Development of an artificial dermis composed of hyaluronic acid and collagen

This study aimed to investigate the efficacy of an artificial dermis composed of hyaluronic acid (HA) and collagen (Col) with or without epidermal growth factor (EGF), both in in vitro and in vivo. The cross-linked high molecular weight HA spongy sheet was prepared by freeze-drying. The spongy sheet was immersed in a mixed solution of high molecular weight HA, low molecular weight HA, and heat-denatured Col, and then lyophilized to obtain a two-layered spongy sheet. Cross-linking among Col molecules was induced by ultraviolet irradiation to prepare the artificial dermis (Type I). In a similar manner, a two-layered artificial dermis containing EGF (Type II) was prepared using a similar mixed solution containing EGF. The in vitro experiments demonstrated that EGF released from the Type II artificial dermis stimulates fibroblasts to produce increased amounts of vascular endothelial growth factor and hepatocyte growth factor. The therapeutic efficacy of artificial dermis was evaluated in animal tests using Sprague Dawley (SD) rats. The dorsal skin of the SD rat was shaved and then exposed to boiling water for 3?s to induce a deep dermal burn. The necrotic tissue was then excised 3?days later. Each artificial dermis was applied to the skin defect for 7?days and assessed for its ability to generate a wound bed. The in vivo experiments demonstrated that the Type II artificial dermis promotes angiogenesis to a greater extent at an early stage (within 3?days), and also suppresses the inflammatory reaction more successfully compared with the Type I artificial dermis. In further animal tests, an autologous skin graft was performed by excising a piece of skin from the abdominal region and then grafting it onto the wound bed prepared using each artificial dermis for 7?days. Although the Type II artificial dermis had the highest potential to promote angiogenesis, in this animal study, each artificial dermis induced excellent wound bed formation acceptable for autologous skin grafting.     

人工真皮治疗创伤后下肢软组织缺损的初步研究

目的探讨人工真皮在修复创伤后下肢软组织缺损中的作用。方法选择下肢创伤后软组织缺损伴有肌肉、肌腱、骨等深部组织外露的患者12例,清创后,将人工真皮移植于伤口覆盖暴露的深部组织,2周后取自体薄皮片移植于其上,1周后换药,观察植皮成活情况。结果所有患者创面愈合良好,深部组织良好覆盖,无明显瘢痕增生,1例因跟骨外露面积大辅以腓肠神经伴行血管岛状皮瓣修复,愈合良好。结论人工合成胶原蛋白海绵治疗创伤后下肢深部组织暴露效果良好,操作简便,局部损伤小,愈合后瘢痕增生不明显。     

Experimental cutaneous wound healing in rabbits: using continuous microamperage low-voltage electrical stimulation

This study was designed to determine the effects of continuous microamperage low-voltage electrical stimulation on cutaneous wound healing. Sixty mature rabbits were randomly divided into three equal groups (experimental, open control, and closed or sutured control groups). After routine surgical preparations, two 3 × 1 cm pieces of lumbosacral skin were excised on both sides in each animal. An incision was made over the fascia and muscle on the right side (deep wounds), and in the left side, only the skin was removed (superficial wound). Continuous direct electrical current (100 μA and 1.5 V) was applied to both wounds of the experimental group for 14 days. All rabbits were kept under observation for a period of 21 days, and their wound contraction and repair were measured daily. The rabbits then were euthanized, and biopsies were taken from the site of initial incisions. There was no significant difference in the rate of wound contraction between experimental group and open control. The yield and ultimate strength of the above mentioned specimens were lower than those of the normal skin, and the differences in biomechanical parameters between all groups were not statistically significant. There was a statistically significant decrease in the biomechanical properties of closed control lesions compared to those of the open control (p < 0.05). Hemorrhages were evident in the upper dermis just below the epidermis, and many macrophages and lymphocytes were infiltrated at the site of injury. Electron microscopic studies showed no significant difference in the collagen fibrils diameter and distribution between different groups. There was no significant difference in the percentage dry weight of the injured skin with those of the normal skin. Results suggest that continuous microamperage low-voltage electrical stimulation, as given, did not significantly improve wound healing.     

