- Review Article
- Open Access
Current progress of skin tissue engineering: Seed cells, bioscaffolds, and construction strategies
Burns & Trauma volume 1, pages 63–72 (2013)
The development of cell biology, molecular biology, and material science, has been propelling biomimic tissue-engineered skins to become more sophisticated in scientificity and more simplified in practicality. In order to improve the safety, durability, elasticity, biocompatibility, and clinical efficacy of tissue-engineered skin, several powerful seed cells have already found their application in wound repair, and a variety of bioactive scaff olds have been discovered to influence cell fate in epidermogenesis. These exuberant interests provide insights into advanced construction strategies for complex skin mimics. Based on these exciting developments, a complete full-thickness tissue-engineered skin is likely to be generated.
As the largest organ, skin covers the entire exterior of the body and takes over about 8% of the total body mass. Due to its direct exposure to potentially harmful microbial, thermal, mechanical, and chemical damages, the loss of skin can occur for many reasons, including disorders, acute trauma, chronic wounds, or even surgical interventions. Tissue-engineered skin (TES) substitutes represent a logical therapeutic option for the treatment of acute and chronic skin wounds. Since the successful isolation and cultivation of human epidermal keratinocytes in 1975, TES has developed from epidermal substitutes to full-thickness skin containing different seed cells [Figure 1]. According to their anatomical structures, skin substitute products could be classified into cellular epithelial autografts, engineered dermal substitutes, and engineered dermo-epidermal composite substitutes. All these products have become a prospective measure for clinical treatment of large full-thickness skin defects.
Recently, highly innovative progress in the development and clinical use of highly sophisticated TES in order to increase their medical safety, durability, elasticity, biocompatibility, and clinical efficacy has been reported. Here, we give a brief introduction to some representative progress in feasible seed cells and supportive biomaterials, as well as their applications in varied TES substitutes in developing our understanding of skin tissue engineering.
Seed cells for skin tissue engineering
Skin-derived seed cells
Up to 1980s, the development of in vitro expansion technique unveils the unlimited proliferation capacity of keratinocytes that, they are capable of being passaged for many hundreds of generations without undergoing senescence. Such exciting characteristics make keratinocyte an ideal alternative treatment for large burns, instead of skin grafting. Since then, numerous clinical trials have demonstrated the efficacious application of cultured keratinocytes in wound healing.[5,6] As the first step of treatment with skin substitutes, single cell suspension and multilayered “keratinocyte sheet” were explored.[7,8] However, due to lack of dermal structures, simple keratinocyte coverage is fragile and the take rate varies depending on the condition of the defected area. These major disadvantages make keratinocytes not optimal for the coverage of full-thickness defects [Table 1]. To further facilitate cell survival and growth, several bioscaffolds were comprised into the keratinocytes containing TES, such as collagen, gelatin, fibrin, chitosan, and various synthetic materials.[13–15] Moreover, in order to overcome some major drawbacks like scar formation and contracture, more dermal lineage seed cells need to be utilized to support the tissue-engineered constructs.
As the major cell type present in the dermis, fibroblast plays an important role in the wound healing process by producing extracellular matrix (ECM) proteins and cytokines. It has been shown that the presence of fibroblasts in three-dimensional (3D) constructs could significantly increase the survival and proliferation rate of keratinocytes. In a bilayered model, the underlying fibroblast promoted the keratinocyte layer into basal, granular, and cornified layers, in addition to promoting keratinocyte proliferation. In addition, the synthesis and deposition collagen and other ECM components form the basis of the granulation tissue, which is a critical process in wound healing. Besides, in response to site-specific microenvironment, fibroblasts from undamaged tissue are able to migrate to the wound and transdifferentiate into myofibroblasts that take on a contractile phenotype. Such characteristics make the fibroblasts a popular candidate for skin substitutes, especially those containing dermal equivalents.
Epidermal stem cells
Epidermal stem cells are a population of adult somatic stem cells specifically located in the basal cell layer of the epidermis. In addition to their low tendency to divide, their extensive and sustained self-renewal capacity plays an essential role in the lifelong epidermogenesis and wound repair of the skin.[20,21] Studies have shown that epidermal stem cells can be inducted into epidermis and its appendages such as sweat gland, hair follicle, and sebaceous follicle under certain microenvironments.[22–24] However, one major technical challenge that hampers the wide application of epidermal stem cells in skin tissue engineering is lack of specific surface antigens for their isolation or enrichment. To overcome this problem, several methods have been used to identify and differentiate them from other types of cells residing in the epidermis.[4,25,26] Since most epidermal stem cells were discovered from the “bulge” region of the hair follicle,[22,27] it is logically predicable to find out that epidermal stem cells have the ability to rebuild skin appendages such as hair follicle and sweat gland when applied in skin tissue engineering. Although the regulation of epidermal stem cell differentiation is not clearly understood, these researches provide hope that functional skin substitutes equipped with appendages can be generated for transplantation in, for example, cases of extensive burns.
Melanocytes are found in the lower layer of the epidermis and provide pigments to determine skin color. Theoretically, they are ideal seed cells to resolve the lacking of natural skin pigmentation which is one major esthetic concern in the current cultured skin grafts. In vitro reconstruction of keratinocytes combined with melanocytes on de-epidermized human dermis could result in a colored equivalent to natural epidermis. Our previous work also revealed the feasibility of melanocytes in skin tissue engineering. After transplantation of constructed skin containing keratinocytes, melanocytes, and dermal fibroblasts, athymic mice generated black skins within 6 weeks. Interestingly, in vivo research conducted by Hachiya et al. found that melanocytes are capable to spontaneously localize to the basal layer from a mixed cell slurry containing keratinocytes, fibroblasts, and melanocytes. In addition, the synthesized melanosome could be correctly transferred to keratinocytes after ultraviolet irradiation. However, functional pigmentation is only the beginning, advanced method on controlling the production of appropriate amount of melanin deserves further investigation, in order to make TES visually indistinguishable with its surrounding tissue.
