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    • Pigment Cell Melanoma Res. 24; 35–50 REVIEW ARTICLEArtificial skin in perspective: concepts and applicationsCarla A. Brohem1, Laura B. da Silva Cardeal1, Manoela Tiago1, Marıa S. Soengas2, ´Silvia B. de Moraes Barros1 and Silvya S. Maria-Engler11 Department of Clinical Chemistry & Toxicology, School of Pharmaceutical Sciences, University of S¼o Paulo, KEYWORDS artificial skin ⁄ skin reconstructs ⁄ skinS¼o Paulo, Brazil 2 Melanoma Group, Centro Nacional de Investigaciones Oncológicas (Spanish National Cancer equivalents ⁄ rafts ⁄ organotypical cultures ⁄ 3D modelsResearch Center) Madrid, Spain PUBLICATION DATA Received 13 July 2010, revisedCORRESPONDENCE S. S. Maria-Engler, e-mail: silvya@usp.br and accepted for publication 27 September 2010, published online 4 October 2010 doi: 10.1111/j.1755-148X.2010.00786.xSummarySkin, the largest organ of the human body, is organized into an elaborate layered structure consisting mainlyof the outermost epidermis and the underlying dermis. A subcutaneous adipose-storing hypodermis layer andvarious appendages such as hair follicles, sweat glands, sebaceous glands, nerves, lymphatics, and bloodvessels are also present in the skin. These multiple components of the skin ensure survival by carrying outcritical functions such as protection, thermoregulation, excretion, absorption, metabolic functions, sensation,evaporation management, and aesthetics. The study of how these biological functions are performed iscritical to our understanding of basic skin biology such as regulation of pigmentation and wound repair.Impairment of any of these functions may lead to pathogenic alterations, including skin cancers. Therefore,the development of genetically controlled and well characterized skin models can have important implications,not only for scientists and physicians, but also for manufacturers, consumers, governing regulatory boardsand animal welfare organizations. As cells making up human skin tissue grow within an organized three-dimensional (3D) matrix surrounded by neighboring cells, standard monolayer (2D) cell cultures do not recapit-ulate the physiological architecture of the skin. Several types of human skin recombinants, also called artificialskin, that provide this critical 3D structure have now been reconstructed in vitro. This review contemplatesthe use of these organotypic skin models in different applications, including substitutes to animal testing. The epidermal layer, derived from embryonic ecto-General architecture of the skin derm, is made up of cells generated by proliferatingEpidermal ⁄ dermal layers keratinocytes of the stratum basale that move upwardsThe organization of the skin into epidermal and dermal while they differentiate (Figure 1). The continuous pro-layers that differ in thickness, strength, and flexibility cess of proliferation, differentiation and, ultimately, cellallows for a structured architecture that provides a vari- death and shedding, allows compartmentalization into aety of skin functions. The outermost layer of the skin, number of strata representing different stages of kerat-known as the epidermis, serves as an impermeable inocyte maturation (Balasubramani et al., 2001; Schulzboundary between the environment and the body. The et al., 2000; Stark et al., 2004a). Besides keratinocytes,underlying dermis is formed of strong connective tissue, which account for about 80% of epidermal cells, thewhich is rich in collagen and confers its characteristic epidermis is also composed of the pigment-producingflexibility to skin. Epidermis and dermis are separated melanocytes, Merkel cells, which are thought to play aby the extracellular matrix (ECM), known as the basal sensory role (Boyce and Warden, 2002; Feliciani et al.,lamina (Ajani et al., 2007; Balasubramani et al., 2001; 1996), and specialized dendritic Langerhans cells,Godin and Touitou, 2007; Horch et al., 2005). which have an essential role in the skin immuneª 2010 John Wiley & Sons A/S 35
    • Brohem et al. Figure 1. Drawing showing skin components and layers. Epidermis: containing melanocytes and keratinocytes that are able to differentiate and form the different strata (corneum, lucidum, granulosum and germinativum). Dermis: formed by fibroblasts embedded in a matrix. Hypodermis: containing the adipose tissue. ´defense system (Phillips, 1998; Regnier et al., 1998, also stabilizes the overlying epidermis mechanically by1999). means of its hemi-desmosomal structure (Balasubrama- As shown in Figure 1, ongoing keratinocyte cell divi- ni et al., 2001; El Ghalbzouri et al., 2005; Marinkovichsion begins in the innermost stratum basal layer of the et al., 1992; Moll and Moll, 1998; Schulz et al., 2000).stratum germinativum and pushes daughter cells api- Basement membrane is mainly made up of collagencally upwards toward the next spinous stratum, where IV, laminin, proteoglycans, and glycosaminoglycans, ascells become dense. As they progress toward the inter- well as growth factors with a wide variety of biologicalface with the environment, they move into the granulo- activities. This dermo-epidermal basement membranesum stratum, where they accumulate lipid granules, strictly controls the traffic of bioactive molecules in bothcritical for maintenance of the water barrier. The loss of directions and is able to bind a variety of cytokines andthe nucleus in differentiating keratinocytes now leads to growth factors, thus representing a reservoir for con-a flattened or horny morphology, with only keratin trolled release during physiological remodeling or repairremaining. Pigmentation is imparted by the addition of processes (Iozzo, 2005). The dermal matrix, which liesmelanin, produced by melanocytes and transferred to underneath the basement membrane, provides energykeratinocytes in the final sublayer of the stratum luci- and nutrition to the overlying epidermis and imparts con-dum. The most external layer, known as the stratum siderable strength to skin by virtue of the arrangementcorneum, represents the final result of keratinocyte dif- of collagen fibers. The collagenous mesh work is inter-ferentiation, and is formed by completely differentiated laced with elastin fibers, fibronectin, proteoglycans, gly-dead keratinocytes interspersed with intercellular lipids cosaminoglycans (GAGs), predominantly hyaluronic acid,(mainly ceramides and sphingolipids) (Ajani et al., 2007). and other components (Balasubramani et al., 2001). In addition to the dense matrix there are dermal fibro-Basement membrane and other constituents blasts, cells of the immune system, nerve endings,of the skin sweat and sebaceous glands, hair follicles, blood ves-Epidermal development during embryogenesis, wound sels, and endothelial cells. The dermal fibroblasts arehealing, and regeneration, as well as normal homeo- known to have numerous functions in the synthesis andstatic maintenance depends on interactions between deposition of ECM components. Moreover, fibroblaststhe epithelia and the connective dermal tissue (Ajani play an important role in proliferation and migration, aset al., 2007; Balasubramani et al., 2001; Schulz et al., well as autocrine and paracrine interactions with neigh-2000; Stark et al., 2004a). The basal stratum of the epi- boring cells (Wong et al., 2007).dermis is intimately related to the underlying dermisthrough a system of protein fiber connections along a The need for in vitro 3D skin modelsmicro-architecture network of rete ridges. These consti-tute a well characterized basement membrane, which Interactions between an individual cell, its immediateanchors the epidermal layer to the loose connective tis- neighbors, and the ECM are responsible for the controlsue of the underlying dermis. This basement membrane of cell behavior (Bissell et al., 1982; Grinnell, 1976,36 ª 2010 John Wiley & Sons A/S