Fabrication and characterization of the porous duck’s feet collagen sponge for wound healing applications

Abstract

There are several artificial dermis commonly use to cover the wound and promote healing. The major goal of wound management is fast and scarless healing. However, there is no ideal skin substitute, that is effective to accelerate wound healing without scar formation. Artificial dermis substitute also has some drawbacks, such as high cost, insufficient available period and donor pathogen infection. To overcome these problems, we developed duck’s feet collagen (DFC) sponge as artificial dermal substitutes for the treatment of full-thickness skin defects. We measured these DFC sponge’s comparative characteristics and performances with an artificial dermis Colladerm by carried out SEM-EDX analyze, water-binding abilities and porosity test. Biocompatibility test was also performed using CCK-8 cytotoxicity assay. We also evaluated its wound healing effects for a full-thickness skin wound and compared with Colladerm in a rat model. Histological studies were carried via hematoxylin and eosin and Masson’s Trichrome staining. Although the wound healing effect of the DFC sponge was almost similar to that of Colladerm, the DFC sponge did not induce scar formation and wound contracture like Colladerm. We suggest that DFC sponge can be used as an ideal dermal substitute to the treatment of full-thickness skin wound.     

Evaluation of a Wound Dressing Composed of Hyaluronic Acid and Collagen Sponge Containing Epidermal Growth Factor in Diabetic Mice

Abstract

This study investigated the effect of a wound dressing composed of hyaluronic acid (HA) and collagen (Col) sponge containing epidermal growth factor (EGF) on wound healing in diabetic mice. High-molecular-weight (HMW) HA aqueous solution, hydrolyzed low-molecular-weight (LMW) HA aqueous solution and heat-denatured Col aqueous solution were mixed, followed by freeze-drying to obtain a spongy sheet. Cross-linkage between Col molecules was induced by UV irradiation to the spongy sheet (Type-I wound dressing). In a similar manner, a spongy sheet containing EGF (Type-II wound dressing) was prepared by freeze-drying the mixed solution of HMW-HA, LMW-HA and Col containing EGF. The efficacy of these products was evaluated in type-II diabetic BKS.Cg-+Lepr^db/+Lepr^db (db/db) mice. Wound dressings were applied to a full-thickness, dorsal skin defect measuring 1.5 cm × 2.0 cm, showing adipose tissue. In the control group, a commercially available artificial dermis composed of collagen spongy sheet (TERUDERMIS^®) was used. A commercially available polyurethane film dressing (Bioclusive^®) was applied over each wound dressing. After 1 week of application, wound conditions were evaluated based on their gross and histological appearances. Type-I and -II wound dressings promoted a decrease in wound size associated with angiogenesis and granulation tissue formation, compared with the artificial dermis. In particular, Type-II wound dressings promoted sufficient re-epithelialization. These findings indicate that the combination of HA, Col and EGF promotes wound healing by stimulating cell activity including cell migration and proliferation on the adipose tissue in a diabetic wound. Type-I and -II wound dressings would be useful to prepare a well-vascularized wound bed acceptable for split-thickness auto-skin grafting.     

Changes in cells' secretory organelles and extracellular matrix during endochondral ossification in the mandibular condyle of the growing rat

The mandibular condyle from 20-day-old rats was examined in the electron microscope with particular attention to intracellular secretory granules and extracellular matrix. Moreover, type II collagen was localized by an immunoperoxidase method. The condyle has been divided into five layers: (1) the most superficial, articular layer, (2) polymorphic cell layer, (3) flattened cell layer, (4) upper hypertrophic, and (5) lower hypertrophic cell layers. In the articular layer, the cells seldom divide, but in the polymorphic layer and upper part of the flattened cell layer, mitosis gives rise to new cells. In these layers, cells produce two types of secretory granules, usually in distinct stacks of the Golgi apparatus: type a, cylindrical granules, in which 300-nm-long threads are packed in bundles which appear “lucent” after formaldehyde fixation; and type b, spherical granules loaded with short, dotted filaments. The matrix is composed of thick banded “lucent” fibrils in a loose feltwork of short, dotted filaments. The cells arising from mitosis undergo endochondral differentiation, which begins in the lower part of the flattened cell layer and is completed in the upper hypertrophic cell layer; it is followed by gradual cell degeneration in the lower hypertrophic cell layer. The cells produce two main types of secretory granules: type b as above; and type c, ovoid granules containing 300-nm-long threads associated with short, dotted filaments. A possibly different secretory granule, type d, dense and cigar-shaped, is also produced. The matrix is composed of thin banded fibrils in a dense feltwork. In the matrix of the superficial layers, the “lucency” of the fibrils indicated that they were composed of collagen I, whereas the “lucency” of the cylindrical secretory granules suggested that they transported collagen I precursors to the matrix. Moreover, the use of ruthenium red indicated that the feltwork was composed of proteoglycan; the dotted filaments packed in spherical granules were similar to, and presumably the source of, the matrix feltwork. The superficial layers did not contain collagen II and were collectively referred to as perichondrium. In the deep layers, the ovoid secretory granules displayed collagen II antigenicity and were likely to transport precursors of this collagen to the matrix, where it appeared in the thin banded fibrils. That these granules also carried proteoglycan to the matrix was suggested by their content of short dotted filaments. Thus the deep layers contained collagen II and proteoglycan as in cartilage; they were collectively referred to as the hyaline cartilage region.     