Embryonic stem cells
Embryonic stem cells (ESCs) are referred as pluripotent cells derived from inner cell mass of a blastocyst. With non-senescing self-renewal and differentiation capability into three germ layers, ESCs might offer a way to meet the challenge of biomimetic complex skin constructs. To initiate epidermal differentiation of ESCs, several protocols have been developed.[32–34] Besides, the transcriptional similarity of ESC-derived keratinocyte lineage and primary foreskin keratinocytes has been proved by microarray analysis, suggesting these cell types respond to specific microenvironment in a similar manner. When grown in specific media and substrate conditions, ectodermal and mesenchymal cells derived from ESCs are capable of generating 3D epithelial tissue. In a preclinical study, ESC-derived keratinocytes formed a pluristratified epidermis when seeded on an artificial matrix and finally regenerated a fully functional mature skin-like structure after in vivo implantation. These studies provide a theoretical basis for the use of ESCs as seed cells for TES. However, embryonic stem cell research is handicapped by moral and ethical restraints, and the source of ESCs is thus problematic.
Inducible pluripotent stem cells
In 2006, inducible pluripotent stem cells (iPSCs) were first discovered by Yamanaka and colleagues, who received The Nobel Prize in Physiology or Medicine in 2012 (jointly with John B. Gurdon) for the finding that mouse fibroblasts could acquire properties similar to those of ESCs after “reprogramming.”[39,40] Since this milestone, iPSCs have become a promising new source of stem cells for skin tissue engineering without any moral and ethical controversies. Recent publications show that procedures differentiating mouse iPSCs into epidermal keratinocytes are similar to keratinocyte differentiation of ESCs.[32,41–43] By using the iPSCs-derived keratinocyte lineage cells, Bilousova et al., have successfully regenerated epidermis, hair follicles, and sebaceous glands in the skin of athymic nude mouse. Though the exciting development has been achieved, utility of iPSCs for medical applications is still pending because of the possibility that the transgene technology may cause carcinogenesis and tumor formation. As novel technologies relating to iPSCs are rapidly being developed, the therapeutic applicability of iPSCs in skin tissue engineering and regenerative medicine will eventually be a reality.
Mesenchymal stem cells
Mesenchymal stem cells are identified from functional tissues after birth (such as bone marrow, adipose, blood, and umbilical cord) and differ from ESCs in that they are more restricted in their proliferation and differentiation potential and cannot spontaneously give rise to complex tissues in vitro. Among these, bone marrow mesenchymal stem cells (BMSCs) and adipose-derived stem cells (ADSCs) are perhaps the most promising candidates for skin regeneration. The potential of such MSCs to differentiate into dermal cells has been described.[49,50] However, unlike many encouraging publications in the past decade that the differentiation of BMSCs across germinal boundaries is crucial for regeneration of injured tissues, recent reports suggest that the growth factors secreted by BMSCs are more important in stimulating proliferation and maturation of endogenous stem cell-like progenitors and decreasing inflammatory reactions.[51,52] Except for the trophic and immunomodulatory functions, BMSCs demonstrated abundant ECM remodeling, matrix contraction, and migration capability when cultured on dermal equivalents.[53,54] Evidences as our work showed that MSCs containing skin equivalent exhibited better healing and keratinization, less wound contraction, and more vascularization in a porcine deep partial-thickness burn wound model. One study of a murine model of full-thickness defect treated with a biograft composed of ADSCs showed increased rate of re-epithelialization and vascularization. Our preliminary work researched the vascularization capacities of two different scaffolds seeded with ADSCs — decellularized small intestinal submucosa (SIS) and acellular dermal matrix (ADM) — and found that both the ADSCs combining biomaterials revealed enhanced angiogenesis and wound healing rate compared with the non-seeded scaffolds. All these findings strongly demonstrate that adult BMSCs/ADSCs can contribute to the healing of injured skin and might provide a cell resource for building TES.
Delivering cells of endothelial origin remains the most systematic approach to enhance neovascularization in TES equivalents, as these cells are destined to directly contribute to vessel formation.[58,59] Moreover, interactions between different seed cells with endothelial cells (ECs) may contribute to vascularization in TES through angiogenesis and/or vasculogenesis. By co-culturing dermal fibroblasts and ECs in collagen sponge in vitro, a complex vascular network with branching structures was formed. In subsequent research using skin fibroblasts and ECs, Kunz-Schughart et al. verified that fibroblast could interact and support the migration, viability, and network formation of ECs. Our result demonstrates an obvious implication of this work that a scaffold-free bilayered TES containing dermal fibroblasts together with dermal microvascular ECs could form capillary-like structures after 20 days of culture. Also, this vascularization process is associated with interactions among keratinocytes, fibroblasts, and ECs. To further facilitate the clinical use of ECs, however, researches on overcoming their disadvantages like limited in vitro expansion capacity and potent immunogenicity are desiderated.
Due to the easy-to-gain and layered pattern, human amniotic membrane (AM) has a long history in skin wound healing. Except for its mechanical support and protection, the epithelial regeneration and immunosuppressive potential of cellular components (namely, amniotic epithelial cells and amniotic mesenchymal cells) in AM have received much attention recently For the first time, our previous research has shown the feasibility of both amniotic epithelial cells and amniotic mesenchymal cells in reconstruction of bilayered skin equivalent and thereafter skin regeneration. Besides, recent work indicates that amniotic fluid-derived cells could act as a substitute seed cell for fibroblast in tissue-engineered dermo-epidermal skin analog. Taken together, these findings suggest amnion might be a new source for obtaining the seed cells to develop new skin products for clinical application.
Bioscaffolds in skin tissue engineering
The selection and optimization of a biomaterial scaffold plays a pivotal role in skin tissue engineering. Except for their mechanically supportive function, efficacious biocompatible scaffolds should assist the successful engraftment of TES, as they expediently accrete wound and promote granulation tissue formation, fibroblast-driven remodeling, angiogenesis, and re-epithelialization. However, novel concepts of skin tissue engineering extend new and specific requirements for biomimetic scaffolds, based on which many interesting natural and synthetic polymers have been used in constructing artificial skin.
