In vitro characterization of an artificial dermal scaffold

The treatment of extensive burn injuries has been enhanced by the development of artificial skin substitutes. Integra Artificial Skin, an acellular collagen-glycosaminoglycan (C-GAG) dermal equivalent requires a two-stage grafting procedure. However, preseeding the C-GAG dermal equivalent with cultured fibroblasts and keratinocytes, with the aim of performing a single-stage grafting procedure, may be beneficial in terms of replacing the requirement for traditional split-skin grafts. In this comparative in vitro study, the interactions of cultured human dermal fibroblasts and epidermal keratinocytes in Integra Artificial Skin in comparison to cadaver deepidermalized dermis (DED) was investigated. An increase in cell proliferation and migration in the C-GAG dermal equivalent was observed over time. Cocultures of fibroblasts and keratinocytes on both dermal equivalents showed positive expression of proliferation, differentiation, and extracellular matrix (ECM) protein markers. Organization of keratinocytes in the epidermal layers of DED composites were better compared to the C-GAG composites. Deposition of ECM proteins was enhanced in the presence of keratinocytes in both dermal equivalents. Results demonstrate that in vitro the C-GAG dermal equivalent is biocompatible for cell attachment, migration, proliferation, and differentiation. Preseeding Integra Artificial Skin with cultured autologous fibroblasts and keratinocytes for in vivo application, as a single-stage grafting procedure, warrants testing. A better clinical outcome may be achieved as shown by our in vitro results of the coculture composites.     

IFATS collection: Using human adipose-derived stem/stromal cells for the production of new skin substitutes

The ability to harvest and culture stem cell populations from various human postnatal tissues is central to regenerative medicine applications, including tissue engineering. The discovery of multipotent mesenchymal stem cells within the stromal fraction of adipose tissue prompted their use for the healing and reconstruction of many tissues. Here, we examined the influence of adipose-derived stem/stromal cells (ASCs) on skin's regenerative processes, from a tissue engineering perspective. Using a self-assembly approach, human skin substitutes were produced. They featured a stromal compartment containing human extracellular matrix endogenously produced from either dermal fibroblasts or adipose-derived stem/stromal cells differentiated or not toward the adipogenic lineage. Human keratinocytes were seeded on each stroma and cultured at the air-liquid interface to reconstruct a bilayered skin substitute. These new skin substitutes, containing an epidermis and a distinctive stroma devoid of synthetic biomaterial, displayed characteristics similar to human skin. The influence of the type of stromal compartment on epidermal morphogenesis was assessed by the evaluation of tissue histology, the expression of key protein markers of the epidermal differentiation program (keratin [K] 14, K10, transglutaminase), the expression of dermo-epidermal junction components (laminins, collagen VII), and the presence of basement membrane and hemidesmosomes. Our findings suggest that adipose-derived stem/stromal cells could usefully substitute dermal fibroblasts for skin reconstruction using the self-assembly method. Finally, by exploiting the adipogenic potential of ASCs, we also produced a more complete trilayered skin substitute consisting of the epidermis, the dermis, and the adipocyte-containing hypodermis, the skin's deepest layer. Disclosure of potential conflicts of interest is found at the end of this article.     

Development of a closed bioreactor system for culture of tissue-engineered skin at an air-liquid interface

A bioreactor has been developed for the production of tissue-engineered skin at an air-liquid interface for clinical and experimental use. In this closed system, scaffold and bioreactor sterilization, cell seeding, and medium perfusion were all performed with a peristaltic pump. Natural and synthetic dermal substitutes were seeded directly with skin cells without opening the bioreactor and fed either by continuous medium perfusion or by batch-feed. The system was validated by monoculture of human dermal fibroblasts and keratinocytes and the coculture of both cell types in acellular human dermis, Azowipes, electrospun polystyrene, and an electrospun composite of polystyrene and poly-DL-lactide fibers. A comparison was made of culture at an air-liquid interface versus submerged culture and of medium change by continuous perfusion versus batch-feed. Fibroblast and endothelial cells showed greater viability under submerged rather than air-liquid conditions whereas keratinocytes favored culture at an air-liquid interface as did cocultured keratinocytes and fibroblasts. Total cellular viability for reconstructed skin with keratinocytes and fibroblasts was greatest with continuous perfusion rather than batch-feed and with electrospun scaffolds compared with acellular human dermis. The bioreactor could also be easily configured to give replicate small areas for experimental use or one continuous area of construct for clinical use.     

