Tissue Engineering of Skin Uses/advantages Limitations ReCell Graft type Cell-seeded scaffold Cryopreserved Silicone backing Synthetic In vitro monolayer of noncultured allogeneic keratinocytes Effective in treating leg ulcers Requires repeated application, takes 24 wks to heal wound, wound infection is the main adverse effect CellTran Ltd. Cell based Freeze-dried lysate Hydrophilic gel d In vitro from cultured allogeneic keratinocytes Lysate carries different growth factors and cytokines to the site of the wound Needs additional investigation Avita Medical Ltd. Cell based Noncultured d d d autologous keratinocyte and melanocyte suspension Contains melanocytes, which help restore skin color in scars and hypopigmented areas Only limited to treatment of <2% total body surface area burn in adults and 4% in children Synthetic d Bio Med Sciences Cell free d Absorbable wound dressing mainly based on DL lactic acid Effective in partial-thickness burns, frostbites and Lyell syndrome, long shelf life, superior antisepsis and less bleeding compared Therapeutic effects decrease with delay of application and increase depth of burn Synthetic In vitro SkinEthic Cell-seeded scaffold Cultured keratinocytes Collagen matrix Laboratories from mammary samples Used in skin irritation testing instead of animal models, used to assess phototoxic potential in vitro Not validated for all toxicity tests Kamel et al Uses/advantages Limitations LyphoDerm Manufacturer Altrika Ltd. 540 Table 1. with other products d 116 118 J Am Coll Surg Brand name Alloderm Uses/advantages Limitations Dermagraft Uses/advantages Limitations Permacol Cell source Biomaterial Scaffold source Cellular loading Lifespan References 541 Allogenic In vivo Permanent Human acellular 119 128 lyophilized dermis with preserved basement membrane processed from cadaveric skin Readily incorporates into wound due to reduced antigenicity, no cases of viral transmission after >100,000 product applications, 2 y shelf life, successfully used in full-thickness skin burns, abdominal wall hernia reconstruction, rhinoplasty, cleft palate repair, temporomandibular joint reconstruction, periodontal surgery, subcutaneous mastectomy and different fistulae reconstruction Possibility of disease transfer and/or graft rejection, uncertain rates of vascularization, poses ethical issues and avoided on moral grounds by some clinicians and patients In vitro Permanent 129 132 Advanced Cell-seeded scaffold Cultured allogenic Silicone film, nylon Xenogeneic and synthetic mesh, GAGs and BioHealing Inc. neonatal porcine dermal fibroblasts collagen form a 3-dimensional bioabsorbable scaffold Neonatal fibroblasts proliferate rapidly to produce collagen, GAGs help wound healing, and has been used in vestibuloplasty and diabetic wounds Possibilities of disease transfer and/or graft rejection, multiple applications, and higher cost Tissue Science Cell free d Porcine-derived Xenogeneic In vivo Permanent 133 Laboratories plc acellular matrix Supports host’s fibroblasts proliferation, nonimmunogenic, has been used in abdominal wall reconstruction Needs overlying epidermal graft, revascularization sometimes not efficient In vivo Temporary Advanced Cell-seeded scaffold Allogenic neonatal Nylon mesh coated Xenogeneic and 134 Synthetic with porcine BioHealing Inc. human dermal dermal collagen fibroblasts and bonded to a silicone membrane Successful in second and third-degree burns, 1.5 y shelf life when frozen Rejection and/or skin disease from fibroblast, mesh not biodegradable Xenogeneic and In vivo Semi-permanent 135 138 Integra Cell free d Polysiloxane synthetic NeuroSciences pseudoepidermis, bovine crosslinked tendon collagen type I, shark chondroitin GAG (Continued) d Tissue Engineering of Skin Uses/advantages Limitations Integra Graft type Cell free Kamel et al Uses/advantages Limitations Transcyte Manufacturer LifeCell Corporation Vol. 217, No. 3, September 2013 Table 2. Examples of Commercially Available and Prospective Dermal Skin Substitutes for Clinical And Laboratory Applications Brand name Uses/advantages SureDerm Uses/advantages Limitations GraftJacket Uses/advantages Limitations Terudermis Uses/advantages Limitations EZDerm Uses/advantages Limitations Cell source Biomaterial Scaffold source Cellular loading Lifespan References Tissue Engineering of Skin Uses/advantages Limitations KaroDerm Graft type Kamel et al Limitations Manufacturer Polysiloxane membrane replaced after 2 