As a major ECM protein of the dermal layer of the skin, collagen is the most widely studied and clinically utilized natural scaffold available for TES substitutes. The advantages include good biocompatibility, proper porous structure, as well as low immunogenicity.[55,67,69] However, the poor mechanical strength and rapid biodegradation rate of natural collagen scaffolds limiting the graft instability are the critical disadvantages that hamper its applications. Therefore, to control the degradation, numerous works have focused on the mechanical properties of collagen, such as chemical and biophysical cross-linking techniques[70,71] or a structural modification method like dense film. For example, addition of matrix protein tropoelastin to type I collagen enhanced the proliferation and migration rates of dermal fibroblasts in vitro. Cross-linking collagen with chitosan after electrospinning resulted in a good potential for keratinocyte migration and wound re-epithelialization. Addition of fibroblast growth factor 2 (FGF-2) or vascular endothelial growth factor (VEGF) to heparin cross-linked collagen scaffold increased its angiogenic potential. Although effective modification and improvement have been made, other discouraging problems in collagen-based polymers for skin tissue engineering still exist, including the high cost of purification process, potential viral and prior contamination, and variability in the physicochemical properties depending on source and processing.
Except for collagen, some other natural polymers have been investigated as scaffolds for skin tissue engineering, including gelatin, hyaluronic acid, fibrin, laminin, and elastin. Under various process conditions, these proteinic polymers could form different phases such as suspensions, gels,[78,79] sponges, films, or sheets. Though these naturally derived molecules have been considered advantageous in their cell interaction and signaling contributions, the mechanical properties of these materials are often poor in comparison to the properties of synthetic materials. Therefore, the noncollagenous biomaterials, like collagen itself, are often organized into synthetic matrices or to retain their stability and mimic the natural ECM.
ADM, which is derived from full-thickness skin by removing cells and cellular components rather than native dermal structure and extracellular proteins, has been successfully used both in pre-clinical animal studies and in human clinical applications.[83,84] Retaining structural and functional proteins that constitute the ECM, including collagen, fibronectin, laminin, and vimentin, ADM provides an intrinsic microenvironment for cell adhesion and proliferation. Previous studies demonstrate that endogenous growth factors such as VEGF, FGF-2, and transforming growth factor β1 remain on ADM and are released into the surrounding tissue to accelerate processes such as angiogenesis, cell recruitment, cell division, and even potential antimicrobial activity.[86,87] Such biological benefits have led to the application of ADM-based skin substitutes in burn coverage for decades.[88,89] Besides its natural structures, our results show that even micronized ADM still facilitates cell adhesion and growth in vitro as well as forms a thick layered tissue when transplanted into subcutaneous muscle.
Compared to biological biomaterials, artificially synthesized polymers such as polyurethane (PU), polypropylene (PP), polyglycolic acid (PGA), polylactic acid (PLA), and their copolymer poly (lactic-co-glycolic acid) (PLGA) display controllable mechanical properties and diverse plasticity. It has been proved that PLGA-knitted matrix exhibits sufficient internal space for tissue ingrowth, in addition to its skeletal role in enhancing natural biopolymers like collagen.[93,94] Other polymeric biomaterials like polyvinyl alcohol (PVA) are capable of increasing structural stability and tensile strength, and improving initial cell proliferation when blended with collagen. Unfavorable cell adhesion materials like poly (dl-lactide) (PDLLA) could interact and integrate well with dermal fibroblasts after addition of 30% poly (ethylene glycol) (PEG) before electrospinning. Polyhydroxybutyrate (PHB) combines with organic-soluble chitosan and also reveals beneficial effect on promoting cell attachment and proliferation. The progress in controlling scaffold property is exciting, but most of the current studies just demonstrate novel materials or methods without making comparison with any other available TES constructs. Predictably, but disappointingly, these works accumulate limited knowledge about the materials and their properties that are most suitable for TES.
To differentiate natural and artificial biomaterials from one another in only one specific aspect, several arrays screening of biomaterial have been developed.[98–100] These efforts provide in vitro pre-screening data for the selection of appropriate set of matrix molecules for skin tissue engineering. However, due to the complexity of in vivo microenvironment, more works such as testing different types of cells and additional modifications need to be extended in order to increase the possibility to successfully predict optimal biomaterials for skin tissue engineering.
Current strategies for TES construction
With various approaches presently being developed in different laboratories and companies, a number of artificial skin equivalents are commercially available and many others are under development. According to the complexity of their anatomical structures, the construction of TES could be classified from single-layered ones to ones with more than two layers.
Based on the achievements made in in vitro cultivation and expansion of human keratinocytes, multiple autologous epidermal substitutes have been developed for re-epithelialization either by cell suspension (e.g. CellSpray), subconfluent layer (e.g. Myskin) or by sheet (e.g. Epicel, EpiDex, and EPIBASE). The application of epidermal substitutes directly mitigates the deficiency of skin biopsy. However, the clinical integration of such cultured epithelial autografts is unpredictable and mostly relies on the condition of the wound bed, rather than the terminally differentiated keratinocytes. Besides, the poor mechanical property and development of contractures due to the lack of dermis are also problematic to the epidermal substitutes. This resulted in further requirements concerning the development of dermal substitutes.
The mechanical stability and elasticity provided by dermal layer could prevent wound contraction and scar hyperplasia. In case of full-thickness burns, both dermal and epidermal equivalents must be applied consecutively, in order to generate suitable nutritional and immunological environment before the application of epidermal layer. A large variety of acellularized dermal substitutes mainly produced from allogeneic, xenogeneic dermal matrices (e.g. AlloDerm, GraftJacket, and Matriderm) or synthetic materials (e.g. Integra, Biobrane, and Hyalomatrix PA) could stimulate the ingrowth of autogeneic ECs and fibroblasts that help defected areas form dermal structure after transplantation, while some of the bioactive dermal substitutes consisting of human neonatal fibroblasts (e.g. Dermagraft and TransCyte) spontaneously show benefits in vascularization, epidermalization, as well as ECM formation.