    • Artificial skinFigure 2. Optical microscopy showingthe differences between human andmouse skin. Note that the murine dermisis thin and the epidermis comprisestypically only 3 layers, whereas humandermis is quite thick and epidermis isgenerally 6–10 layers thick. HE staining.60· magnification. Mouse tissue Human tissue2008; Lin and Bissell, 1993; Smalley et al., 2006; Yang questions that cannot be answered solely in the contextet al., 1987). Therefore, cells grown in 2D monolayers of monolayer tissue culture. Models that recapitulatecannot capture the relevant complexity of the in vivo the basic architecture of the human skin would supplymicro-environment (Mazzoleni et al., 2009). For exam- sites for studying cell–cell interactions and effects ofple, it has long been suggested that cells cultured on the stromal environment in the regulation of melanogen-2D substrates such as culture plates, lose a myriad of esis, proliferation, and differentiation of keratinocytes,important signals, key regulators, and tissue pheno- as well as re-epithelialization processes after wounding.types. Cells growing in 3D have different cell surface These skin models also present a relevant platform forreceptor expression, proliferative capacity, extracellular the creation of cosmetic, photoaging, and cancer mod-matrix synthesis, cell density, and metabolic functions els, as well as an excellent system for pharmacological(Bissell et al., 1982; Grinnell, 1976, 2008; Horning et al., analyses. Furthermore, the human skin equivalents can2008; Lin and Bissell, 1993; Mazzoleni et al., 2009; be engineered easily with specific genetic alterations inSmalley et al., 2006; Yang et al., 1987). Thus, 2D mono- either dermal or epidermal compartments to determinelayer models not only fail to reproduce the complex and how the outcome of these modifications may be modu-dynamic environments of in vivo tissues, but can also to lated by the stromal environment. These skin modelssome degree trigger false findings by forcing cells to also present time- and cost-effective alternatives to theadapt to an artificial, flat, and rigid surface. Growing use of laboratory animals that are currently being usednumbers of studies report differences in phenotype, cel- for such studies.lular signaling, cell migration, and drug responses when While murine animals have routinely been used asthe same cells are grown under 2D as opposed to 3D experimental models for skin biology and skin cancerculture conditions (reviewed by Mazzoleni et al., 2009). (Mancuso et al., 2009; Paulitschke et al., 2010; Youssef In vitro studies have shown that dermal fibroblasts et al., 2010), the dissimilar architecture of mouse andsecrete soluble factors that diffuse to the overlying epi- human skin severely limits this approach. To begin with,dermis and can influence keratinocytes to induce the the epithelium of fur-covered mouse models is denselyproduction of basement membrane proteins or melano- packed with hair follicles that are synchronized for thegenic factors (Balasubramani et al., 2001; El Ghalbzouri first few months of life. In contrast, humans possesset al., 2002a; Wong et al., 2007). Keratinocytes in mono- larger interfollicular regions with sparse hair follicles thatculture produce only a thin epidermal layer and, without are asynchronous in nature. Furthermore, adult murinemesenchymal support, undergo apoptosis after about dermis is thin and the epidermis typically comprises2 weeks in culture (Wong et al., 2007). Dermal fibro- only three layers, with a high rate of turnover, whereasblasts promote not only keratinocyte proliferation, but human dermis is quite thick and epidermis is generallyalso the development of identifiable keratinocyte layers. 6–10 layers thick (Figure 2). Human skin melanoctyesConsequently, upon combination of postmitotic dermal reside in the basal layer of the epidermis, whereas theyfibroblasts (feeder cells) and epidermal keratinocytes, are located mainly in dermal hair follicles in mice. Addi-properly stratified epithelia fail to form in simple 2D fee- tionally, a cutaneous muscle layer, the panniculous car-der-layer co-cultures. Only in advanced 3D in vitro sys- nosus, which is present in mice but non-existent intems do keratinocytes develop well ordered epithelia (El humans, has led to findings that are not consistentGhalbzouri et al., 2002a; Stark et al., 2004a; Wong et al., among species (Donahue et al., 1999).2007), thus offering an opportunity to more closely reca- Skin responsiveness and functionality are also distinctpitulate artificial human skin. in mouse and humans. For instance, wounding Therefore, strategies that allow the reconstruction of responses in mouse skin allow effective regeneration ofartificial human skin equivalents in a 3D setting, includ- tissue, whereas damage in human skin leads to scaring both dermal and epidermal components, would pro- tissue or hypertrophic dermal skin lesions known asvide versatility as well as answers to physiological keloids, which are absent in mouse skin (Khorshid,ª 2010 John Wiley & Sons A/S 37
    • Brohem et al.2005). The function of epithelium is also dissimilar ultrastructures and markers of epidermal differentiationbetween mouse and human skin. Mouse skin provides (e.g. keratins, fillagrin, involucrin) (El Ghalbzouri et al.,less of a water barrier and displays higher percutaneous 2002a,b, 2008).absorption than that of human skin, thus limiting topical In an editorial about the development of new skindrug-delivery studies (Menon, 2002). equivalents, Phillips (1998) describes the historical evo- lution of skin substitutes. In 1975, Rheinwald and Green described a methodology for the in vitro cultivation andGeneration of skin equivalents serial subculture of epidermal cells that were able toMultiple approaches produce viable keratinocyte sheets. This technique wasSeveral types of human skin equivalents that closely critical in the development of tissue culture technology,resemble normal human skin tissue have now been and has improved the cultivation of large quantities ofsuccessfully reconstructed in vitro (Table 1). These skin keratinocytes in vitro (Phillips, 1998). Once keratinocytesequivalents consist mainly of keratinocytes that are cul- could be propagated in large quantities, the first steptured on a dermal substitute for approximately 2 weeks towards successful epidermal differentiation was theat the air–liquid interface, which allows for epidermal exposure of normal human keratinocyte cultures atdifferentiation and development of stratified layers the air–liquid interface, in order to induce terminal differ- ´(Figure 3) (El Ghalbzouri et al., 2002a,b, 2008; Prunieras entiation resulting in a multilayered stratified tissueet al., 1983; Stark et al., 2004a). Although major impor- ´ (Figure 4) (Regnier et al., 1998, 1999). Keratinocytetance is assigned to the epidermal layer of skin equiva- differentiation and consequent programmed cell deathlents, dermal substitutes are widely used in clinics for ultimately led to formation of the uppermost stratumsevere burns and chronic wounds (Boyce and Warden, corneum in the skin equivalent models, which is formed2002; Fimiani et al., 2005). These dermal substitutes continuously during