wks with an autograft, dermal component enhances ingrowth of fibroblasts and keratinocytes, used in >10,000 patients and in many clinical scenarios, including burns, diabetic ulcers, and auricular reconstruction, good cosmetic outcomes, moderate shelf life, good barrier function 3 wks for preparation, needs meticulous surgical preparation of wound bed to guarantee good outcomes, requires a second surgery to permanently close wounds HANS BIOMED Cell free d Human lyophilized Allogenic 139,140 In vivo Permanent Corporation pre-meshed dermis Used in replacement of damaged soft tissue as hypertrophic scar and burn, used in correction of nasal septal perforation, long shelf time (up to 2 y) Needs subsequent skin grafting, should be rehydrated before application Karocell Tissue Cell free d Human acellular Allogenic In vivo Permanent d Engineering AB dermis Provides a dermal bed for subsequent skin grafting Possibility of disease transfers and/or graft rejection; requires subsequent grafting 141 144 Allogenic In vivo Permanent Wright Medical Cell free d Human acellular Technology pre-meshed dermis Successfully used in tendon and lower extremity wound repair (superficial and deep) Information about thermal injuries treatment is limited d Lyophilized cross- Xenogeneic and Olympus Terumo Cell free In vivo Semi-Permanent 145 149 linked collagen Biomaterial Synthetic sponge from Corp. bovine heatdenatured collagen and Silicone Useful in deep burns where bone and muscle exposure is present, also in skin flap donor-site regeneration, otological surgery and post-traumatic deformity correction, investigated for potential use in scarred vocal folds Requires split-thickness skin grafting (STSG) complementarily 150 152 Xenogeneic In vivo Temporary Brennen Medical, Cell free d Aldehyde crossInc. linked collagen reconstituted from porcine dermal collagen Bioactive wound dressing used in partial-thickness burns No difference with petroleum nonadherent dressing for partial-thickness burns (Continued) 542 Table 2. Continued J Am Coll Surg Brand name Matriderm Uses/advantages Limitations Srattice Uses/advantages Limitations OASIS Wound Matrix Limitations Pelnac Uses/advantages Biomaterial Scaffold source Cellular loading Lifespan References Collagen types I, III Xenogeneic In vivo Permanent 153 158 and V from non cross-linked lyophilized bovine dermis coated with a elastin hydrolysate Promising results when applied simultaneously with split-thickness skin grafts, single-stage surgical intervention; used in necrotizing fasciitis defects, in the management of exposure of Achilles tendon secondary to burn injury Requires graft from patient LifeCell Cell free d Porcine dermis and Xenogeneic and In vivo Permanent 159 161 Corporation polysorbate synthetic Used in breast reconstruction, investigated for potential use in ventral hernia, promising results especially in potentially contaminated fields, better than Alloderm in terms of avoiding use of cadaveric human donor tissue, with more ethical acceptance, suitable tensile properties for reconstruction of fascial defects Must be in contact with healthy tissues to permit regeneration, showed decrease in thickness over time and lesser tensile strength when compared with Permacol in abdominal wall defects Xenogeneic In vivo Permanent 162 165 Cook Biotech Inc. Cell free d Collagen matrix material from lyophilized porcine small intestine submucosa Used in chronic wounds, with faster healing rate and less recurrence achieved, shown to support in vitro epidermal differentiation and basement membrane formation, minimal contraction of full-thickness wounds when investigated in vivo in rodents Additional clinical trials needed to study its effect in full-thickness wounds Xenogeneic and In vivo Semi-permanent 166 168 Gunze Ltd, Medical Cell free d Collagen from synthetic Mateials Center porcine tendon and silicone Can be stored up to 3 y, used in repair of lower limb skin after necrotizing fasciitis and skin defects, easy to use, safe with excellent long-term results Requires a 2-stage operative procedure 169 173 UDL Laboratories, Cell free d Pseudoepidermal Xenogeneic and In vivo Temporary Inc. semi-permeable Synthetic silicone membrane attached to nylon fabric and porcine collagen Used in partial-thickness burns in children, toxic epidermal necrolysis, paraneoplastic pemphigus and chronic wounds, effective for vapor loss control, faster healing compared with conventional dressings, reduced