Double-layered skin substitutes combine both epidermal and dermal layers to histologically mimic the structure of normal skin. Without economic consideration, currently the epidermal/dermal composite TES (e.g. Apligraf) represents the best treatment for skin repair, when compared with single-layered products. In skin tissue engineering, the interaction between parenchyma and stroma appears to be instructive in programming tissue structure and function during epidermogenesis. It is widely accepted that the inductive effects of fibroblasts on epithelial morphogenesis are mediated by cell-cell interactions and ECM secretion.[118,119] Due to the interplays between multiple seed cell types, epidermal/dermal substitutes show enhanced wound closure and keratinization capability. A full-thickness living skin analogue (Activskin) developed by our team has been used to treat refractory ulcers successfully in clinical applications. The treatment of refractory ulcers has drawn greater attention in recent decades owing to the increase in life expectancy in the industrialized world and the associated increase in the prevalence of comorbid conditions such as diabetes and vascular disease.
More complicated substitutes
In addition to the clinically available TES, great efforts have been made exploring optimized 3D constructs to repair skin tissue. Recent advances in skin biology have stressed the importance of cell-cell interactions during epidermal morphogenesis. In particular, progress in the field of skin tissue engineering has contributed tremendously to our knowledge about in vitro epidermal morphogenesis, resulting in the reconstruction of highly sophisticated and innovative 3D skin equivalents that mimic human skin in terms of tissue architecture and function, including hair follicle, capillary network,[59,62] sensory innervation, adipose tissue, and pigment production.[30,125] When grown in the environment that mimics the anatomical position of skin, these bioengineered skins present definite differences in epidermal regeneration according to the types of seed cells used in the dermal substitutes. An exhilarating advancement is the elucidation of the crucial role of MSCs in skin regeneration. Apart from the morphogenesis potential, recent experimental evidence also demonstrates that BMSCs combined with epidermal stem cells could accelerate wound re-epithelialization and have better therapeutic potential in activating blood vessel and hair follicle formation than epidermal stem cells alone. Like BMSCs, ADSCs are capable of differentiating into various skin cells which subsequently contribute to the wound healing. The result obtained in our recent study in a bilayered TES system shows that a mixture of dermal fibroblasts and ADSCs in a ratio of 1:1 is superior to fibroblasts or ADSCs alone in promoting keratinocyte proliferation and differentiation. With the steps into wound healing echanisms, proper combinations of cells and their interplays in initiating healing may be a way out for permanent TES construction.
Challenges and perspectives
Currently, most of the TES could only function as temporary substitutions in skin wound healing. Lack of immediate blood supply and activation of immune rejection are the two major problems preventing the permanent integration of allogeneic TES. Evidences have shown that most of the cells contained in TES do not survive for 1 month after transplantation. This temporary nature, whether the substitutes are degradable or have to be removed, makes TES predominantly serve as “coverings” instead of a real organic regenerator. However, it is reasonable to believe that with the development of skin biology, material science, and engineering technology, the TES will finally have an equivalent structural and functional therapeutic outcome as autogeneic skin transplantation does.
Shevchenko RV, James SL, James SE. A review of tissue-engineered skin bioconstructs available for skin reconstruction. J R Soc Interface 2010;7:229–58.
Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells. Cell 1975;6:331–43.
Bottcher-Haberzeth S, Biedermann T, Reichmann E. Tissue engineering of skin. Burns 2010;36:450–60.
Abbas O, Mahalingam M. Epidermal stem cells: Practical perspectives and potential uses. Br J Dermatol 2009;161:228–36.
Wood FM, Kolybaba ML, Allen P. The use of cultured epithelial autograft in the treatment of major burn wounds: Eleven years of clinical experience. Burns 2006;32:538–44.
Clugston PA, Snelling CF, Macdonald IB, Maledy HL, Boyle JC, Germann E, et al. Cultured epithelial autografts: Three years of clinical experience with eighteen patients. J Burn Care Rehabil 1991;12:533-9.
Lootens L, Brusselaers N, Beele H, Monstrey S. Keratinocytes in the treatment of severe burn injury: An update. Int Wound J 2013;10:6–12.
Cuono C, Langdon R, McGuire J. Use of cultured epidermal autografts and dermal allografts as skin replacement after burn injury. Lancet 1986;1:1123–4.
Butler CE, Orgill DP. Simultaneous in vivo regeneration of neodermis, epidermis, and basement membrane. Adv Biochem Eng Biotechnol 2005;94:23–41.
Duan H, Feng B, Guo X, Wang J, Zhao L, Zhou G, et al. Engineering of epidermis skin grafts using electrospun nanofibrous gelatin/ polycaprolactone membranes. Int J Nanomedicine 2013;8:2077–84.
Currie LJ, Sharpe JR, Martin R. The use of fibrin glue in skin grafts and tissue-engineered skin replacements: A review. Plast Reconstr Surg 2001;108:1713–26.
Howling GI, Dettmar PW, Goddard PA, Hampson FC, Dornish M, Wood EJ. The effect of chitin and chitosan on the proliferation of human skin fibroblasts and keratinocytes in vitro. Biomaterials 2001;22:2959–66.
Kao B, Kadomatsu K, Hosaka Y. Construction of synthetic dermis and skin based on a self-assembled peptide hydrogel scaffold. Tissue Eng Part A 2009;15:2385–96.
Blackwood KA, McKean R, Canton I, Freeman CO, Franklin KL, Cole D, et al. Development of biodegradable electrospun scaffolds for dermal replacement. Biomaterials 2008;29:3091–104.
Sarkar SD, Farrugia BL, Dargaville TR, Dhara S. Chitosan-collagen scaffolds with nano/microfibrous architecture for skin tissue engineering. J Biomed Mater Res A 2013.
Sun T, Jackson S, Haycock JW, MacNeil S. Culture of skin cells in 3D rather than 2D improves their ability to survive exposure to cytotoxic agents. J Biotechnol 2006;122:372–81.
Sun T, Mai S, Norton D, Haycock JW, Ryan AJ, MacNeil S. Self-organization of skin cells in three-dimensional electrospun polystyrene scaffolds. Tissue Eng 2005;11:1023–33.
el-Ghalbzouri A, Gibbs S, Lamme E, Van Blitterswijk CA, Ponec M. Effect of fibroblasts on epidermal regeneration. Br J Dermatol 2002;147:230–43.