Effects of serum-free culture at the air-liquid interface in a human tissue-engineered skin substitute

Previous studies have reported that well-defined culture conditions can improve keratinocytes terminal differentiation and reproducibility. The aim of our study was to compare skin substitutes cultured in a complete medium with those cultured in a serum-free medium at the air-liquid interface to optimize the self-assembly method. Skin substitutes, cultured in a serum-free medium over 7, 14, and 21 days, were compared with others cultured in a complete medium (5% serum) over the complete culture period. Masson's Trichrome staining showed that the substitutes cultured in a serum-free medium generated a well-developed and differentiated epidermis. Immunolabeling analyses between the substitutes cultured without serum and those cultured in complete serum showed similar expression of epidermal differentiation markers, dermo-epidermal junction, and dermal extracellular matrix components. On the basis of our Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) results, the skin substitutes cultured in serum-free condition over 21 days of culture at the air-liquid interface showed lower frequencies of the CH(2) symmetric mode of vibrations, which means a better lipid organization of the stratum corneum. No significant difference in hydrocortisone penetration was observed between serum-free medium substitutes and the controls. Results demonstrate that the absence of serum does not compromise the characteristics of the skin substitutes observed in this study.     

Cytotoxicity tests for antimicrobial agents using cultured skin substitutes fixed at interface of air and culture medium

The present study is focussed on a new cytotoxicity test using cultured dermal and epidermal sheets, which are fixed at the air and medium interface as a wound surface model. The cultured dermal sheet is composed of human fibroblasts and a collagen matrix, and the cultured epidermal sheet is composed of human keratinocytes and a collagen matrix. Each cultured sheet was fixed at the air and medium interface, over which a piece of test specimen was placed. The in vitro system created, provides a mimetic wound surface since during wound repair, fibroblasts are embedded in an extracellular matrix, while keratinocytes migrate and proliferate on provisional granulation tissue. The results thus obtained in this cytotoxicity test are useful for determining the efficacious amount of antimicrobial agent used in clinical cases.     

Insulin-like growth factor-binding protein contributes to the proliferation of less proliferative cells in forming skin equivalents

In this study, the effects and the mediating factors of dermal cells on the epidermal regenerative ability were investigated. Human epidermal cells were separated into rapidly adhering (RA) cells and slowly adhering (SA) cells and used for culturing skin equivalents (SEs). For dermal part, normal human fibroblasts, dermal sheath cells (DSCs), and dermal papilla cells were used. SEs produced using SA cells and DSCs showed a thicker epidermis and higher expressions of alpha(6)- and beta1-integrin than SEs using SA cells and normal fibroblasts showed. We hypothesized that DSCs may secrete specific cytokines that can influence the regenerative potential of epidermal cells, and compared cytokine secretion by DSCs and normal human fibroblasts. Using RayBio human cytokine antibody array C (series 1000), 120 cytokines were tested. Results showed that DSCs produced a much greater amount of insulin-like growth factor-binding protein (IGFBP-2), angiogenin, and BMP-6 than normal human fibroblasts produced. On the basis of the cytokine antibody array, we next investigated whether IGFBP-2, angiogenin, or BMP-6 has effects on SEs reconstruction. The addition of IGFBP-2 induced a thicker and more mature epidermis and higher expressions of alpha(6)- and beta1-integrin, whereas BMP-6 exhibited little effect. Thus, the SEs with IGFBP-2 showed almost the same morphology of the SEs using DSCs. Further, p63, a putative keratinocyte stem cell marker, was more frequently observed in the basal layer of SE with IGFBP-2. In conclusion, IGFBP-2 is a major factor from DSCs that affects epidermal regenerative capacity of skin and may play an important role for stemness maintenance in human epidermal keratinocytes.     