pain Limited results when compared with Orcel (Continued) d 543 Limitations Cell source Tissue Engineering of Skin Uses/advantages Limitations Biobrane/ Biobrane-L Graft type Cell free Kamel et al Uses/advantages Manufacturer Dr Suwelack Skin and HealthCare AG Vol. 217, No. 3, September 2013 Table 2. Continued HA, hyaluronic acid. Uses/advantages Limitations Uses/advantages Limitations Hyalograft 3D Lifespan Cellular loading Scaffold source Biomaterial In vivo Semi-permanent 54,174 HYAFF (HA ester) Allogenic and Synthetic covered by a temporary silicone layer serving as epidermis Safe, used in dermabrasion and deep partial-thickness wounds, investigated in porcine model for full-thickness wounds Expensive, cannot be used on infectious wounds, it takes on a greenish color on jellifying, which can be misinterpreted as a symptom of infection Fidia Advanced Cell-seeded scaffold Autologous cultured HA ester Allogenic In vitro Permanent 175 178 Biopolymers fibroblasts Safe, contributes to fast formation of basement membrane, specific for deep dermal lesions, used successfully in a case of sclerodermal cutaneous ulcers No statistically significant difference in treatment of diabetic neurotrophic foot ulcers when compared with nonadherent paraffin gauze Ulcers resistant to conventional healing The healing of chronic wounds depends primarily on the condition of the wound bed, irrespective of the cellular delivery method or the condition of the cells delivered.88 It is suggested that augmentation of local wound microenvironment, with a stable provisional matrix formed by proteolysis-resistant angiogenic peptide nanofibers, will attenuate inflammation, enhance neovascularization, and improve wound healing.201 d Cell source J Am Coll Surg Engineered skin presents an alternative88 to traditional split-thickness skin grafts,193,195 especially because burns >4 cm require grafts for closure and healing.196 Engineered skin has been shown to provide at least similar efficacy with conventional meshed split-thickness autografts and is crucial in restoring lost barrier function.88 In addition, ES has been used in treating extensive burns for at least 3 decades,92 with animal models providing a substantial basis for its use.135,197,198 In the time since, a number of commercially available skin constructs have been developed.199 Engineered skin has reduced the need for donor skin harvesting,186 with lesser donor thickness and increased donor site healing.92 In addition, when compared with meshed split-thickness autograft controls, Waymack and colleagues determined that 22 of 38 Apligraf recipient sites were superior in terms of overall cosmetic appearance. There was also better closure, pigmentation, vascularity, pliability, graft height, and Vancouver burn scar assessments in the Apligraf group.181 However, additional randomized controlled studies with long-term follow-up would increase the use of ES.200 Cell free Graft type Tissue Engineering of Skin Fidia Advanced Biopolymers Manufacturer Brand name Table 2. Continued Kamel et al Hyalomatrix PA References 544 Venous ulcers Venous insufficiency presents as the largest underlying cause of lower peripheral ulcers,202-205 making it the impetus to provide better alternatives to traditional skin grafting. For instance, Apligraf has improved median wound closure time and increased complete healing at 6 months in comparison with an active control.206 In a larger, multicenter trial randomizing 293 patients, Falanga and colleagues207 demonstrated a higher proportion of patients achieving complete wound closure (63% vs 49%; p ¼ 0.02). In addition, median time to complete wound closure was significantly higher as well (61 days vs 181 days; p ¼ 0.003).207 In a second, prospective randomized controlled trial of 120 patients, Apligraf treatment with compression therapy was compared with standard compression therapy. Again, at 6 months, the Apligraf treatment resulted in increased complete wound closure (47% vs 19%; p ¼ 0.005) at a higher rate of recovery (181 days Examples of Commercially Available and Prospective Composite Skin Substitutes for Clinical and Laboratory Applications Brand name Apligraf Uses/advantages Limitations Orcel Uses/advantages Limitations Permaderm Uses/advantages Uses/advantages Limitations Cell source Biomaterial Scaffold source Cellular loading Lifespan References Bovine collagen sponge Xenogeneic In vitro Temporary 96,179 184 Allogenic cultured