this process (Figure 4).include cell-free dermal matrices such as de-epider- Studies on the effectiveness, metabolic transforma-mized dermis (DED), inert filters, fibroblast-populated tion, and potential pathologic effects of a huge varietycollagen matrices, and lyophilized collagen-GAG mem- of topical products have only been possible in vitro duebranes (Boyce and Warden, 2002; El Ghalbzouri et al., to the presence of the stratum corneum in the recon-2002a,b, 2008; Fimiani et al., 2005; Stark et al., 2004a). structed epidermis of artificial skin (Ajani et al., 2007; Engineered skin bioproducts containing biopolymers Boyce and Warden, 2002; El Ghalbzouri et al., 2008; Po-are also being used as grafts in the treatment of nec, 2002). For safety reasons, multiple tests for topicalpatients with excised burns, burn scars, and congenital products have been developed to detect changes in theskin lesions (Boyce and Warden, 2002). In addition, integrity, morphology, viability, and release of pro-inflam-materials such as collagen–glycosaminoglycan matrices, matory mediators in this layer (Ponec, 2002). One pointallogeneic dermis, and synthetic polymers have been to be mentioned is that after a prolonged time in cultureused as replacements for specific skin layers (Balasubra- (6–7 weeks), the in vitro epidermis remains viable andmani et al., 2001). Summarizing, there are a variety of displays all the signs of a normal differentiation pro-‘skin models’ that can be broadly categorized into (i) gram, but the thickness of the stratum corneum gradu-those containing only epidermal components, (ii) grafts ally increases with time. This can be a critical issue inconsisting of dermal components alone, and (iii) full- some tests, as the gradual thickening of the stratumthickness composite grafts containing both epidermal corneum may lead to higher resistance to environmentaland dermal components. In the literature, these sys- stimuli (Ponec, 2002). Thus, these models remaintems have been referred to as 3D skin, reconstructed imperfect, and may be intrinsically variable. Neverthe-skin, skin equivalents, artificial skin, organotypic culture less, despite these limitations, they have already pro-of skin, skin substitutes, or skin grafts. It should be vided much information about dermal–epidermalnoted that the last two terms are most commonly used interactions, cell–cell and cell–matrix interactions,for bioengineered products that use human or animal responses of dermal and epithelial cells to biological sig-components such as cultured cells or collagen. nals and pharmacological agents, as well as effects of The past few years have seen an increase in the drugs and growth factors on skin reconstruction pro-number of commercially available skin models: e.g. Epi- cesses (Fimiani et al., 2005).DermTM (MatTek, Ashland, MA, USA), EpiskinTM (pre-viously manufactured by Episkin, Chaponost, France, Importance of the microenviromentand now by L’Oreal, SkinEthic, Nice, France), ApligrafÒ(Organogenesis Inc., Canton, MA, USA) and the models ECMengineered by SkinEthic (Boelsma et al., 2000; Ponec, The ECM, a gel-like medium produced by the surround-2002; Welss et al., 2004). As these models are used for ing fibroblasts, is the largest component of normal skincommercial purposes, morphological studies have been and confers to the skin its unique properties of elastic-performed to demonstrate a relevant multilayered ity, tensile strength, and compressibility. Both dermalepithelium, and the presence of characteristic epidermal fibroblasts and epidermal cells secrete the two critical38 ª 2010 John Wiley & Sons A/S
    • Table 1. Different strategies to generate artificial skin Dermal layer Epidermal layer Cellular Cellular Time of Presence Type components components differentiation Supplemental factors References + DED NHF (in collagen NHK 1–3 weeks 5% bovine calf serum Boelsma et al., 1999 Filter inserts matrices) HaCaT 0.5 lM hydrocortisone Collagen gels 0.1 lM isoproterenol 0.5 lg ⁄ ml insulinª 2010 John Wiley & Sons A/S 1 ng ⁄ ml EGF 0.1 nM cholera toxin + Collagen type I NHF NHK 7 days Asselineau et al., 1985 Bernerd and Asselineau, 1997, 1998; Pageon and Asselineau, 2005 + Rat tail collagen MDCK for basement REK (neonatal rat 5 days Ajani et al., 2007 type I membrane epidermal formation (removed kerationcytes) before keratinocyte seeding) + NHF mouse 3T3 fibroblast Newborn NHK 2–3 weeks 24.3 lg ⁄ ml adenine ´ Dube et al., 2010 5% Fetalclone II 5 lg ⁄ ml insulin 0.4 lg ⁄ ml hydrocortisone 1 nM cholera toxn 10 ng ⁄ ml EGF ) Filter insert NHK 17 days 5% calf serum El Ghalbzouri et al., 2008 (covered or not 1 lM hydrocortisone with collagen 1 lM isoproterenol type IV) 0.1 lM insulin 100 lM L-serine 1 lMdL-a-tocopherol-acetate and a lipid supplement containing 25 lM palmitic acid, 15 lM linoleic acid, 7 lM arachidonic acid and 24 lM bovine serum albumin 0.01 lM L-carnitine 1 lM DL-a-tocopherol-acetate 25 lM palmitic acid 30 lM linoleic acid 7 lM arachidonic acid 50 lg ⁄ ml ascorbic acid 1 ng ⁄ ml EGF Artificial skin 39
    • 40 Brohem et al. Table 1. (Continued) Dermal layer Epidermal layer Cellular Cellular Time of Presence Type components components differentiation Supplemental factors References + Calf collagen type I NHF NHK + NHM (20:1) Over 1 week Liu et al., 2007 + Collagen type I NHF NHK + NHM (4:1) 7 days Inserts immersed Souto et al., 2009 (Gen-col block collagen) + DED NHF NHK + NHM (20:1) 8 days SCF ´ Cario-Andre et al., 2006 Collagen type I NMF human HGF Endothelin 1 or 3 + Collagen type I NHF NHK 7–10 days (human) Calf serum Stark et al., 2004a 3T3-MEF HaCaT 10–14 days (mouse) Insulin EGF Cholera toxin Adenine Hydrocortisone Ascorbic acid TGF-a (only in HaCaT) + Hyalograft 3-D NHF NHK Up to 12 weeks FAD medium Stark et al., 2004b, 2006; 10% fetal bovine serum Boehnke et al., 2007 0.1 M cholera toxin 0.4 lg hydrocortisone 50 lg L-ascorbic acid 200 U ⁄ ml ⁄ day aprotinin + Collagen type I NHK 2–3 weeks Aprotinin solution Sobral et al., 2007 Matrigelª 2010 John Wiley & Sons A/S
    • Artificial skinFigure 3. Optical microscopy of humanfacial skin and human artificial skin. Thethickness of the epidermis and dermis isvery similar. Human artificial skin presentsall epidermal layers, showing thedifferentiation of this epithelium. HEstaining. 60· magnification. Human skin Human artificial skinFigure 4. Preparation model of artificial skin. First step is the preparation of the dermal equivalent, consisting of type I collagen andfibroblasts. After polymerization of this layer, keratinocytes (K) and melanocytes (M) are plated. At 24 h after plating and after the contractionof collagen gel, the entire structure is transfered to a steel grid to form the air–liquid interface. After 2 weeks on the interface there is theformation of artificial skin. HE staining. 