Self-organization of skin cells in three-dimensional electrospun polystyrene scaffolds

Much research in tissue engineering focuses on the synthesis of complex three-dimensional polymer scaffolds containing functional biomolecules to which cells are introduced. Typical scaffolds for skin tissue engineering are macroscopically porous with struts or fibers approximately 10 microm thick at a packing fraction of approximately 0.1. We made a polystyrene scaffold without cell signaling or spatial information by electrospinning and studied the growth of skin fibroblasts, keratinocytes, and endothelial cells, as single and cocultured populations in the presence and absence of fetal calf serum. In the absence of serum, keratinocytes, fibroblasts, and endothelial cells did not grow when cultured alone. However, when fibroblasts were cocultured with keratinocytes and endothelial cells, expansion of keratinocytes and endothelial cells occurred even in the absence of serum. Furthermore, cells displayed native spatial three-dimensional organization when cultured at an air-liquid interface, even when all three cell types were introduced at random to the scaffold. This study shows that coculture with fibroblasts enables keratinocytes and endothelial cells to proliferate without serum, but also to self-organize according to the native epidermal-dermal structure given the symmetry-breaking field of an air-liquid interface.     

Production of a novel fibroblast-populated platelet matrix cocultured with keratinocytes

We have developed a new method for the production of a dermal matrix equivalent. Human platelets were used to dilute human fibroblasts. The platelet mix was placed in a cell culture well. Addition of 200 microL of a thrombin solution caused gel formation. Gels were overlaid with standard Iscove's growth medium supplemented with 10% fetal bovine serum, insulin, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer. Medium was exchanged regularly. Keratinocytes were plated on top of selected gels and elevated to the air-liquid interface. The gels were harvested weekly, fixed, cut, and stained with hematoxylin and eosin stains and immunostains for collagens I, III, and IV and cytokeratins. Digital image analysis was used to quantitate collagen production. Growth factors, including transforming growth factor-beta (TGF-beta), platelet-derived growth factor, and vitamin C were added. Staining identified fibroblasts within the gels with a surrounding fibrous matrix. Immunostaining for cytokeratin identified keratinocytes on the gel surface. Immunostaining revealed the fibrous matrix to be composed of collagen I and III and some collagen IV. Digital image analysis demonstrated that greater TGF-beta concentration resulted in greater collagen production. These differences were statistically significant. With development of this construct, a viable dermal/epidermal replacement may be possible. TGF-beta enhances collagen production by fibroblasts in this matrix.     

Fibroblasts facilitate re-epithelialization in wounded human skin equivalents

The re-epithelialization of the wound involves the migration of keratinocytes from the edges of the wound. During this process, keratinocyte migration and proliferation will depend on the interaction of keratinocytes with dermal fibroblasts and the extracellular matrix. The present study aimed to investigate (1) the role of fibroblasts in the re-epithelialization process and on the reconstitution of the dermal-epidermal junction (DEJ) and (2) differential protein expression during re-epithelialization. For both purposes, three-dimensional human skin equivalents (HSE) were used. A full-thickness wound in HSE was introduced by freezing with liquid nitrogen and a superficial wound by linear incision with a scalpel. The closure of the wound in the absence or presence of exogenous growth factors was followed by monitoring the rate of re-epithelialization and regeneration of the DEJ. The results obtained in this study demonstrate that fibroblasts facilitate wound closure, but they differentially affected the deposition of various basement membrane components. The deposition of laminin 5 at the DEJ was delayed in superficial wounds as compared to the full-thickness wounds. During freeze injury, some basement membrane (BM) components remain associated with the dermal compartment and probably facilitate the BM reconstitution. The re-epithelialization process in full-thickness but not in superficial wounds was accelerated by the presence of keratinocyte growth factor and especially by epidermal growth factor. In addition, we have examined the deposition of various basement membrane components and the differences in protein expression in a laterally expanding epidermis in uninjured HSE. Laminin 5, type IV and VII collagen deposition was decreased in the laterally expanding epidermis, indicating that the presence of these proteins is not required for keratinocyte migration to occur in vitro. Using two-dimensional polyacrylamide gel electrophoresis, we have identified DJ-1, a protein not earlier reported to be differently expressed during the epithelialization process of the skin.     