Sonnemann KJ, Bement WM. Wound repair: Toward understanding and integration of single-cell and multicellular wound responses. Annu Rev Cell Dev Biol 2011;27:237–63.
Blanpain C. Stem cells: Skin regeneration and repair. Nature 2010;464:686–7.
Ikuta S, Sekino N, Hara T, Saito Y, Chida K. Mouse epidermal keratinocytes in three-dimensional organotypic coculture with dermal fibroblasts form a stratified sheet resembling skin. Biosci Biotechnol Biochem 2006;70:2669–75.
Zhang CP, Fu XB. Therapeutic potential of stem cells in skin repair and regeneration. Chin J Traumatol 2008;11:209–21.
Fernandes KJ, McKenzie IA, Mill P, Smith KM, Akhavan M, Barnabe-Heider F, et al. A dermal niche for multipotent adult skin-derived precursor cells. Nat Cell Biol 2004;6:1082–93.
Ito M, Liu Y, Yang Z, Nguyen J, Liang F, Morris RJ, et al. Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis. Nat Med 2005;11:1351–4.
Dunnwald M, Tomanek-Chalkley A, Alexandrunas D, Fishbaugh J, Bickenbach JR. Isolating a pure population of epidermal stem cells for use in tissue engineering. Exp Dermatol 2001;10:45–54.
Lei XH, Ning LN, Cao YJ, Liu S, Zhang SB, Qiu ZF, et al. NASA-approved rotary bioreactor enhances proliferation of human epidermal stem cells and supports formation of 3D epidermis-like structure. PloS One 2011;6:e26603.
Stenn KS, Cotsarelis G Bioengineering the hair follicle: Fringe benefits of stem cell technology. Curr Opin Biotechnol 2005;16:493–7.
Gagnon V, Larouche D, Parenteau-Bareil R, Gingras M, Germain L, Berthod F Hair follicles guide nerve migration in vitro and in vivo in tissue-engineered skin. J Invest Dermatol 2011;131:1375–8.
Rehder J, Souto LR, Issa CM, Puzzi MB. Model of human epidermis reconstructed in vitro with keratinocytes and melanocytes on dead de-epidermized human dermis. Sao Paulo Med J 2004;122:22–5.
Liu Y, Suwa F, Wang X, Takemura A, Fang YR, Li Y, et al. Reconstruction of a tissue-engineered skin containing melanocytes. Cell Biol Int 2007;31:985–90.
Hachiya A, Sriwiriyanont P, Kaiho E, Kitahara T, Takema Y, Tsuboi R. An in vivo mouse model of human skin substitute containing spontaneously sorted melanocytes demonstrates physiological changes after UVB irradiation. J Invest Dermatol 2005;125:364–72.
Metallo CM, Ji L, de Pablo JJ, Palecek SP. Retinoic acid and bone morphogenetic protein signaling synergize to efficiently direct epithelial differentiation of human embryonic stem cells. Stem Cells 2008;26:372–80.
Aberdam E, Barak E, Rouleau M, de LaForest S, Berrih-Aknin S, Suter DM, et al. A pure population of ectodermal cells derived from human embryonic stem cells. Stem Cells 2008;26:440–4.
Inanc B, Elcin AE, Unsal E, Balos K, Parlar A, Elcin YM. Differentiation of human embryonic stem cells on periodontal ligament fibroblasts in vitro. Artif Organs 2008;32:100–9.
Metallo CM, Azarin SM, Moses LE, Ji L, de Pablo JJ, Palecek SP. Human embryonic stem cell-derived keratinocytes exhibit an epidermal transcription program and undergo epithelial morphogenesis in engineered tissue constructs. Tissue Eng Part A 2010;16:213–23.
Hewitt KJ, Shamis Y, Carlson MW, Aberdam E, Aberdam D, Garlick JA. Three-dimensional epithelial tissues generated from human embryonic stem cells. Tissue Eng Part A 2009;15:3417–26.
Guenou H, Nissan X, Larcher F, Feteira J, Lemaitre G, Saidani M, et al. Human embryonic stem-cell derivatives for full reconstruction of the pluristratified epidermis: A preclinical study. Lancet 2009;374:1745–53.
Han YF, Tao R, Sun TJ, Chai JK, Xu G, Liu J. Advances and opportunities for stem cell research in skin tissue engineering. Eur Rev Med Pharmacol Sci 2012;16:1873–7.
Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663–76.
Qi H, Pei D. The magic of four: Induction of pluripotent stem cells from somatic cells by Oct4, Sox2, Myc and Klf4. Cell Res 2007;17:578–80.
Bilousova G, Chen J, Roop DR. Differentiation of mouse induced pluripotent stem cells into a multipotent keratinocyte lineage. J Invest Dermatol 2011;131:857–64.
Sakurai M, Hayashi R, Kageyama T, Yamato M, Nishida K. Induction of putative stratified epithelial progenitor cells in vitro from mouse-induced pluripotent stem cells. J Artif Organs 2011;14:58–66.
Tolar J, Xia L, Riddle MJ, Lees CJ, Eide CR, McElmurry RT, et al. Induced pluripotent stem cells from individuals with recessive dystrophic epidermolysis bullosa. J Invest Dermatol 2011;131:848–56.
Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S. Generation of mouse induced pluripotent stem cells without viral vectors. Science 2008;322:949–53.
Uitto J. Regenerative medicine for skin diseases: iPS cells to the rescue. J Invest Dermatol 2011;131:812–4.
Vogel G. Stem cells. Diseases in a dish take off. Science 2010;330:1172–3.
Badylak SF, Taylor D, Uygun K. Whole-organ tissue engineering: Decellularization and recellularization of three-dimensional matrix scaffolds. Annu Rev Biomed Eng 2011;13:27–53.
Hodgkinson T, Bayat A. Dermal substitute-assisted healing: Enhancing stem cell therapy with novel biomaterial design. Arch Dermatol Res 2011;303:301–15.