40· magnification.classes of ECM molecules – fibrous proteins and prote- et al., 1991; Phillips, 1998). In the case of skin recon-oglycans. Strength, flexibility, and resilience are structs, a living dermal component can be vital to theimparted by fibrous structural proteins, including colla- cultured skin, and therefore the addition of a dermal ele-gens, elastin, and laminin. In turn, highly hydrated prote- ment should be carefully considered in the design ofoglycans provide cushioning support to cells in the any such product. For example, the composite graftECM. (epidermal and dermal elements) showed significant The ECM can contribute to the microenvironment advantages over the epidermal sheet graft in the closurespecifically through its mechanical features, providing of full-thickness wounds (Cooper et al., 1991).support and anchorage for cells. The ECM also influ-ences tissue segregation as well as regulation of intra- Dermal substratescellular communication via signaling pathways and its Two types of dermal substrates are used for the gener-ability to bind growth factors, enzymes, and other dif- ation of reconstructed epidermis: (i) acellular structures,fusible molecules. Interactions between cells and the which can be either inert filters or de-epidermized der-ECM are extremely important for processes such as mis (DED), and (ii) a cellular substrate composed ofnormal cell growth and differentiation. For example, in fibroblast-populated collagen matrix (Ponec et al., 1997).acute wounds, the provisional wound matrix, containing Skin reconstructs have been described as containingfibrin and fibronectin, provides a scaffolding to direct only acellular dermal substrate, forming a stratified epi-cells into the injury, as well as stimulating them to pro- dermis of three to four viable cell layers, and must beliferate, differentiate, and synthesize new ECM. supplemented with various growth factors including epi- An increasing number of studies have identified that dermal growth factor (EGF), keratinocyte growth factorit is essential to maintain the composition and structural (KGF), and ⁄ or insulin-like growth factor (IGF). However,organization of the ECM (i.e. the number of cells in the keratinocyte proliferation is stimulated and epidermaldermal layer) to obtain normal skin tissue organization in morphology is improved in the presence of fibroblasts3D models (Chioni and Grose, 2008). The cells of the (El Ghalbzouri et al., 2002a,b; Wong et al., 2007)underlying dermal layer do not act simply as a structural (Table 1). In skin reconstructs, viable fibroblasts arepassive framework, but rather influence epithelial migra- shown to increase epidermal growth and spreading, astion, differentiation, growth, and attachment (Cooper well as enhance the formation of basement membranesª 2010 John Wiley & Sons A/S 41
    • Brohem et al.proteins, thus improving the attachment of the epider- different body sites have different functional properties,mal layer (Cooper et al., 1991). which may affect their suitability for dermal substitutes. As Nolte et al. (2008) pointed out, it is important to con-Fibroblast and its cellular interactions sider these facts in future in vivo human studies in tis-Another important point to consider when manufactur- sue-engineered dermal substitutes.ing skin reconstructs for a long period of time is thecontraction of the collagen matrix in the presence of fi- Additional cellular componentsbroblasts (Bell et al., 1979). The contractility of this Fibroblasts are not the only cells that are important inmatrix depends on the number of fibroblasts and the the dermal layer of a skin reconstruct. The 3D culturetime-frame from the generation of the dermal layer to can also contain other cell types that will enrich the der-the subsequent seeding of keratinocytes (El Ghalbzouri mal microenvironment, such as myofibroblasts, endo-et al., 2002b). Some groups have been trying to improve thelial cells, inflammatory cells, and adipocytes. Athe skin model by avoiding collagen contraction, while wound-healing study used human mesenchymal stemretaining the presence of fibroblasts. El Ghalbzouri et al. cells seeded together with dermal fibroblasts in recon-(2002b) used a centrifugal seeding method to incorpo- structed skin to show that human mesenchymal stemrate different numbers of fibroblasts into the DED. They cells could contribute to the wound-healing processobserved that fibroblast initially had a stimulatory effect (Chioni and Grose, 2008).on induced keratinocyte proliferation, which decreasedat later time points in cell culture. They also showed Addressing skin model limitationsthat the interaction between keratinocytes and fibro-blasts induced the production of growth factors by the Phototypesfibroblasts that provided key signals for the induction of Comparisons of cells in monolayer cultures versus skinappropriate epidermal differentiation. The factors pro- reconstructs indicate that the 3D models may alsoduced by fibroblasts (influencing keratinocyte prolifera- address aspects of skin pigmentation. Melanocytes intion in either a negative or a positive manner) were skin reconstructs can be present as single cells at thedependent on fibroblast density and culture time (El basal layer of the epidermis, reflecting the distributionGhalbzouri et al., 2002b). of human skin (Meier et al., 2000). Furthermore, mela- In an attempt to preserve the epidermal homeostasis nin production is more active in melanocytes growing inand increase the lifespan of skin, avoiding dermal shrink- 3D than in 2D (Nakazawa et al., 1998), demonstratingage and increasing tissue stability, some authors used autocrine and paracrine signaling networks mainlymatrices other rather than collagen as sponges or scaf- between both melanocyte and keratinocyte cell–cellfolds (Auxenfans et al., 2008; Boehnke et al., 2007; interactions in the skin (Costin and Hearing, 2007).Stark et al., 2004b, 2006). In a system that used a der- Melanocytes, however, have not been included inmal equivalent constituted by an esterified hyaluronic standard skin grafts. In these cases, even after success-material (Hyalograft-3D) colonized with fibroblast, Stark ful transplantation, skin grafts remain depigmented andet al. (2004b, 2006) improved long-term growth and dif- thus present an aesthetical dilemma (Balasubramaniferentiation for at least 12 weeks. Differentiation and et al., 2001; Boyce and Warden, 2002; Liu et al., 2007).ultrastructure markers were used to show the viability As cutaneous pigmentation is a result not only of mela-of the system in studies such as skin regeneration nin synthesis by melanocytes, but also of the transfer(Boehnke et al., 2007) and homeostasis (Stark et al., 2006). of melanosomes to neighboring keratinocytes, it is criti- Other studies have shown the effect of fibroblast cal that melanocytes establish correct interactions withdensity on keratinocyte proliferation and correct epider- ´ surrounding keratinocytes (Cario-Andre et al., 2006; Fitz-mal proliferation, by demonstrating that fibroblasts pro- ´ patrick and Szabo,1959; Nakazawa et al., 1998; Regnierduce collagen types IV and VII, laminin 5 and nidogen, et al., 1999).all of which contribute to basement membrane forma- One fact that remains to be discussed is that epider-tion. Furthermore, fibroblasts secrete cytokines such as mal pigmentation is dependent upon the phototype oftransforming growth factor beta (TGF-b), which stimu- melanocytes. In basal conditions, intra-individual skinlates keratinocytes to synthesize basement membrane pigmentation varies according to the phototype of mela-components, including collagen types IV and VII (Wong nocytes, which is regulated mainly by melanocortin-1et al., 2007). receptor (MCIR) and remains the major determinant of The importance of the role of fibroblasts in keratino- the pigmentation phenotype in skin (Rees, 2003). Thecyte differentiation was also demonstrated, showing MC1R gene encodes a seven-transmembrane G-protein-that fibroblasts produce different patterns of cytokine coupled receptor that regulates the quantity and qualityrelease during their differentiation. Moreover, differenti- of melanin produced via activation of adenyl cyclase andated fibroblasts induce the production of the highest lev- production of cyclic AMP (Mountjoy et al., 1992).els of keratinocyte growth factor and TGF-b1 (Nolte Activation of this signal transduction pathway ultimatelyet al., 2008). It was also shown that fibroblasts from leads to activation of various genes, most notably42 ª 2010 John Wiley & Sons A/S