应用胶原海绵构建组织工程皮肤的实验研究

目的探讨以胶原海绵为支架材料构建组织工程皮肤的方法。方法体外分离、培养、鉴定角朊细胞和成纤维细胞,制备胶原海绵作为组织工程支架材料;在成功构建人工真皮的基础上,种植表皮细胞,构建人工复合皮肤,通过大体观察、HE染色与免疫组织化学进行组织学检测。结果制备的胶原海绵孔径平均100~150μm,孔隙率89%,组织相容性良好;以胶原海绵构建的皮肤替代物免疫组织化学染色显示Ⅳ型胶原、纤维连接蛋白和层粘连蛋白阳性,HE染色可见均具有表皮和真皮双层结构,在形态结构上与正常皮肤相似。结论培养的人角朊细胞和成纤维细胞种植于胶原海绵支架上培养可构建出具有类似天然皮肤结构的组织工程皮肤。     

The influence of microtextured basal lamina analog topography on keratinocyte function and epidermal organization

The rational design of future bioengineered skin substitutes requires an understanding of the mechanisms by which the three-dimensional microarchitecture of tissue scaffolds modulates keratinocyte function. Microtextured basal lamina analogs were developed to investigate the relationship between the characteristic topography at the dermal-epidermal interface of native skin and keratinocyte function. Microfabrication techniques were used to create master patterns, negative replicates, and collagen membranes with ridges and channels of length scales (e.g., grooves of 50-200 microm in depth and width) similar to the invaginations found in basal lamina at the dermal-epidermal junction of native skin. Keratinocytes were seeded on the surfaces of basal lamina analogs, and histological analyses were performed after 7 days of tissue culture at the air-liquid interface. The keratinocytes formed a differentiated and stratified epidermis that conformed to the features of the microtextured membranes. Morphometric analyses of immunostained skin equivalents suggest that keratinocyte stratification and differentiation increases as channel depth increases and channel width decreases. This trend was most pronounced in channels with the highest depth-to-width ratios (i.e., 200 microm deep, 50 microm wide). It is anticipated that the findings from these studies will elucidate design parameters to enhance the performance of future bioengineered skin substitutes.     

The use of PEGT/PBT as a dermal scaffold for skin tissue engineering

Human skin equivalents (HSEs) were engineered using biodegradable-segmented copolymer PEGT/PBT as a dermal scaffold. As control groups, fibroblast-populated de-epidermized dermis, collagen, fibrin and hybrid PEGT/PBT-collagen matrices were used. Two different approaches were used to generate full-thickness HSE. In the 1-step approach, keratinocytes were seeded onto the fibroblast-populated scaffolds and cultured at the air-liquid (A/L) interface. In the 2-step approach, fully differentiated epidermal sheets were transferred onto fibroblast-populated scaffolds and cultured at the A/L. In a 1-step procedure, keratinocytes migrated into the porous PEGT/PBT scaffold. This was prevented by incorporating fibroblast-populated collagen into the pores of the PEGT/PBT matrix or using the 2-step procedure. Under all experimental conditions, fully differentiated stratified epidermis and basement membrane was formed. Differences in K6, K16, K17, collagen type VII, laminin 5 and nidogen staining were observed. In HSE generated with PEGT/PBT, the expression of these keratins was higher, and the deposition of collagen type VII, laminin 5 and nidogen at the epidermal/matrix junction was retarded compared to control HSEs. Our results illustrate that the copolymer PEGT/PBT is a suitable scaffold for the 2-step procedure, whereas the incorporation of fibroblast-populated collagen or fibrin into the pores of the scaffold is required for the 1-step procedure.     