human keratinocytes and fibroblasts Delivers cytokines and growth factors to wound bed, originally used for diabetic and venous ulcers, but also used in cases of burns and pyoderma gangrenosum, take almost resembles autografts, cosmetically favorable, minimal rejection Requires repeated applications, cells of the construct do not survive after 1 to 2 mos in vivo, product shelf life of 5 d, difficult in handling, risk of disease transfer, high cost, large-scale clinical trials still needed to confirm its use in burns Ortec international Cell-seeded scaffold Allogenic cultured Bovine collagen sponge Xenogeneic In vitro Temporary 169,185 Inc. human keratinocytes and fibroblasts Used in recessive dystrophic epidermolysis bullosa, provides cytokines and growth factors leading to a favorable environment, reduced scarring, shorter healing time, 9-mo shelf life cryopreserved Risk of rejection; biological dressing rather than a permanent skin substitute Absorbable biopolymer Xenogeneic In vitro Permanent Regenecin Inc. Cell-seeded scaffold Autologous cultured 186 keratinocytes and substrate fabricated fibroblasts from bovine collagen Reduce requirements for donor skin harvesting for grafting in cases of full-thickness burns >50% total body surface area, good outcomes compared with meshed grafts, reduced time to wound closure, morbidity and mortality in extensive deep burns Additional studies needed to prove efficacy and confirm use for different clinical indications Elastomeric and HC Implants BV Cell-seeded scaffold Autologous cultured Synthetic In vitro Temporary 59,187,188 biodegradable keratinocytes and polyethylene oxide fibroblasts terephthalate/ polybutylene terephthalate copolymer Reduced risk of immunogenicity and/or infection; used in partial-thickness wounds. Nonbiodegradable, cannot be used as a permanent skin substitute, is not “off-the-shelf,” increased cost compared with other composite substitutes Recombinant In vitro Permanent 189,190 Dermal substitute Fidia Advanced Cell-seeded scaffold Autologous cultured including Biopolymers keratinocytes and hyaluronic acid fibroblasts matrix Used in treatment of diabetic foot ulcers, many of which were full-thickness with an area >5 cm2, low recurrence rate of ulcers, used in a case of parotid surgery to prevent neck scarring Not a “true” bilayered substitute, requires the grafting of 2 products, difficult to use clinically Tissue Engineering of Skin Uses/advantages Limitations TissueTech Autograft System (Laserskin and Hyalograft 3D) Graft type Cell-seeded scaffold Kamel et al Limitations PolyActive Manufacturer Organogenesis Inc. Vol. 217, No. 3, September 2013 Table 3. 545 546 Kamel et al Tissue Engineering of Skin vs not attained; p < 0.005). In addition, in a model presented by Schonfeld and colleagues,208 the annual medical cost for managing patients with “hard-to-heal” venous leg ulcers was lower for Apligraf-treated patients than with the conventional Unna’s boot ($20,041 vs $27,493). At the end of the year-long follow-up, 48.1% of patients treated with Apligraf remained healed compared with 25.2% for Unna’s boot. Diabetic ulcers Bioengineered skin constructs have also shown considerable improvement in closure of diabetic foot ulcers.209 In a large prospective randomized controlled trial conducted by Marston and colleagues, 245 patients with chronic ulcers (longer than 6 weeks’ duration) were followed. Closure of Dermagraft-treated wounds was determined to be 1.6 to 1.7 times faster than conventionally treated wounds.129 This validates the favorable foundation provided by teams, including Gentzkow and colleagues,210 Naughton and colleagues,211 Mansbridge and colleagues,212 Margolis and colleagues,213 and Brem and colleagues,214 for the use of ES in diabetic ulcers. Despite these successes, the current state of the art still needs improvement.199 More effort is needed in finetuning the molecular basis for wound closure, including growth factors and wound healing modulators, such as PDGF, EGF, and TGF-b215 (see section on molecular signaling). Decubitus ulcers Cultured skin substitutes have been used in treating pressure ulcers. In a small trial of 23 patients conducted by Brem and colleagues, 13 of 21 pressure ulcers treated with Apligraf healed in a mean of 29 days.216 Likewise, in developing a single-application, autologous, fullthickness skin substitute, Gibbs and colleagues demonstrated its successful use in 2 pressure ulcer