    • Artificial skinmicrophthalmia transcription factor (MITF), responsible Schoop et al., 1999). These HaCaT cells grown at thefor the expression of numerous enzymes and differenti- air–liquid interface initially develop a multilayered epithe-ation factors of the melanogenic cascade (Levy et al., lium. However, during the course of culture, marked2006) as well as the survival of migrating melanoblasts alterations in tissue architecture become apparent, with ´(Steingrımsson et al., 2004). disordered tissue organization, including the presence of The different skin phototypes (types I–VI) can to rounded cells with abnormally shaped nuclei (Boelsmasome extent be reproduced in vitro by selecting the et al., 1999; Schoop et al., 1999; Stark et al., 2004a).donor for melanocyte isolation. Melanocytes derived Although many studies used HaCaT cells in skinfrom a darkly pigmented individual will generally give reconstruction procedures, several indicated that HaCaTrise to a reconstructed epidermis phenotypically pig- cells have a limited ability to produce an organizedmented, independent of the origin of keratinocytes. For mature cornified epithelium (Boelsma et al., 1999).this reason, the use of melanocytes has to be taken into Despite this, this reconstructed epidermis is often con-consideration not only in skin grafts for clinical applica- sidered functional and is used as a model for elucidationtions but also for the modulation of melanogenesis by of the molecular mechanisms regulating keratinocytepro-pigmenting or de-pigmenting agents (Cario-Andre ´ growth and differentiation, making it popular for use in ´et al., 2006; Regnier et al., 1999). The use of pigmented pharmacotoxicological studies (Schoop et al., 1999;skin equivalents has also been adopted to obtain a bet- Stark et al., 2004a). Nevertheless, the inability to gener-ter understanding of congenital hyperpigmentary disor- ate stratum corneum must be considered when cosme-ders (Okazaki et al., 2005). tologic applications are the main focus. ´ Cario-Andre et al. (2006) have shown that there canalso be a dermal modulation of human epidermal pig- The immune system: how to reconstitute thismentation. Epidermal reconstructs from phototypes II– response in vitro?III were grafted on the back of immunotolerant Swiss Another important element to be considered in skinnu ⁄ nu mice; depending on the presence of colonizing equivalent models is the lack of cells of the immunehuman or mouse fibroblasts, they developed a non-regu- system. In addition to keratinocytes, supra-basal layerslar pigmentation pattern. The authors also demonstrated in the human skin contain Langerhans cells (LC), in thethat when human white Caucasoid split-thickness skin proportion of approximately 30 000 cells ⁄ cm2. Thesewas xenografted onto the Swiss nu ⁄ nu mouse strain it cells correspond to CD34+ dendritic cells of hematopoi-phenotypically appeared black within 3 months, reveal- etic origin, displaying Birkbeck granules and CD1aing a phototype VI pattern of melanin distribution. This ´ surface antigens (Facy et al., 2004; Regnier et al.,demonstrates that melanocyte proliferation and melanin 1997). LC cells are responsible for immune systemdistribution ⁄ degradation can be influenced by fibroblast functions in the skin, capturing allergens of low molecu-secretion and acellular dermal connective tissue (Cario- lar weight that bind to the skin for possible antigen ´Andre et al., 2006). processing and T-cell recruitment. Allergen-induced epidermis cytokines, such as tumor necrosis factorImmortalized cell lines versus primary cells alpha (TNF-a) and interleukin (IL)-1b, are involved in thein artificial skin migration of these Langerhans cells, which, whenPrimary cells can be used to evaluate differences in the stimulated, present CD86 surface markers (Facy et al.,epithelial maturation depending on the phototype of the 2004; Tiznado-Orozco and Orea-Solano, 2004).donor and on the anatomical location of the tissue of However, the integration of LC into in vitro modelsorigin. However, primary cells have a limited lifespan, has remained a challenge, as these cells, unlike kerati-and the number of cells cannot be increased to produce nocytes and melanocytes, cannot be subcultured andthe large amounts that may be needed to generate mul- ´ expanded in vitro (Regnier et al., 1998, 1999). A skintiple reconstructs for statistical analyses. Immortalized reconstruction composed of both dermal and epidermalcell lines could improve the reproducibility and consis- layers containing not only keratinocytes, but also thetency of skin models, reducing intra- and inter-laboratory pigmented and immune system constituents, would bevariations. Thus, established lines would make tight reg- an excellent model to assess not only mechanisms ofulatory procedures possible for biological and clinical epidermal and dermal cell–cell interactions but also thestudies, as well as industrial applications (Boelsma role of each cell type in the skin immune responseet al., 1999, Stark et al., 2004a). ´ (Regnier et al., 1998). The spontaneously immortalized human keratinocyte ´ In 1998, Regnier and collaborators demonstrated thatline, HaCaT, is one of the most frequently used kerati- in vitro-generated dendritic cells ⁄ Langerhans cells fromnocyte cell lines because of its highly preserved differ- CD34+ hematopoietic progenitors seeded into a recon-entiation capacity. HaCaT cells are able to form a structed epidermis gave rise to resident dendritic epider-differentiated, ordered, structured, and functional epider- mal LC, expressing major histocompatibility complexmis when transplanted onto subcutaneous tissue of (MHC) class II and CD1a antigens, also containing char-athymic mice (Boelsma et al., 1999; Ponec, 2002; acteristic Birbeck granules. Confocal laser scanningª 2010 John Wiley & Sons A/S 43
    • Brohem et al.microscopy also revealed a cell morphology identical to tee on Alternatives to Animal Testing, 2002; Welssthat observed for LC in vivo. Interestingly, when purified et al., 2004).CD34+ hematopoietic progenitor cells not exposed to Skin reconstructs are now being proposed as an alter-factors such as granulocyte-macrophage colony-stimulat- native method to animal testing for assessing irritation,ing factor (GM-CSF) and TNF-a, were co-seeded directly corrosiveness, phototoxicity, and genotoxicity of variouswith keratinocytes and melanocytes onto the dermal reagents (Curren et al., 2006; Hu et al., 2009; Welssequivalent, the keratinocytes were able to induce the et al., 2004). These reconstructs are favored overmaturation of these hematopoietic progenitors into epi- monolayer keratinocyte cultures for multiple reasons, ´dermal LC (Regnier et al., 1998). including the ability to test compounds with low water- Facy et al. (2004) have now proposed that in a com- solubility, as well as the fact that the concentrations ´plete reconstructed epidermis, similar to Regnier et al. inducing irritant responses in monolayer keratinocyte(1998), the reactivity of Langerhans cells from CD34+- cell cultures are extremely different than concentra-derived dendritic cells is comparable to that in vivo. The tions in vivo, and the cultures typically obtained fromauthors also call attention to the fact that