Conjugation of extracellular matrix proteins to basal lamina analogs enhances keratinocyte attachment

The dermal-epidermal junction of skin contains extracellular matrix proteins that are involved in initiating and controlling keratinocyte signaling events such as attachment, proliferation, and terminal differentiation. To characterize the relationship between extracellular matrix proteins and keratinocyte attachment, a biomimetic design approach was used to precisely tailor the surface of basal lamina analogs with biochemistries that emulate the native biochemical composition found at the dermal-epidermal junction. A high-throughput screening device was developed by our laboratory that allows for the simultaneous investigation of the conjugation of individual extracellular matrix proteins (e.g. collagen type I, collagen type IV, laminin, or fibronectin) as well as their effect on keratinocyte attachment, on the surface of an implantable collagen membrane. Fluorescence microscopy coupled with quantitative digital image analyses indicated that the extracellular matrix proteins adsorbed to the collagen-GAG membranes in a dose-dependent manner. To determine the relationship between extracellular matrix protein signaling cues and keratinocyte attachment, cells were seeded on protein-conjugated collagen-GAG membranes and a tetrazolium-based colorimetric assay was used to quantify viable keratinocyte attachment. Our results indicate that keratinocyte attachment was significantly enhanced on the surfaces of collagen membranes that were conjugated with fibronectin and type IV collagen. These findings define a set of design parameters that will enhance keratinocyte binding efficiency on the surface of collagen membranes and ultimately improve the rate of epithelialization for dermal equivalents.     

Skin basement membrane and extracellular matrix proteins characterization and quantification by real time RT-PCR

Three-dimensional gelatin-chondroitin 6 sulphate-hyanuronic acid (gelatin-C6S-HA) biomatrices were used as the scaffold to investigate the phenotypic and molecular expression of basement membrane (BM) and extracellular matrix (ECM) proteins in vitro. The cells were cultured in three different culture conditions: keratinocytes (K) monoculture, or dermal fibroblasts (FB) monoculture, or organotypic keratinocytes and dermal fibroblasts (K&FB) coculture model. The deposition of BM proteins and ECM proteins secreted by these two kinds of cells was quantitatively characterized by real time RT-PCR and examined by immunohistochemistry. The results showed that K expressed specific keratin and E-cadherin proteins, while type I collagen was secreted by FB. FB were shown to synthesize and deposit laminin 5, type IV collagen, and type VII collagen, whereas K dominantly produced integrin alpha 6 and integrin beta 4 as well as laminin 5. Interestingly, the integrin beta 4 was expressed neither in K monoculture nor in FB monoculture, but was seen in organotypic K&FB coculture model in the early culture stage. The histology studies revealed numerous features of epidermalization including a well organized basal layer of distinct cylindrical cells, granular and a horny layer, as well as complete BM formation. These results indicated that K and FB not only kept their phenotype when culturing on 3D scaffold, but also worked together to reconstruct dermal-epidermal basement membrane zone. In brief, our results directly provide the quantification in the expression of BM and ECM proteins by using real time RT-PCR in mRNA level and morphological appearance by immunostain in protein level.     