patients.217 As seen, these initial successes require additional, largerscale trials to substantiate the efficacy of ES. Dermatologic conditions Pyoderma gangrenosum The cause of this condition is currently unclear218 and without general recommendations and guidelines for its management.219 Conventional therapy includes highdose corticosteroids and immunosuppressants.220,221 In a 26-year-old woman presenting with a left anterior tibial ulcer, Apligraf treatment resulted in 30% to 40% wound closure rate within 2 weeks, and complete closure and revascularization by 6 weeks.222 J Am Coll Surg Vitiligo Vitiligo is the most common depigmenting disorder globally, with an estimated prevalence of 0.5%.223 It presents as both segmental and nonsegmental, with a clinical definition proposed only recently.224 Surgical methods are available for treatment, with Andreassi and colleagues grafting autologous keratinocyte cultures on a Laserskin HA membrane onto 11 patients. Follow-up at 3, 6, 12, and 18 months showed good results, including 6 patients with near complete (90% to 100%) repigmentation.225 Laboratory applications Tissue-engineered skin provides 3-dimensional models for nonclinical research. This has led to reduction of animal use in research. In addition, ES provides a platform for more meaningful study of cellular interactions with the ECM, given the differences in skin characteristics between animal models and human skin. Psoriasis skin model Psoriasis is a skin condition characterized on a histological level by thickened epidermis that extends deeply into dermis.226 In 1981, Krueger and colleagues observed that skin both involved and uninvolved with psoriasis, when removed from an affected individual, became equally hyperproliferative on transplantation to nude mice.227 Krueger and Jorgensen proposed an in vitro psoriasis model to track the progress of fibroblasts in the disease, especially in relation to excess keratinocyte proliferation and differentiation.228 Other instances of in vitro models include epidermal reconstruction in culture for staining with keratin monoclonal antibodies,229 and the adoption of a dermal equivalent in suspension to elucidate the mechanisms underpinning keratinocyte fibroblast hyperproliferation.230 More recently, Barker and colleagues reconstructed skin models from keratinocyte and fibroblast cultures, seeded from normal, involved psoriatic, and uninvolved psoriatic biopsies.231 However, in the case of Jean and colleagues, a self-assembly method was used in developing the skin construct, exploiting the capability of mesenchymal cells, such as fibroblasts, in producing their own ECM.232 This method allowed for improved dissection of the complex interactions in the causes of psoriasis, by removing select elements such as immunocytes. Study of skin pigmentation and skin melanoma model Reconstructed skin models are also useful in the study of pigmentation, melanocyte function, and the factors that regulate skin pigmentation.233-235 In an attempt to understand and qualify the pigmentation process at a cellular Vol. 217, No. 3, September 2013 level, there has been a shift from single and bilayered constructs to 3-dimensional models.233,234,236 In this model, new evidence postulated that fibroblasts might suppress melanocyte pigmentation in vivo.236 Similarly, when reviewing human skin models in relation to pigmentation, Berking and Herlyn highlighted the increased flexibility of skin constructs given the option of transplantation onto immunodeficient laboratory animals.233 The authors provided examples of the now-possible longterm carcinogenesis studies and the tracking of histology and immunochemical processes in skin grafts. Wound healing model The current gold standard in analysis of skin morphology, including the tracking of fibroblasts and keratinocytes, is the processing of a small biopsy.237,238 However, this is an invasive procedure that alters the original morphology of the sampled tissue.237 In addition, when studying the healing of an existing wound, creating a new wound through the biopsy procedure is not ideal.238 To avoid this, advanced, noninvasive imaging modalities are being used in tandem with traditional wound-healing ES models. In addition, these high-resolution techniques afford increased granularity in tracking fibroblasts and keratinocytes. Allergen and skin irritation model In light of criticisms of animal model data reproducibility in humans, Spiekstra