this complete monolayer cell tests have been difficult to correlate withmodel has the potential to be produced at an industrial the in vivo situation (MacNeil, 2007; de Silva and Euro-level and may be an alternative model for estimating pean Federation of the Cosmetics Industry, Steeringthe sensitizing potential of new chemicals. Facy et al. Committee on Alternatives to Animal Testing, 2002;(2004) pointed out the importance for research studies Welss et al., 2004). Percutaneous absorption, whichto investigate Langerhans cell biology in vitro. cannot be properly assessed in monolayer conditions, is another critical factor in considering the safety assess- ment of cosmetic ingredients, as this governs the mar-Applications gin of safety for the ingredients studied (de Silva andAlternatives to animal testing and regulations European Federation of the Cosmetics Industry, Steer-One major concern in the production and use of novel ing Committee on Alternatives to Animal Testing, 2002).chemical reagents that are to be applied to the skin is Once skin reconstructs appeared as an alternativetheir capacity to cause acute skin irritation upon contact. method to animal testing, various companies providedHence, new procedures have been established for the reconstituted human epidermal in vitro skin equivalents,safe handling, packaging and transport, as well as for and the number of distributors is increasingly growing.general safety assessment of these reagents (Fentem During 1999 and 2000, ECVAM commissioned a vali-et al., 2001; Ponec, 2002). These reagents are often eval- dation study of five in vitro tests for acute skin irritation.uated for their irritant potential by the application to ani- This study specifically addressed aspects of protocolmals, followed by observations of visible changes refinement (phase I), protocol transfer (phase II), andincluding erythema and edema. However, testing for skin protocol performance (phase III), in accordance with theirritation in animals is not always predictive of human scheme defined by ECVAM (Botham, 2004; Fentemresponses. Furthermore, these irritability tests may et al., 2001; Portes et al., 2002). Results indicated ancause pain and discomfort to the experimental animals acceptable intra-laboratory reproducibility. However,(Boelsma et al., 1999; Ponec, 2002; Welss et al., 2004). inter-laboratory reproducibility was of concern, and the The European Cosmetics Association (COLIPA) is predictive ability of all methods was also inadequate.committed to replacing animal testing as well as improv- One explanation for the insufficient reproducibility anding the prediction of irritants in the cosmetic and toiletry false positives is the common formation of an imperfectindustry without compromising human safety or envi- barrier function in reconstructed epidermis. Further-ronmental protection (de Silva, 2002; Welss et al., more, the use of various experimental protocols which2004). In 1992, COLIPA coordinated the efforts of the differ in the concentrations of the applied irritants used,cosmetics industry to develop alternative methods to the time of exposure, and the time of incubation madeanimal testing for the safety assessment of cosmetics it difficult to create a standardized protocol for assessingand hence created the Steering Committee on Alterna- irritation in vitro (Welss et al., 2004). Protocol refine-tives to Animal Testing (SCAAT) (de Silva and European ments and improvements in 2007 enabled two skinFederation of the Cosmetics Industry, Steering Commit- reconstruct models to be validated by ECVAM for usetee on Alternatives to Animal Testing, 2002). In 1994, as alternative methods in skin irritation tests (Botham,the European Centre for the Validation of Alternative 2004; Fentem and Botham, 2002; Fentem et al., 2001;Methods (ECVAM) organized a workshop on the poten- Hoffmann et al., 2008; Portes et al., 2002).tial use of non-invasive methods in the safety assess- A promising new use of 3D human skin models forment of cosmetic products, and COLIPA created a the evaluation of genotoxicity of topically applied com-specific task force to create guidelines that would pounds and formulations is also underway. Curren et al.reflect the protocols and practices of industry (Boelsma (2006) developed a micronucleus assay employing theet al., 2000; Fentem et al., 2001; de Silva and European EpiDerm 3D human skin model, which is now in theFederation of the Cosmetics Industry, Steering Commit- process of validation (Hu et al., 2009).44 ª 2010 John Wiley & Sons A/S
    • Artificial skinPhotoaging model: the UV effect The skin reconstructs are also an efficient model notSun exposure causes various deleterious cutaneous only to test the SFP of a sunscreen, but also to studyeffects, leading to short-term responses such as ery- cell and ECM modifications provoked by photoaging.thema, sunburn, and suntan. Long-term effects include Photoaged skin contains notorious changes observed inskin cancers and premature photoaging (Bernerd and the uppermost epidermal compartment, but also in theAsselineau, 1998; Bernerd et al., 2000). deep dermal compartment of the skin, such as degrada- Solar UV light reaching Earth is a combination of both tion of the connective tissue, decrease in collagen con-UVB (290–320 nm) and UVA (320–400 nm) wave- tent, and accumulation of abnormal elastic tissuelengths. Although UVB irradiation has received more characterizing solar elastosis (Bernerd and Asselineau,attention, an increasing number of studies are now 1998; Bernerd et al., 2000). Moreover, photoaging isemphasizing the harmful effects of UVA (Bernerd and associated with the appearance of advanced glycationAsselineau, 1998, 2008). However, for ethical reasons, end products (AGEs). AGEs are new residues createdexperimental chronic UV exposure assays pose a chal- by cross-linked formations that are produced by a non-lenge in humans. enzymatic glycation reaction in the extracellular matrix Conventional monolayer cultures do not reproduce of the dermis. AGEs are now considered one of the fac-accurately the physiological conditions for studying UV tors responsible for loss of elasticity and other proper-exposure (Bernerd and Asselineau, 1998, 2008). ties of the dermis during aging (Pageon and Asselineau,Instead, the full 3D skin model composed of dermal and 2005). In a study that compared the histological resultsepidermal equivalent layers may be suitable for deter- obtained within the reconstructed skin containing nativemining specific biological effects induced by UVB and collagen and collagen modified by glycation, no signifi-UVA irradiation (Bernerd and Asselineau, 1998; Bernerd cant differences were observed in morphological struc-et al., 2000). As the reconstructed skin model is able to ture except for the reduction of dermal thicknesses indifferentiate epidermal horny layers, compounds or sun- the glycated sample. Pageon and Asselineau (2005) alsoscreens can be topically applied on the skin, mimicking concluded that this system is a promising model toa more realistic situation (Bernerd et al., 2000). For a observe the effects of aging on ECM elements and mayproper UV-irradiation study of topically applied sunsc- provide a tool for evaluating the efficacy of AGE inhibi-reens considering the UV effects on photoaging, the tors (Pageon and Asselineau, 2005).use of pigmented reconstructed epidermis containing Other studies involving aging, the ECM, and evalua- ´melanocytes is crucial (Nakazawa et al., 1998; Regnier tion of new molecules are also utilizing skin