Wu Y, Chen L, Scott PG, Tredget EE. Mesenchymal stem cells enhance wound healing through differentiation and angiogenesis. Stem Cells 2007;25:2648–59.
Li H, Fu X, Ouyang Y, Cai C, Wang J, Sun T. Adult bone-marrow-derived mesenchymal stem cells contribute to wound healing of skin appendages. Cell Tissue Res 2006;326:725–36.
Caplan AI. Why are MSCs therapeutic? New data: New insight. J Pathol 2009;217:318–24.
Rasmusson I, Le Blanc K, Sundberg B, Ringden O. Mesenchymal stem cells stimulate antibody secretion in human B cells. Scand J Immunol 2007;65:336–43.
Schneider RK, Neuss S, Stainforth R, Laddach N, Bovi M, Knuechel R, et al. Three-dimensional epidermis-like growth of human mesenchymal stem cells on dermal equivalents: Contribution to tissue organization by adaptation of myofibroblastic phenotype and function. Differentiation 2008;76:156–67.
Schneider RK, Puellen A, Kramann R, Raupach K, Bornemann J, Knuechel R, et al. The osteogenic differentiation of adult bone marrow and perinatal umbilical mesenchymal stem cells and matrix remodelling in three-dimensional collagen scaffolds. Biomaterials 2010;31:467–80.
Liu P, Deng Z, Han S, Liu T, Wen N, Lu W, et al. Tissue-engineered skin containing mesenchymal stem cells improves burn wounds. Artif Organs 2008;32:925–31.
Huang SP, Hsu CC, Chang SC, Wang CH, Deng SC, Dai NT, et al. Adipose-derived stem cells seeded on acellular dermal matrix grafts enhance wound healing in a murine model of a full-thickness defect. Ann Plast Surg 2012;69:656–62.
Liu S, Zhang H, Zhang X, Lu W, Huang X, Xie H, et al. Synergistic angiogenesis promoting effects of extracellular matrix scaffolds and adipose-derived stem cells during wound repair. Tissue Eng Part A 2011;17:725–39.
Hendrickx B, Vranckx JJ, Luttun A. Cell-based vascularization strategies for skin tissue engineering. Tissue Eng Part B Rev 2011;17:13–24.
Zhang X, Yang J, Li Y, Liu S, Long K, Zhao Q, et al. Functional neovascularization in tissue engineering with porcine acellular dermal matrix and human umbilical vein endothelial cells. Tissue Eng Part C Methods 2011;17:423–33.
Hudon V, Berthod F, Black AF, Damour O, Germain L, Auger FA. A tissue-engineered endothelialized dermis to study the modulation of angiogenic and angiostatic molecules on capillary-like tube formation in vitro. Br J Dermatol 2003;148:1094–104.
Kunz-Schughart LA, Schroeder JA, Wondrak M, van Rey F, Lehle K, Hofstaedter F, et al. Potential of fibroblasts to regulate the formation of three-dimensional vessel-like structures from endothelial cells in vitro. Am J Physiol Cell Physiol 2006;290:C1385–98.
Liu Y, Luo H, Wang X, Takemura A, Fang YR, Jin Y, et al. In vitro construction of scaffold-free bilayered tissue-engineered skin containing capillary networks. Biomed Res Int 2013;2013:561410.
Kesting MR, Wolff KD, Hohlweg-Majert B, Steinstraesser L. The role of allogenic amniotic membrane in burn treatment. J Burn Care Res 2008;29:907–16.
Shimmura S, Shimazaki J, Ohashi Y, Tsubota K. Antiinflammatory effects of amniotic membrane transplantation in ocular surface disorders. Cornea 2001;20:408–13.
Li H, Chu Y, Zhang Z, Zhang G, Jiang L, Wu H, et al. Construction of bilayered tissue-engineered skin with human 71 amniotic mesenchymal cells and human amniotic epithelial cells. Artif Organs 2012;36:911–9.
Hartmann-Fritsch F, Hosper N, Luginbuhl J, Biedermann T, Reichmann E, Meuli M. Human amniotic fluid derived cells can competently substitute dermal fibroblasts in a tissue-engineered dermo-epidermal skin analog. Pediatr Surg Int 2013;29:61–9.
Ruszczak Z. Effect of collagen matrices on dermal wound healing. Adv Drug Deliv Rev 2003;55:1595–611.
Shin H, Jo S, Mikos AG. Biomimetic materials for tissue engineering. Biomaterials 2003;24:4353-64.
Nam K, Kimura T, Funamoto S, Kishida A. Preparation of a collagen/polymer hybrid gel designed for tissue membranes. Part I: Controlling the polymer-collagen cross-linking process using an ethanol/water co-solvent. Acta Biomater 2010;6:403–8.
Weadock KS, Miller EJ, Keuffel EL, Dunn MG. Effect of physical crosslinking methods on collagen-fiber durability in proteolytic solutions. J Biomed Mater Res 1996;32:221–6.
Wang HM, Chou YT, Wen ZH, Wang ZR, Chen CH, Ho ML. Novel biodegradable porous scaffold applied to skin regeneration. PloS One 2013;8:e56330.
Faraj KA, van Kuppevelt TH, Daamen WF Construction of collagen scaffolds that mimic the three-dimensional architecture of specific tissues. Tissue Eng 2007;13:2387–94.
Rnjak-Kovacina J, Wise SG, Li Z, Maitz PK, Young CJ, Wang Y, et al. Electrospun synthetic human elastin: Collagen composite scaffolds for dermal tissue engineering. Acta Biomater 2012;8:3714–22.
Nillesen ST, Geutjes PJ, Wismans R, Schalkwijk J, Daamen WF, van Kuppevelt TH. Increased angiogenesis and blood vessel maturation in acellular collagen-heparin scaffolds containing both FGF2 and VEGF. Biomaterials 2007;28:1123–31.
Lynn AK, Yannas IV, Bonfield W. Antigenicity and immunogenicity of collagen. J Biomed Mater Res B Appl Biomater 2004;71:343–54.
Lee CH, Singla A, Lee Y. Biomedical applications of collagen. Int J Pharm 2001;221:1–22.