Epidermis promotes dermal fibrosis: role in the pathogenesis of hypertrophic scars

Hypertrophic scarring is a pathological process characterized by fibroblastic hyperproliferation and by excessive deposition of extracellular matrix components. It has been hypothesized that abnormalities in epidermal-dermal crosstalk explain this pathology. To test this hypothesis, a tissue-engineered model of self-assembled reconstructed skin was used in this study to mimic interactions between dermal and epidermal cells in normal or pathological skin. These skin equivalents were constructed using three dermal cell types: normal wound (Wmyo) or hypertrophic wound (Hmyo) myofibroblasts and normal skin fibroblasts (Fb). Epidermis was reconstructed with normal skin keratinocytes (NK) or hypertrophic scar keratinocytes (HK). In the absence of keratinocytes, Hmyo formed a thicker dermis than Wmyo. When seeded with NK, the dermal thickness of Hmyo (121.2 +/- 31.4 microm vs 196.2 +/- 27.8 microm) and Fb (43.7 +/- 7.1 microm vs 83.6 +/- 16.3 microm) dermis was significantly (p < 0.05) reduced, while that of Wmyo (201.5 +/- 15.7 microm vs 160.7 +/- 21.1 microm) was increased. However, the presence of HK always induced significantly thicker dermis formation than observed with NK (Wmyo: 238.8 +/- 25.9 microm; Hmyo: 145.5 +/- 22.4 microm; Fb: 74.2 +/- 11.2 microm). These results correlated with collagen and MMP-1 secretion and with cell proliferation, which were increased when keratinocytes were added, except for the collagen secretion of Hmyo and Fb in the presence of NK. The level of dermal apoptosis was not different when epidermis was added to the dermis (<1% in each category). These observations strongly suggest that hypertrophic scar keratinocytes play a role in the development of pathological fibrosis by influencing the behaviour of dermal cells.     

Safety evaluation of human living skin equivalents.

Human living skin equivalents (LSEs) offer an alternative to the use of split-thickness autografts for the treatment of hard-to-heal wounds. LSEs consist of 4 active components: a well-differentiated stratum corneum derived from epidermal keratinocytes, dermal fibroblasts, and an extracellular collagen matrix. Neonatal foreskins are used as the source of keratinocytes and dermal fibroblasts for the manufacture of LSEs. Following isolation and expansion in vitro, the cells are cultured on a 3-dimensional scaffold to give an upper epidermal layer and supporting dermal layer. The resulting product has the appearance and handling characteristics of human skin. Safety evaluation of LSEs begins with insuring that foreskins are obtained only from healthy infants whose mothers are negative for a panel of adventitious agents. Keratinocyte and fibroblast cell banks are characterized using morphologic, biochemical, and histologic criteria; checked for the absence of contaminating cell types such as melanocytes, macrophages, lymphocytes, and Langerhans cells; subjected to rigorous microbiological testing (with any production materials of biological origin); and evaluated for in vivo tumorigenicity. The consistency of certain key morphologic and functional characteristics are regularly assessed. Because an LSE represents an allogeneic graft, preclinical safety studies include in vitro and in vivo determinations of its potential immunogenicity. Immunocompromised (SCID) mice reconstituted with human leukocytes or engrafted with human fetal hematolymphoid organs have been useful animal models for assessing possible immunologic responses to LSEs. Additional preclinical studies are being conducted to show that LSEs are noncytotoxic and lack allergenic, sensitizing, or irritation potential.     

组织工程皮肤的构建及组织形态学观察

背景：应用于临床的组织工程皮肤具有血管化速度慢、力学强度差以及无法永久性保留等局限,因此,需要对相关技术环节进行改进以制备一种合适的永久性皮肤替代物。 目的：建立一种构建有活性的双层组织工程皮肤的方法,并对其组织形态学进行观察。 方法：以Ⅳ型胶原和成纤维细胞联合修饰的同种脱细胞真皮基质为支架,与表皮干细胞复合后依次经浸没式培养和气液分离界面培养构建组织工程皮肤,利用光镜和扫描电镜对其组织学特点进行观察和分析。 结果与结论：以表皮干细胞和同种脱细胞真皮基质制备的组织工程皮肤具有表、真皮双层结构,其中表皮由多层不同分化程度的表皮细胞组成,真皮为天然三维孔隙结构,胶原纤维完整,且表皮层与真皮层紧密连接,形成整体结构,可满足全层皮肤替代物的基本组织学要求。     