recon-et al., 1999). structs as a study model. Sok et al. (2008) showed that The solar protection factor (SPF) that corresponds to a C-xylopyranoside derivative, C-b-D-xylopyranoside-2-erythema prevention is one of first features for evaluat- hydroxy-propane (C-xyloside), induced the neo-synthesising sunscreen protection. After 24 h of UVB exposure, of matrix proteins such as glycosaminoglycans and hep-typical sun-burned cells (SBC) are formed in the epider- aran sulfate proteoglycans, and also restored dermalmis, corresponding to the clinical appearance of ery- epidermal junction integrity, suggesting that it may havethema. SBC correspond to apoptotic keratinocytes, beneficial effects on aged skin (Sok et al., 2008).which allow elimination of cells strongly damaged byUVB irradiation. Previously, erythema could not be Pharmacological applicationsassessed in vitro, but sunscreen efficiency can now be In the field of pharmacology, drug discovery is generallymeasured in reconstructed skin in vitro by observation dependent upon the predictive capacity of cell-basedand counting of UVB-induced SBC (Bernerd et al., assays (Mazzoleni et al., 2009). Most frequently, the2000). efficacy of anti-cancer drugs is tested in 2D monolayer The use of a full skin reconstruct model in UV studies cells cultured on plates during the initial drug develop-is essential as the major skin target of UVB is the epi- ment and discovery phase. However, huge differencesdermis, whereas UVA exposure mainly affects the der- are observed when these drugs are tested in vivo.mis. UVB causes significant alterations in keratinocyte These differences may be the result of different cell sur-differentiation processes, whereas UVA induces apopto- face receptors, proliferation kinetics, ECM components,sis in fibroblasts located in the superficial area of the cellular densities, and metabolic functions of 2D-main-dermal equivalent (Bernerd and Asselineau, 1998, tained cells (Horning et al., 2008).2008). As mentioned above, pharmacological penetration is Skin penetration is a key point in the evaluation of a an important limitation in pharmacological compoundspotential skin sunscreen. The ability of these com- that need to reach deep layers of the skin (Godin andpounds to penetrate the skin will depend on the time ´ Touitou, 2007; Regnier et al., 1993). Skin from cadaversand concentration required to reach the desired target has previously been used in drug transport studies, butsite. The stratum corneum is the main barrier against limited availability and large variations between speci-skin penetration, and efficient penetration of this barrier mens have now increased the application potentialdepends on specific processes (Ponec, 2002). of skin reconstruct models (Pasonen-Seppa ¨nen et al.,ª 2010 John Wiley & Sons A/S 45
    • Brohem et al.2001). The 3D model has permeability characteristics duced with the basic fibroblast growth factor gene. Inand metabolic activity resembling that of native skin. the vertical growth phase the cells were able to invadeThis is critical, as metabolic activity may affect the per- the dermis and an irregular basement membrane wasmeability of some drugs and their potential for research formed. In metastatic melanoma, cells rapidly prolifer-on irritation, toxicity, and keratinocyte differentiation ated and aggressively invaded deep into the dermis, in(Godin and Touitou, 2007; Pasonen-Seppanen et al., ¨ a growth pattern very similar to the pattern in vivo ´2001; Regnier et al., 1993). (Meier et al., 2000). One of the concerns regarding the skin reconstruct These skin cultures are excellent models to assessmodel is its lack of skin appendages, including piloseba- melanoma–keratinocyte interactions (Hsu et al., 2000),ceous units, hair follicles, and sweat glands. Because of as well as study keratinocyte-derived lesions.this lack, this model provides much lower barrier proper- A study characterized the migratory pattern of a squa-ties than that found in whole skin. Consequently, while mous cell carcinoma cell line (HaCaT-II-4) when E-cadh-the skin reconstruct model is superior to a monolayer erin expression was suppressed. The authors were ablemodel, the kinetic parameters of skin permeation to show that loss of cell adhesion enabled migration ofobtained from these studies must still be considered an single, intra-epithelial tumor cells between normal kerati-overestimation when compared to the flux across nocytes that were essential for initial stromal invasionhuman skin (Godin and Touitou, 2007). (Alt-Holland et al., 2008). Boccardo et al. (2004) used or- ganotypic cultures of human keratinocytes to evaluateSkin cancer the effects of TNF-a in cells that expressed HPV-18 onc-Cancer is a heterogeneous disease whose initiation and ogenes. Another example of the utilization of organotypicprogression is tightly modulated by cell–cell and cell– culture in epithelial tumor models is described bymatrix interactions. For these reasons, the use of 3D Hoskins et al. (2009), who studied Fanconi anemia (FA).culture models has been steadily increasing in studies They described the growth and molecular properties ofof tumor biology (Chioni and Grose, 2008). FA-associated cancers (FANCA)-deficient versus FAN- In invasive skin cancers such as melanoma, skin CA-corrected HPV E6 ⁄ E7 immortalized keratinocytesreconstructs are very convenient for modeling not only in monolayer and organotypic epithelial raft culturesthe growth and progression of melanoma cells in a 3D (Hoskins et al., 2009).microenvironment, but also for studying the communi-cation among melanoma cells and surrounding epider- Skin disorders and clinical applicationsmal keratinocytes and dermal fibroblasts (Berking and Skin reconstructs are currently being tested and used inHerlyn, 2001; Smalley et al., 2006). The use of artificial clinics for several skin pathologies. Disorders which mayskin has shown that surrounding fibroblasts are benefit from the development of human skin equivalentsrecruited by the primary melanoma and provide survival include psoriasis (Barker et al., 2004; Gazel et al., 2006;signals in the form of altered ECM deposition and Konstantinova et al., 1996; Tjabringa et al., 2008), vitiligogrowth factors, as well as stimulating the production of ´ (Bessou et al., 1997; Cario-Andre et al., 2007), keloidsmatrix metalloproteinases, promoting tumor cell inva- (Butler et al., 2008; Chiu et al., 2005), nevus (Gontiersion (Haass and co-workers 2005; Smalley et al., 2006). et al., 2002, 2004) and genodermatoses such as xero- ´Fernandez et al. (2006) used human artificial skin to test derma pigmentosum (Bergoglio et al., 2008; Bernerdthe efficacy of bortezomib, an anti-tumoral drug acting et al., 2001; Ergun et al., 2002; Herlin et al., 2009) and ¨on the proteasome, in a 3D model. Yu and co-workers epidermolysis bullosa (De Luca et al., 2009; Ferrari(2009) evaluated the role of BRAF mutation and p53 et al., 2006; Mavilio et al., 2006; Wong et al., 2007).inactivation during transformation of a subpopulation of The best temporary skin replacement for full-thicknessprimary human melanocytes, and observed the forma- burns, considering structure and function, is the cadavertion of pigmented lesions reminiscent of in situ mela- allograft skin (Hansen et al., 2001; Phillips, 1998; Wongnoma in artificial skin reconstructs. In addition, artificial et al., 2007). However, difficulties associated with han-skin has been used to screen the therapeutic potential dling and transport of the material, as well as the possi-of