Khor HL, Ng KW, Schantz JT, Phan TT, Lim TC, Teoh SH, et al. Poly (?-caprolactone) films as a potential substrate for tissue engineering an epidermal equivalent. Mater Sci Eng C 2002;20:71–5.
Sun T, Haycock J, MacNeil S. In situ image analysis of interactions between normal human keratinocytes and fibroblasts cultured in three-dimensional fibrin gels. Biomaterials 2006;27:3459–65.
Wang TW, Sun JS, Wu HC, Tsuang YH, Wang WH, Lin FH. The effect of gelatin-chondroitin sulfate-hyaluronic acid skin substitute on wound healing in SCID mice. Biomaterials 2006;27:5689–97.
Lee SB, Kim YH, Chong MS, Hong SH, Lee YM. Study of gelatin-containing artificial skin V: Fabrication of gelatin scaffolds using a salt-leaching method. Biomaterials 2005;26:1961–8.
Meana A, Iglesias J, Del Rio M, Larcher F, Madrigal B, Fresno M, et al. Large surface of cultured human epithelium obtained on a dermal matrix based on live fibroblast-containing fibrin gels. Burns 1998;24:621–30.
Hodgkinson T, Bayat A. Dermal substitute-assisted healing: Enhancing stem cell therapy with novel biomaterial design. Arch Dermatol Res 2011;303:301–15.
Chun YS, Verma K, Rosen H, Lipsitz S, Morris D, Kenney P, et al. Implant-based breast reconstruction using acellular dermal matrix and the risk of postoperative complications. Plast Reconstr Surg 2010;125:429–36.
Askari M, Cohen MJ, Grossman PH, Kulber DA. The use of acellular dermal matrix in release of burn contracture scars in the hand. Plast Reconstr Surg 2011;127:1593–9.
Ge L, Zheng S, Wei H. Comparison of histological structure and biocompatibility between human acellular dermal matrix (ADM) and porcine ADM. Burns 2009;35:46–50.
Derwin KA, Badylak SF, Steinmann SP, Iannotti JP. Extracellular matrix scaffold devices for rotator cuff repair. J Shoulder Elbow Surg 2010;19:467–76.
Reing JE, Zhang L, Myers-Irvin J, Cordero KE, Freytes DO, Heber-Katz E, et al. Degradation products of extracellular matrix affect cell migration and proliferation. Tissue Eng Part A 2009;15:605–14.
Chen RN, Ho HO, Tsai YT, Sheu MT. Process development of an acellular dermal matrix (ADM) for biomedical applications. Biomaterials 2004;25:2679–86.
Callcut R, Schurr M, Sloan M, Faucher L. Clinical experience with Alloderm: A one-staged composite dermal/epidermal replacement utilizing processed cadaver dermis and thin autografts. Burns 2006;32:583–8.
Zhang X, Deng Z, Wang H, Yang Z, Guo W, Li Y, et al. Expansion and delivery of human fibroblasts on micronized acellular dermal matrix for skin regeneration. Biomaterials 2009;30:2666–74.
Sanders J, Stiles C, Hayes C. Tissue response to single-polymer fibers of varying diameters: Evaluation of fibrous encapsulation and macrophage density. J Biomed Mater Res 2000;52:231–7.
Dai W, Kawazoe N, Lin X, Dong J, Chen G. The influence of structural design of PLGA/collagen hybrid scaffolds in cartilage tissue engineering. Biomaterials 2010;31:2141–52.
Chen X, Qi YY, Wang LL, Yin Z, Yin GL, Zou XH, et al. Ligament regeneration using a knitted silk scaffold combined with collagen matrix. Biomaterials 2008;29:3683–92.
Chen G, Sato T, Ohgushi H, Ushida T, Tateishi T, Tanaka J. Culturing of skin fibroblasts in a thin PLGA-collagen hybrid mesh. Biomaterials 2005;26:2559–66.
Lin HY, Kuo YJ, Chang SH, Ni TS. Characterization of electrospun nanofiber matrices made of collagen blends as potential skin substitutes. Biomed Mater 2013;8:025009.
Cui W, Zhu X, Yang Y, Li X, Jin Y. Evaluation of electrospun fibrous scaffolds of poly (dl-lactide) and poly (ethylene glycol) for skin tissue engineering. Mater Sci Eng C 2009;29:1869–76.
Ma G, Yang D, Wang K, Han J, Ding S, Song G, et al. Organic?soluble chitosan/polyhydroxybutyrate ultrafine fibers as skin regeneration prepared by electrospinning. J Appl Polym Sci 2010;118:3619–24.
Lammers G, Tjabringa GS, Schalkwijk J, Daamen WF, van Kuppevelt TH. A molecularly defined array based on native fibrillar collagen for the assessment of skin tissue engineering biomaterials. Biomaterials 2009;30:6213–20.
Yliperttula M, Chung BG, Navaladi A, Manbachi A, Urtti A. High-throughput screening of cell responses to biomaterials. Eur J Pharm Sci 2008;35:151–60.
Kennedy SB, Washburn NR, Simon CG Jr, Amis EJ. Combinatorial screen of the effect of surface energy on fibronectin-mediated osteoblast adhesion, spreading and proliferation. Biomaterials 2006;27:3817–24.
MacNeil S. Progress and opportunities for tissue-engineered skin. Nature 2007;445:874–80.
Navarro FA, Stoner ML, Park CS, Huertas JC, Lee HB, Wood FM, et al. Sprayed keratinocyte suspensions accelerate 72 epidermal coverage in a porcine microwound model. J Burn Care Rehabil 2000;21:513–8.
Haddow DB, Steele DA, Short RD, Dawson RA, Macneil S. Plasma-polymerized surfaces for culture of human keratinocytes and transfer of cells to an in vitro wound-bed model. J Biomed Mater Res A 2003;64:80–7.
Wright KA, Nadire KB, Busto P, Tubo R, McPherson JM, Wentworth BM. Alternative delivery of keratinocytes using a polyurethane membrane and the implications for its use in the treatment of full-thickness burn injury. Burns 1998;24:7–17.