Physical plasma therapy accelerates wound re-epithelialisation and enhances extracellular matrix formation in cutaneous skin grafts

Skin grafting is a surgical method of cutaneous reconstruction, which provides volumetric replacement in wounds unable to heal by primary intention. Clinically, full-thickness skin grafts (FTSGs) are placed in aesthetically sensitive and mechanically demanding areas such as the hands, face, and neck. Complete or partial graft failure is the primary complication associated with this surgical procedure. Strategies aimed at improving the rate of skin graft integration will reduce the incidence of graft failure. Cold atmospheric plasma (CAP) is an emerging technology offering innovative clinical applications. The aim of this study was to test the therapeutic potential of CAP to improve wound healing and skin graft integration into the recipient site. In vitro models that mimic wound healing were used to investigate the ability of CAP to enhance cellular migration, a key factor in cutaneous tissue repair. We demonstrated that CAP enhanced the migration of epidermal keratinocytes and dermal fibroblasts. This increased cellular migration was possibly induced by the low dose of reactive oxygen and nitrogen species produced by CAP. Using a mouse model of burn wound reconstructed with a full-thickness skin graft, we showed that wounds treated with CAP healed faster than did control wounds. Immunohistochemical wound analysis showed that CAP treatment enhanced the expression of the dermal–epidermal junction components, which are vital for successful skin graft integration. CAP treatment was characterised by increased levels of Tgfbr1 mRNA and collagen I protein in vivo, suggesting enhanced wound maturity and extracellular matrix deposition. Mechanistically, we show that CAP induced the activation of the canonical SMAD-dependent TGF- β 1 pathway in primary human dermal fibroblasts, which may explain the increased collagen I synthesis in vitro. These studies revealed that CAP improved wound repair and skin graft integration via mechanisms involving extracellular matrix formation. CAP offers a novel approach for treating cutaneous wounds and skin grafts. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The structure of the osteoderms in the gekko: Tarentola mauritanica

Histological and cytological analysis reveals that the osteoderms of Tarentola mauritanica are composed of an outer part superimposed on a basal region. The structure of both parts can be related to that of the surrounding dermis. The basal part of the osteoderms, inserted in the dense dermis, is made up of abundant closely packed collagen fibrils that orient the mineral deposit. The outer part, located in the superficial loose dermis, is crossed by few bundles of mineralized collagen fibrils arising from the basal part. These bundles connect the osteoderm to the overlying loose dermis. The outer superficial part is characterized by the presence of mineralized globules surrounding the mineralized collagen bundles. In these globules, the crystals are deposited on a microfibrillar matrix rich in acidic mucosubstances and composed of radially oriented, tangled microfilaments that lie among the collagen bundles. The two different mineralizing systems in the osteoderms of Tarentola mauritanica may reflect two different organic matrices. The mineral is deposited in a preexisting dermal tissue, as a “metaplastic ossification,” and is another expression of the potential retained by the reptilian dermis to form mineralized structures.     

A comparison of the usefulness of nuclear beta-catenin in the diagnosis of desmoid-type fibromatosis among commonly used anti-beta-catenin antibodies

Desmoid-type fibromatosis (DF) is a locally aggressive but non-metastatic (myo)fibroblastic neoplasm. A hallmark of the tumor is nuclear positivity for beta-catenin in immunohistochemistry due mostly to CTNNB1 mutations. However, a recent study has reported that even beta-catenin ‘nuclear-negative’ DFs can harbor CTNNB1 mutations and that the positive ratio of nuclear beta-catenin in DF is different among antibodies. Here, we reviewed soft tissue lesions for which the possibility of DF was considered and compared the sensitivity and specificity of nuclear beta-catenin for the diagnosis of DF among commonly used anti-beta-catenin antibodies, i.e., clone beta-catenin 1, 17C2 and 14. We analyzed 26 cases of DF, 28 cases of benign fibroblastic lesions, and 27 cases of other soft tissue tumors. The sensitivity and specificity of nuclear beta-catenin for the diagnosis of DF were different among antibodies; 54% and 98% in clone beta-catenin 1, 85% and 84% in 17C2, and 96% and 62% in 14. IHC of LEF1 showed comparable results with IHC of beta-catenin, with a sensitivity of 88% and specificity of 76%. Additionally, when beta-catenin 1 was used, DFs showed characteristic dotted cytoplasmic staining, often appearing as rings. Our results might be helpful for making a correct diagnosis of DF.     