and colleagues239 and Tornier and colleagues240 have demonstrated increased reproducibility with their in vitro skin models when compared with animal models. Spiekstra and colleagues reconstructed a neonatal foreskin epidermal keratinocyte model, and claimed a higher correlation between irritant and sensitizer potency when testing 2,4-di-nitro-chloro-benzene and cinnamaldehyde in comparison with a guinea pig model.241 Tornier and colleagues tested 50 compounds for their skin irritation potential on 3-dimensional reconstituted human epidermal cultures developed by SkinEthic, achieving good reproducibility, increased convenience, and reduced costs. CHALLENGES The time required for cells to replicate in vitro in sufficient quantities for clinical use, which averages 2 or 3 weeks, is a major challenge facing ES. Unfortunately, this necessary delay in the application of ES limits its practicality. In addition, the need for adherence to basic surgical principles cannot be overemphasized. Along with the time-frame issues of cellular replication, wound bed preparation is a key challenge facing any ES substitute requiring revascularization. It is also during this Kamel et al Tissue Engineering of Skin 547 time frame of engineered inosculation or vascular ingrowth that the skin substitute is most vulnerable to infection. A systematic review and meta-analysis by Ho and colleagues concludes that acellular dermal matrixassisted breast reconstruction shows higher incidence of infection and cellulitis.242 Wound infection has also been reported as one of the main adverse effects of cryopreserved human keratinocytes in the treatment of venous ulcers.111 Therefore, with ES substitutes, wound bed preparation can be even more of an important prerequisite than in the autograft skin treatment methodologies. In addition, without meticulous surgical debridement to achieve a vascularized dermis or dermal equivalent, it becomes very difficult for the keratinocytes to attach to the wound bed. This is especially so in the case of deep wounds with fat and granulation tissue that have less architecture to support dermal and epidermal cell growth. Some skin substitutes, for the reasons outlined here, need a 2-stage application procedure (see Tables 2 and 3) to achieve wound bed preparation and successful application with vascular ingrowth. This led to the idea of a synthesized reconstructed skin in the laboratory, in which attachment of the keratinocytes to the dermis is achieved before application.93 Inadequate angiogenesis can result in a poor take of the skin substitute. One proposed solution is to culture human dermal microvascular endothelial cells from the same host giving keratinocytes and fibroblasts.243 Another solution involves pretreatment of the wound to achieve vascularization before grafting.93 It has been suggested that early anastomosis between graft and wound bed vessels occurs within the central area of the graft.90 This indicates that ES with prefabricated vessels could potentially simulate autologous skin grafts with regard to their take.244 However, the current view is that the partial replacement of graft vasculature by endothelial and endothelial progenitor cells from the recipient along preexisting channels is the predominant mechanism for skin graft revascularization.245 This knowledge places more emphasis on wound bed preparation than the prefabrication of vessels in skin substitutes from a revascularization standpoint, as mentioned here. Another problem is hypopigmentation due to either the absence of melanocytes in many ES products57 or melanocyte retention in the cultured skin substitute, with the development of pigmented foci that are insufficient in restoring uniform distribution of skin color.246 Some skin substitutes, such as Apligraf, rely on allogenic skin cells, demonstrating the ability to aid wound healing and be readily available.206 Cell age in vitro is thought to have an effect on its survival time after application in vivo. Allogenic fetal fibroblasts show 548 Kamel et al Tissue Engineering of Skin a persistence pattern similar to that of syngeneic cells.247 However, persistence of cells after their application to the host is an issue affecting the success of ES. Scars at the margins of the skin substitute are of inferior functional and aesthetic quality compared with normal skin tissue, and are also less resistant to mechanical tension. To overcome this, future generations