oncolytic adenoviruses in melanocytic cells. Organo- ble risk of disease transmission, have limited thetypic 3D culture models are also used for different availability and use of this model (Hansen et al., 2001;tumor types including breast, prostate, and ovarian can- Phillips, 1998; Wong et al., 2007). There has thereforecer (Chioni and Grose, 2008). been a rapid increase in the use of split-thickness skin Skin models have also been used for genetic and grafts or cultured epidermal grafts in clinical proceduresfunctional analyses of early stages of tumor develop- in the past few years (Phillips, 1998; Wong et al., 2007).ment. Normal melanocytes in this model remained sin- While the main use of homologous skin grafts, recon-gly distributed at the basement membrane. In the radial structs or related bio-products has traditionally been ingrowth phase of melanoma, proliferation and migration the treatment of severe burns and skin disorders, vari-of the cancer cells in the dermal reconstruct and tumori- ous new clinical and experimental approaches in whichgenicity in vivo were observed when cells were trans- this type of skin substitute can be useful have recently46 ª 2010 John Wiley & Sons A/S
    • Artificial skinemerged. The main new clinical indications for skin allo- dimensional organotypic epidermal model. Exp. Cell Res. 313,grafts include skin loss, surgical wounds, and genoder- 3005–3015. Alt-Holland, A., Shamis, Y., Riley, K.N., DesRochers, T.M., Fusenig,matoses (Bernerd et al., 2001; De Luca et al., 2009; N.E., Herman, I.M., and Garlick, J.A. (2008). E-cadherin suppres-Ergun et al., 2002; Ferrari et al., 2006; Fimiani et al., ¨ sion directs cytoskeletal rearrangement and intraepithelial tumor2005; Herlin et al., 2009; Wong et al., 2007). cell migration in 3D human skin equivalents. J. Invest. Dermatol. Two key factors were considered essential for the uti- 128, 2498–2507.lization of skin substitutes in clinical applications: the Asselineau, D., Bernard, B.A., Bailly, C., and Darmon, M. (1985).ability to grow keratinocytes in vitro and the increasing Epidermal morphogenesis and induction of 67kD keratin polypep-practice of early wound excision in the extensively tide by culture at the liquid-air interface. Exp. Cell Res. 159, 536– 539.burned patient (Cooper et al., 1991). Auxenfans, C., Fradette, J., Lequeux, C., Germain, L., Kinikoglu, B., Human skin substitutes are now commercially avail- Bechetoille, N., Braye, F., Auger, F.A., and Damour, O. (2008).able from different companies with different composi- Evolution of three dimensional skin equivalent models recon-tions; however, they present a number of challenges structed in vitro by tissue engineering. Eur. J. Dermatol. 19,regarding preclinical safety evaluation. In particular, for 107–113.clinical use, the companies must demonstrate that the Balasubramani, M., Kumar, T.R., and Babu, M. (2001). Skin substi-skin grafts do not carry pathogens, that they lack immu- tutes: a review. Burns 27, 534–544. Barker, C.L., McHale, M.T., Gillies, A.K., Waller, J., Pearce, D.M.,nogenicity, that they show normal physiological func- Osborne, J., Hutchinson, P.E., Smith, G.M., and Pringle, J.H.tions, and that they have no potential for tumorigenicity (2004). The development and characterization of an in vitro(Nemecek and Dayan, 1999). model of psoriasis. J. Invest. Dermatol. 123, 892–901. Bell, E., Ivarsson, B., and Merrill, C. (1979). Production of a tissue- like structure by contraction of collagen lattices by human fibro-Final comments blasts of different proliferative potential in vitro. Proc. Natl Acad. Sci. U S A 76, 1274–1278.As artificial skin reconstructs are becoming robust and Bergoglio, V., Warrick, E., Chevallier-Lagente, O., and Magnaldo, T.have been validated for many applications, there has (2008). Cutaneous gene therapy: the graft takes. Med. Sci.been a push towards their use in the clinic (i.e. skin (Paris) 24, 607–614.repair following wounding or burning). Besides these Berking, C., and Herlyn, M. (2001). Human skin reconstruct mod-applications, organotypic skin provides an amenable set- els: a new application for studies of melanocyte and melanomating for functional analyses of genetically altered cells in biology. Histol. Histopathol. 16, 669–674.the context of physiological cell–cell and cell–matrix Bernerd, F., and Asselineau, D. (1997). Successive alteration and recovery of epidermal differentiation and morphogenesis afterinteractions. There is also a wide range of possibilities specific UVB-damages in skin reconstructed in vitro. Dev. Biol.for studies of UV-induced carcinogenesis and aging, as 183, 123–138.well as efficacy and selectivity of chemotherapeutic Bernerd, F., and Asselineau, D. (1998). UVA exposure of humanagents. In addition to melanoma, or frequent tumors of skin reconstructed in vitro induces apoptosis of dermal fibro-keratinocytes (i.e. basal cell or squamous cell carcino- blasts: subsequent connective tissue repair and implications inmas), other skin disorders such as vitiligo and psoriasis, photoaging. Cell Death Differ. 5, 792–802.in which cell–cell contact is a preponderant factor, can Bernerd, F., and Asselineau, D. (2008). An organotypic model of skin to study photodamage and photoprotection in vitro. J. Am.also be studied using three-dimensional skin modes. Acad. Dermatol. 58, S155–S159.Advances in the stem cell field offer the exciting possi- Bernerd, F., Vioux, C., and Asselineau, D. (2000). Evaluation of thebility of generating large numbers of donor-matched protective effect of sunscreens on in vitro reconstructed humanbackgrounds for autologous transplants, as well as a skin exposed to UVB or UVA irradiation. Photochem. Photobiol.platform for in depth analyses of the physiological impact 71, 314–320.of various skin cell precursors in health and disease. Bernerd, F., Asselineau, D., Vioux, C., Chevallier-Lagente, O., Bouadjar, B., Sarasin, A., and Magnaldo, T. (2001). Clues to epidermal cancer proneness revealed by reconstruction of DNAAcknowledgements repair-deficient xeroderma pigmentosum skin in vitro. Proc. Natl Acad. Sci. U S A 98, 7817–7822.We are especially grateful to Dr. Monique Verhaegen (Dermatology ` Bessou, S., Gauthier, Y., Surleve-Bazeille, J.E., Pain, C., and Taıeb, ¨Department, University of Michigan, USA), for the critical reading A. (1997). Epidermal reconstructs in vitiligo: an extrinsic factor isof the manuscript and her precious suggestions. This study was needed to trigger the disease. Br. J. Dermatol. 137, 890–897.supported by FAPESP (2006 ⁄ 50479-7 and 2008 ⁄ 58817-4), CNPq, Bissell, M.J., Hall, H.G., and Parry, G. (1982). How does theCAPES and PRP-USP. M.S. is supported by NIH R01 CA107237, extracellular matrix direct gene expression? J. Theor. Biol. 99,CA125017; Spanish Ministry of Science and Innovation SAF 2008- 31–68.1950 and institutional grants from the Spanish Association against Boccardo, E., Noya, F., Broker, T.R., Chow, L.T., and Villa, L.L.Cancer. (2004). HPV-18 confers resistance to TNF-alpha in organotypic cultures of human keratinocytes. Virology 328, 233–243. Boehnke, K., Mirancea, N., Pavesio, A., Fusenig, N.E., Boukamp,References P., and Stark, H.J. (2007). Effects of fibroblasts and microenvi-Ajani, G., Sato, N., Mack, J.A., and Maytin, E.V. (2007). Cellular ronment on epidermal regeneration and tissue function in long- responses to disruption of the permeability barrier in a three- term skin equivalents. Eur. J. Cell Biol. 86, 731–746.ª 2010 John Wiley & Sons A/S 47
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