Utilizing Human Dermal Fibroblast Heterogeneity in Autologous Dermal Fibroblast Therapy: An Overcomplicated Strategy or a Promising Approach?

Although human dermal fibroblast heterogeneity has been acknowledged for several decades and a large body of in vitro studies has been performed with zonal dermal fibroblast, current autologous dermal fibroblast therapies do not reflect human dermal fibroblast heterogeneity. To determine if the utilization of human dermal fibroblast heterogeneity in autologous dermal fibroblast therapy is more of a translational perspective that may thus be more likely to make it to the clinic, this article critically reviews the previous studies on dermal fibroblast heterogeneity performed to date. We found that in vitro studies of human dermal fibroblast heterogeneity have run nearly parallel to the in vivo study of autologous dermal fibroblast therapy. Although several human to nude mice xenotransplantation experiments have been performed in different layers of human dermal fibroblast, their clinical significance remains to be considered. We conclude that there is still a great gap between basic experiments and the clinical employment of human dermal fibroblast heterogeneity. To overcome this, it is necessary to conduct clinical trials, which might be restricted by ethical issues. Alternatively, it might be easier to conduct in vivo studies in animal models. Based on our previous study of dermal fibroblast heterogeneity in pigs, we propose the use of pigs as a good animal model for dermal fibroblast heterogeneity. Time will show whether the utilization of human dermal fibroblast heterogeneity in autologous dermal fibroblast therapy is an overcomplicated strategy or a promising approach. Anat Rec, 302:2126–2131, 2019. © 2019 American Association for Anatomy     

Relationship Between the Dose of Ascorbic Acid and its Structural Analogs and Proline Hydroxylation in Various Biological Systemst

The effect of ascorbic acid on the rate of in vitro collagen formation was examined in several guinea pig tissues having different rates of collagen metabolism and in chick embryo skin after various conditions of ascorbic acid supplementation.

It was found that in embryonic skin, carrageenan granuloma and liver the extent of C¹⁴-hydroxyproline forniation was markedly influenced by the amount of ascorbic acid present in the tissue, which, in turn, reflected the dose of ascorbic acid administered in vitro. The increase of collagen synthesis above the normal rate was directly proportional to the increase in concentration of ascorbic acid in the tissue. This was observed not only in tissues from animals dependent on an exogenous supply of ascorbic acid but also in tissues from organisms capable of ascorbic acid synthesis. However, the rate of collagen synthesis was not affected by ascorbic acid in organs which normally exhibit a low metabolic turnover of collagen (lung, muscle); in these collagen synthesis was found to be independent of the amount of available ascorbic acid. The presence of additional ascorbic acid in the incubation medium had a stirnulatory effect on collagen synthesis in chick embryo skin and carrageenan granuloma, but this immediate effect was less pronounced than that of the endogenous ascorbic acid, elevated by in viao supplementation. It is suggested that the stimulatory effect of ascorbic acid on collagen synthesis may be dependent on the availability of the precursor form of proline hydroxylase.

Comparison of the in vitro and in vivo effect of some ascorbic acid analogs upon proline hydroxylation in carrageenan granuloma from scorbutic guinea pigs indicates that although some of the derivatives were effective in vitro, their efficiency in vivo was not comparable to the effect of L-ascorbic acid.

Using the measure of radioactivity of ultrafilterable hydroxproline as an index of collagen degradation, it was found that ascorbic acid deficiency did not accelerate degradation of collagen which had been hydroxylated.     