Tausche AK, Skaria M, Bohlen L, Liebold K, Hafner J, Friedlein H, et al. An autologous epidermal equivalent tissue-engineered from follicular outer root sheath keratinocytes is as effective as split-thickness skin autograft in recalcitrant vascular leg ulcers. Wound Repair Regen 2003;11:248–52.
Vaillant L. Treatment of venous leg ulcers with Epibase. A prospective study. Preliminary results. Ann Dermatol Venereol 2002;129:1245–6.
Heimbach DM. A nonuser’s questions about cultured epidermal autograft. J Burn Care Rehabil 1992;13:127–9.
Wainwright D. Use of an acellular allograft dermal matrix (AlloDerm) in the management of full-thickness burns. Burns 1995;21:243-8.
Bond JL, Dopirak RM, Higgins J, Burns J, Snyder SJ. Arthroscopic replacement of massive, irreparable rotator cuff tears using a GraftJacket allograft: Technique and preliminary results. Arthroscopy 2008;24:403–9 e1.
Ryssel H, Gazyakan E, Germann G, Öhlbauer M. The use of MatriDerm® in early excision and simultaneous autologous skin grafting in burns-A pilot study. Burns 2008;34:93–7.
Heimbach DM, Warden GD, Luterman A, Jordan MH, Ozobia N, Ryan CM, et al. Multicenter postapproval clinical trial of Integra dermal regeneration template for burn treatment. J Burn Care Rehabil 2003;24:42–8.
Feldman DL, Rogers A, Karpinski RH. A prospective trial comparing Biobrane, Duoderm and xeroform for skin graft donor sites. Surg Gynecol Obstet 1991;173:1–5.
Gravante G, Delogu D, Giordan N, Morano G, Montone A, Esposito G. The use of Hyalomatrix PA in the treatment of deep partial-thickness burns. J Burn Care Res 2007;28:269–74.
Gentzkow GD, Iwasaki SD, Hershon KS, Mengel M, Prendergast JJ, Ricotta JJ, et al. Use of dermagraft, a cultured human dermis, to treat diabetic foot ulcers. Diabetes Care 1996;19:350–4.
Noordenbos J, Dore C, Hansbrough JF. Safety and efficacy of TransCyte for the treatment of partial-thickness burns. J Burn Care Rehabil 1999;20:275–81.
Falanga V, Sabolinski M. A bilayered living skin construct (APLIGRAF) accelerates complete closure of hard-to-heal venous ulcers. Wound Repair Regen 1999;7:201–7.
El Ghalbzouri A, Commandeur S, Rietveld MH, Mulder AA, Willemze R. Replacement of animal-derived collagen matrix by human fibroblast-derived dermal matrix for human skin equivalent products. Biomaterials 2009;30:71–8.
Dong R, Liu X, Liu Y, Deng Z, Nie X, Wang X, et al. Enrichment of epidermal stem cells by rapid adherence and analysis of the reciprocal interaction of epidermal stem cells with neighboring cells using an organotypic system. Cell Biol Int 2007;31:733–40.
Aoki S, Takezawa T, Uchihashi K, Sugihara H, Toda S. Non-skin mesenchymal cell types support epidermal regeneration in a mesenchymal stem cell or myofibroblast phenotype-independent manner. Pathol Int 2009;59:368–75.
Nie X, Cai JK, Yang HM, Xiao HA, Wang JH, Wen N, et al. Successful application of tissue-engineered skin to refractory ulcers. Clin Exp Dermatol 2007;32:699–701.
Martinez-Santamaria L, Guerrero-Aspizua S, Del Rio M. Skin bioengineering: Preclinical and clinical applications. Actas Dermosifiliogr 2012;103:5–11.
Mahjour SB, Ghaffarpasand F, Wang H. Hair follicle regeneration in skin grafts: Current concepts and future perspectives. Tissue Eng Part B Rev 2012;18:15–23.
Blais M, Grenier M, Berthod F. Improvement of nerve regeneration in tissue-engineered skin enriched with schwann cells. J Invest Dermatol 2009;129:2895–900.
Monfort A, Soriano-Navarro M, Garcia-Verdugo JM, Izeta A. Production of human tissue-engineered skin trilayer on a plasma-based hypodermis. J Tissue Eng Regen Med 2013;7:479–90.
Liu F, Luo XS, Shen HY, Dong JS, Yang J. Using human hair follicle-derived keratinocytes and melanocytes for constructing pigmented tissue-engineered skin. Skin Res Technol 2011;17:373–9.
Peng LH, Mao ZY, Qi XT, Chen X, Li N, Tabata Y, et al. Transplantation of bone-marrow-derived mesenchymal and epidermal stem cells contribute to wound healing with different regenerative features. Cell Tissue Res 2013;352:573–83.
Altman AM, Matthias N, Yan Y, Song YH, Bai X, Chiu ES, et al. Dermal matrix as a carrier for in vivo delivery of human adipose-derived stem cells. Biomaterials 2008;29:1431–42.
Lu W, Yu J, Zhang Y, Ji K, Zhou Y, Li Y, et al. Mixture of fibroblasts and adipose tissue-derived stem cells can improve epidermal morphogenesis of tissue-engineered skin. Cells Tissues Organs 2012;195:197–206.
Griffiths M, Ojeh N, Livingstone R, Price R, Navsaria H. Survival of Apligraf in acute human wounds. Tissue Eng 2004;10:1180–95.
This work was supported by a grant from National High Technology Research and Development Program of China (863 Program) (2012AA020507).
How to cite this article: Bi H, Jin Y. Current progress of skin tissue engineering: Seed cells, bioscaffolds, and construction strategies. Burn Trauma 2013;1:63–72.
Source of Support: Supported by a grant from National High Technology Research and Development Program of China (863 Program) (2012AA020507). Conflict of Interest: None declared.
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Bi, H., Jin, Y. Current progress of skin tissue engineering: Seed cells, bioscaffolds, and construction strategies. Burn Trauma 1, 63–72 (2013). https://doi.org/10.4103/2321-3868.118928
- Regenerative medicine
- wound healing
- seed cells
- tissue-engineered skin