of ES should incorporate anti-scarring technologies.248 One other important problem limiting the practicality of skin substitutes is the transmission of infection through the manufacturing process. Although thorough precautions against pathogens are taken during the manufacturing of collagen from cadaveric skin, the question of contamination looms large.249 Engineered skin products relying on nonautologous donor cells exclusively need cells screened from cell banks to avoid viral infections. In xenobiotic products, bovine serum and porcine fibroblasts are used and the screening of these cell banks can be even more complex. Cell culture, whether of human or xenogeneic sources, is a predominant methodology to grow different types of cells for ES. However it is during the cell culture phase that bacterial and fungal contamination can occur, despite meticulous control.89 Although these are irradiated throughout the manufacturing process to decrease the risk of iatrogenic harm through transmission of micro-organisms, increased public concern about bovine spongioform encephalitis and prion disease suggests a limit of widespread use of xenobiotic culture products. Skin substitutes do not provide a complex system of differentiated structures that are present in normal skin, such as sweat glands, sebaceous glands, and hair follicles. Similarly, the absence of adipose tissue does not provide insulation. The lack of these elements results in poor temperature control. Engineered skin also lacks nerve endings, resulting in loss of sensation. In a recent study,250 authors suggest that dissociated mammalian skin cells can form a new hair follicle in vivo and that this mechanism has been highly conserved. The application of such technique could ameliorate the function of future skin substitutes. From a commercial point of view, TE skin substitutes have not achieved success. A systematic review by Langer and Rogowski concludes that cell-derived products used in diabetic and venous ulcers along with the standard care are costly in comparison with non-TE efforts.251 However, according to their study, economic evidence postulates that despite high initial costs, the use of TE wound care products can be cost effective or even cost saving when restricted to recalcitrant, unresponsive ulcers. Although relative biological effectiveness (RBE) assays have a range of laboratory applications, they are not necessarily able to replicate the pathological progression of the disease afforded by an animal model in melanoma J Am Coll Surg studies. It is believed that animal testing with different biological end points is still a necessary step on the path to clinical safety validation.252 Examples of the limitations specific to each ES product are mentioned in Tables 1 to 3. CONCLUSIONS Skin cells are of central importance to the future efforts for the complete healing of wounds. However, they survive poorly without ECM, which provides the structural template necessary for their growth. Given the lack of variety of cells in most of the current skin substitutes, keratinocytes alone do not lead to fully functional, regenerated skin. Unfortunately, these substitutes survive poorly without vascularization, which is reflected by the poor efficacy of skin cells in the treatment of burns that extend into the supportive layers of the dermis and hypodermis.253 There are many approaches to improving cellular therapy of skin. These include the choice of the type of cells, their source (autologous vs allogenic), and techniques aimed at enhancing cellular survival and physiological function post seeding. Future research should focus on how to decrease the risks of disease transmission in patients receiving cultured cells through development of reliable xenobiotic-free culture protocols. Additional research on the molecular basis of scar wound healing and simulation of scarless fetal healing will continue to contribute to skin TE strategies. Author Contributions Study conception and design: Kamel, Caterson Acquisition of data: Kamel, Ong Analysis and interpretation of data: Kamel, Ong, Junker Drafting of manuscript: Kamel, Ong, Junker Critical revision: Eriksson, Junker, Caterson REFERENCES 1. Skalak R, Fox CF. Tissue Engineering. New York: Alan R. Liss, Inc.; 1988. 2. Patil S, Li Z, Chan C. Cellular to tissue informatics: approaches to optimizing cellular function of engineered tissue. 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