Differential collagen-glycosaminoglycan matrix remodeling by superficial and deep dermal fibroblasts: potential therapeutic targets for hypertrophic scar

Skin substitutes are the preferred treatment option in the case of extensive skin loss following burns or other injuries. Among skin substitutes, cultured skin substitutes containing autologous fibroblasts and keratinocytes on collagen-glycosaminoglycan (C-GAG) matrix are most preferred for wound repair. A significant negative outcome of wound healing is hypertrophic scarring (HTS), a dermal fibroproliferative disorder, that leads to considerable morbidity. To examine the role of superficial and deep dermal fibroblasts in HTS, and determine if they differentially remodel C-GAG matrices, fibroblasts were isolated from superficial and deep dermis of lower abdominal tissue of abdominoplasty patients and cultured on C-GAG matrices for four weeks. Over time, deep fibroblasts contracted and stiffened the matrices significantly more and decreased their ultimate tensile strength compared to superficial fibroblasts. Differential remodeling of C-GAG matrices by fibroblasts obtained from different locations of the same organ has not been reported before. Deep fibroblasts were found to express significantly more osteopontin, angiotensin-II, peroxisome proliferator-activated receptor (PPAR)-α, and significantly less tumor necrosis factor-α, PPAR-β/δ, PPAR-γ, and the proteoglycan, fibromodulin compared to superficial fibroblasts. These molecular targets could potentially be used in therapeutic strategies for treatment of HTS.     

Skin aging in patients with acquired immunodeficiency syndrome

To evaluate the histomorphometric skin changes over aging patients with autopsied acquired immunodeficiency syndrome (AIDS). In 29 skin fragments of autopsied elderly (older than 50 years) and nonelderly patients with AIDS, epidermal thickness, the number of layers, the diameter of cells, the percentage of collagen and elastic fibers in the dermis, and the number and morphology of Langerhans cells were assessed. Statistical analysis was performed by SigmaStat 2.03 program. The thickness of the epidermis (92.55 × 158.94 μm), the number of layers (7 × 9 layers), and the diameter of the cells (13.27 × 17.6 μm) were statistically lower among the elderly. The quantity of collagen fibers (9.68 × 14.11%) and elastic fibers (11.89 × 15.31%) was also significantly lower in the elderly. There was a decrease in total (10.61 × 12.38 cel/mm²) and an increase in immature Langerhans cells (6.31 × 4.98 cel/mm²) in elderly patients with AIDS. The aging of the skin of patients with AIDS is amended in different histomorphometric aspects, the epidermis constituents suffer less pronounced changes in normal aging, and the dermis has more intense changes in elastic fibers and collagen.     

裸鼠尾的一般形态结构及淋巴管

裸鼠尾的一般形态结构和淋巴管的资料对多种裸鼠尾动物模型的建立具有重要意义。本文应用光镜、透射电镜及透明标本对10只雄性裸鼠尾进行了有关研究,获得了裸鼠尾的一般形态结构和淋巴管的有关资料。裸鼠尾的中央是一条29节尾椎组成的骨链。尾椎链的外面覆以三层结构,即皮肤、皮下组织和肌层。裸鼠尾的淋巴管网有浅、深两层。深淋巴管位于皮下组织内。浅淋巴管位于真皮内,呈网格状。浅淋巴管网的构型与皮肤纹理的构型具有相似性。     

Stereological analysis of collagen and elastic fibers in the normal human dermis: Variability with age,sex, and body region

Normal human dermis has been analyzed using sterological methods to estimate the quantitative modifications of collagen and elastic fibers in relation to age, sex, and body region. Forty-five skin biopsies from the trunk or the limbs of 26 males and 19 females of different age were fixed in glutaraldehyde and osmium tetroxide and embedded in epoxy resin. The relative volumes of collagen and elastic fibers were calculated by the point counting method on 1 μm semithin sections. Photographic sampling was performed on four consecutive dermis layers: the papillary layer and three consecutive layers of reticular dermis. The data were subjected to analysis of variance which showed that all the factors studied exert a significant influence on the relative amounts of collagen and elastic fibers. The fractional volume of collagen fibers is constant throughout all dermis layers analyzed and is always higher in females than in males, except for the second and third decades of life. Collagen fiber density increases with age in both sexes up to 30–40 years, when it starts decreasing. Both the relative volumes and the diameters of elastic fibers increase from papillary to deep reticular dermis. In reticular dermis of both sexes there is an increment of elastic fiber density in the first decade of life, followed by a drop particularly marked in males. After 20 years, the relative volume of elastic fibers displays a decreasing trend in females, whereas it increases in males, attaining the highest values beyond the 40s. © 1994 Wiley-Liss, Inc.     