An in vitro examination of an extracellular matrix scaffold for use in wound healing

This paper describes evidence that an extracellular matrix (ECM) secreted by human umbilical vein endothelial cells (HUVECs) assembled on gelatin coated plates overlaid by a mixed matrix secreted by human dermal microvascular endothelial cells (HDMECs) and human dermal fibroblasts provides a viable acellular scaffold for use in wound healing. Trypsinized epidermal keratinocytes or colonies from Dispase-digested fresh and cadaver skin tissue adhered and proliferated on either HUVECs ECM/gelatin or mixed matrix overlaid on HUVECs ECM/gelatin. An epithelial-mesenchymal interaction, previously thought to be tissue-specific, was exposed as well as concomitant integrin versatility. Furthermore, heterologous HDMECs and dermal fibroblasts attached and proliferated on the mixed matrix as well as HUVECs ECM. The conditioned medium from HUVECs (HUVECs CM) was found to neutralize the lingering after effects of Dispase, and could be used for the tissue culture of epidermal keratinocytes, HDMECs and dermal fibroblasts, which share related extracellular secretions. Taken together, these results indicate that cultured epithelial autografts can be redesigned to include both epithelial and dermal elements, and advances the acellular 'sandwich' ECM scaffold as a possible structural replacement for the lamina densa and lamina lucida, damaged or completely missing in some wounds and burns.     

Replacement of animal-derived collagen matrix by human fibroblast-derived dermal matrix for human skin equivalent products

Reconstructed human skin equivalents (HSEs) are representative models of human skin and widely used for research purposes and clinical applications. Traditional methods to generate HSEs are based on the seeding of human keratinocytes onto three-dimensional human fibroblast-populated non-human collagen matrices. Current HSEs have a limited lifespan of approximately 8 weeks, rendering them unsuitable for long-term studies. Here we present a new generation of HSEs being fully composed of human components and which can be cultured up to 20 weeks. This model is generated on a primary human fibroblast-derived dermal matrix. Pro-collagen type I secretion by human fibroblasts stabilized during long-term culture, providing a continuous and functional human dermal matrix. In contrast to rat-tail collagen-based HSEs, the present fibroblast-derived matrix-based HSEs contain more continuity in the number of viable cell layers in long-term cultures. In addition, these new skin models exhibit normal differentiation and proliferation, based on expression of K10/K15, and K16/K17, respectively. Detection of collagen types IV and VII and laminin 332 was confined to the epidermal-dermal junction, as in native skin. The presence of hemidesmosomes and anchoring fibrils was demonstrated by electron microscopy. Finally, we show that the presented HSE contained a higher concentration of the normal moisturizing factor compared to rat-tail collagen-based skin models, providing a further representation of functional normal human skin in vitro. This study, therefore, demonstrates the role of the dermal microenvironment on epidermal regeneration and lifespan in vitro.     

Cell proliferation and differentiation in a model of human skin equivalent

Recent advances in culturing technology has permitted the production of organotypic models that may be referred to as human skin equivalents (HSE). We have studied histochemical, ultrastructural, and kinetic aspects of an HSE composed by an epidermal equivalent and a dermal equivalent separated by a basement membrane. Only keratinocytes and fibroblasts were present in the epidermal and dermal equivalents, respectively; cells of other lineages were lacking. Keratinocyte stratification and differentiation seemed similar to natural skin. Evidence is shown that such an HSE may also release growth factors such as vascular endothelial growth factor that are believed to play a role in skin grafting. The distribution of cycling cells as well as the values of the growth fraction are comparable to those observed in natural skin. Although the absence of several cells populations that reside in natural skin is a remarkable feature of this HSE, the high levels of tissue organization and cell differentiation lead us to believe that such an HSE may be considered a candidate substitute of human skin in biological, pharmacologic, and clinical applications.