正常皮肤中单核巨噬细胞和树枝状细胞的分布规律

目的：观察正常真皮内的单核-巨噬细胞和树枝状细胞的分布、排列规律，探讨单核-巨噬细胞在皮肤免疫防御中的作用。方法：正常皮肤8例，取面部、躯干、四肢近端、四肢远端、手掌和足跖6个部位皮肤，进行纵行与水平切片。CD1a和CD68单克隆抗体染色，观察朗格汉斯细胞（LC）和单核．巨噬细胞的分布形态和密度。结果：真皮浅层CD68阳性细胞呈网状分布，其密度为361-562个／mm^2。真皮内血管周围及附属器周围亦见CD68阳性细胞。真皮深层CD68阳性细胞多为树枝状，散在分布于粗大的胶原纤维之间。不同解剖部位真皮浅层CD68阳性细胞密度分别为：四肢远端562个／mm^2，腹部517个／mm^2，面部509个／mm^2，手掌507个／mm^2，四肢近端472个／mm^2，足跖361个／mm^2。真皮浅层CD68阳性细胞在手掌和足跖部位高于相应部位的表皮内CD1a和CD68细胞。结论：在真皮浅层形成数层单核．巨噬细胞网，此网在接近真表皮交界处较致密。真皮内单核-巨噬细胞的这种分布形式说明这些细胞在真皮内有明确的方向性，其防御的方向是穿透表皮进入真皮的入侵物。     

Quantifying light scattering with single-mode fiber -optic confocal microscopy

Background  

Confocal microscopy has become an important option for examining tissues in vivo as a diagnostic tool and a quality control tool for tissue-engineered constructs. Collagen is one of the primary determinants of biomechanical stability. Since collagen is also the primary scattering element in skin and other soft tissues, we hypothesized that laser-optical imaging methods, particularly confocal scattered-light scanning, would allow us to quantify scattering intensity and determine collagen content in biological layers.     

Multiphoton microscopy study of the morphological and quantity changes of collagen and elastic fiber components in keloid disease

Multiphoton microscopy was used to study the extracellular matrix of keloid at the molecular level without tissue fixation and staining. Direct imaging of collagen and elastin was achieved by second harmonic generation and two-photon excited fluorescence, respectively. The morphology and quantity of collagen and elastin in keloid were characterized and quantitatively analyzed in comparison to normal skin. The study demonstrated that in keloid, collagen content increased in both the upper dermis and the deep dermis, while elastin mostly showed up in the deep dermis and its quantity is higher compared to normal skin. This suggests the possibility that abnormal fibroblasts synthesized an excessive amount of collagen and elastin at the beginning of keloid formation, corresponding to the observed deep dermis, while after a certain time point, the abnormal fibroblast produced mostly collagen, corresponding to the observed upper dermis. The morphology of collagen and elastin in keloid was disrupted and presented different variations. In the deep dermis, elastic fibers showed node structure, while collagen showed obviously regular gaps between adjacent bundles. In the upper dermis, collagen bundles aligned in a preferred direction, while elastin showed as sparse irregular granules. This new molecular information provided fresh insight about the development process of keloid.     

A bilayer scaffold prepared from collagen and carboxymethyl cellulose for skin tissue engineering applications

Abstract

Treatment of chronic skin wound such as diabetic ulcers, burns, pressure wounds are challenging problems in the medical area. The aim of this study was to design a bilayer skin equivalent mimicking the natural one to be used as a tissue engineered skin graft for use in the treatments of problematic wounds, and also as a model to be used in research related to skin, such as determination of the efficacy of transdermal bioactive agents on skin cells and treatment of acute skin damages that require immediate response. In this study, the top two layers of the skin were mimicked by producing a multilayer construct combining two different porous polymeric scaffolds: as the dermis layer a sodium carboxymethyl cellulose (NaCMC) hydrogel on which fibroblasts were added, and as the epidermis layer collagen (Coll) or chondroitin sulfate-incorporated collagen (CollCS) on which keratinocytes were added. The bilayer construct was designed to allow cross-talk between the two cell populations in the subsequent layers and achieves paracrine signalling. It had interconnected porosity, high water content, appropriate stability and elastic moduli. Expression of vascular endothelial growth factor (VEGF), basic-fibroblast growth factor (bFGF) and Interleukin 8 (IL-8), and the production of collagen I, collagen III, laminin and transglutaminase supported the attachment and proliferation of cells on both layers of the construct. Attachment and proliferation of fibroblasts on NaCMC were lower compared to performance of keratinocyte on collagen where keratinocytes created a dense and a stratified layer similar to epidermis. The resulting constructs succesfully mimicked in vitro the natural skin tissue. They are promising as grafts for use in the treatment of deep wounds and also as models for the study of the efficacy of bioactive agents on the skin.