Native Human Skin Research Articles

Screening for a new Streptomyces strain capable of efficient keratin degradation

Keratinous wastes could be degraded by some microorganisms in nature. Native human foot skin (NHFS) was used as sole nitrogen source to screen microorganisms with keratin-degrading capability. From approximately 200 strains, a strain of Streptomyces sp. strain No.16 was found to possess the strongest keratinolytic activity, and the total activity in the culture was 110 KU/ml with specific activity of 2870 KU/mg protein (KU: keratinase unit). Substrate specificity test indicated that the crude keratinase could degrade keratin azure, human hair, cock feathers and collagen. The optimal pH of the crude keratinase ranged from 7.5 to 10 and the temperature ranged from 40°C to 55°C. Metal chelating agent ethylenediamine tetraacetic acid obviously stimulated the keratinolytic activity but suppressed the proteolytic activity. To our knowledge, this is the first report on specific induction of keratinases by NHFS from an actinomycete. Moreover, excellent characteristics of its crude keratinase may lead to the potential application in waste treatment and recovery, poultry and leather industry, medicine, and cosmetic development.

Improvement of Epidermal Barrier Properties in Cultured Skin Substitutes after Grafting onto Athymic Mice

Barrier function in cultured skin substitutes (CSS) prepared from human cell sources was measured by noninvasive (surface hydration, transepidermal water loss) and invasive methods (water permeation, niacinamide flux) before and after grafting onto athymic mice. In vitro measurements were made on days 7 and 14. Although three of the four measures of barrier function improved markedly from day 7 to 14, the values obtained were still far from those obtained with native human skin controls. Additional CSS were grafted onto athymic mice on day 14, and skin was harvested 2 and 6 weeks after grafting. Grafting brought about a substantial decrease in all measurements by 2 weeks and almost complete normalization of barrier function after 6 weeks. The most sensitive measure of this recovery was niacinamide permeability, which decreased from (280 ± 40) × 10^–4 cm/h in vitro to (17 ± 30) × 10^–4 cm/h 2 weeks after grafting and (5 ± 2) × 10^–4 cm/h 6 weeks after grafting, versus control values of (2 ± 2) × 10^–4 cm/h in human cadaver skin and (0.6 ± 0.4) × 10^–4 cm/h in human epidermal membrane prepared from freshly excised breast skin. These results demonstrate the reformation of epidermal barrier function after transplantation and provide insights for the development of a functional epidermal barrier in CSS in vitro.

Comparative study between reconstructed and native human epidermis using nuclear microscopy

The physiological status of native skin is suffering from large inter-individual variations, especially in terms of inorganic ions content. For this reason, together with the advent of ethic laws on animal experimentation, reconstructed skin or epidermis models are extensively employed nowadays in penetration studies for cosmetic or pharmacological applications.It has been already verified that reconstructed human epidermis (RHE) has similar physiological mechanisms to native human skin, but until now, there are few studies where the elemental concentrations of both skins, reconstructed and native, are compared. In this work, freeze-dried thin sections of human native skin obtained from surgery have been characterized using PIXE, RBS and STIM at the CENBG nuclear microprobe. RHE samples were treated and analyzed in the same conditions for comparison. The combination of the different imaging and analysis techniques made possible a clear delimitation and identification of skin ultrastructure. The elemental concentrations of P, S, Cl, K and Ca were measured in the different strata. For both skins, concentrations have been compared and significant differences in terms of elemental concentrations have been determined using statistical approaches. Similar physiological characteristics were pointed out in both skin models, in particular the Ca gradient presumably involved in the regulation of the barrier effect.

Keratin expression in cultured skin substitutes suggests that the hyperproliferative phenotype observed in vitro is normalized after grafting

Cultured skin substitutes, consisting of fibroblasts and keratinocytes in a biopolymer matrix, are an adjunctive treatment for full thickness burn wounds. Previous studies revealed that cultured skin substitutes in vitro exhibit a gene expression profile similar to hyperproliferative skin or wounded normal skin. In the present study, we sought to determine whether this hyperproliferative phenotype is maintained after healing of grafted cultured skin in vivo. Immunohistochemistry was used to localize multiple keratin proteins in native human skin, and in cultured skin substitutes in vitro and after grafting to athymic mice. Keratin 6, keratin 16, and keratin 17, which are known to be upregulated during keratinocyte activation and in hyperproliferative epidermis, were highly expressed in cultured skin substitutes in vitro. These proteins were low or undetectable in native human skin, and were reduced in cultured skin after grafting. Conversely, keratin 15, which is downregulated in activated keratinocytes, was not detected in cultured skin substitutes in vitro but was upregulated after grafting to mice. The results confirm previous observations suggesting a hyperproliferative or activated phenotype in cultured skin substitutes in vitro, similar to wounded native skin. After grafting to athymic mice, the expression patterns suggest a normalization of cultured skin substitutes to a phenotype more closely resembling uninjured human skin.

210. Permanent Correction of Leptin Deficiency through Cutaneous Gene Therapy. Lentivirus but Not Retrovirus-Transduced Human Keratinocyte Orthotopic Grafts Achieve Therapeutic Systemic Leptin Levels

Top of pageAbstract Leptin deficiencies are involved in rare cases of inherited human obesity and in various forms of lipodystrophy. Previous results from our laboratory showed the feasibility of leptin deficiency correction in the ob/ob mice model using skin grafts from transgenic mice expressing leptin from the epidermis. In spite of epidermal stem cell targeting, several attempts to achieve permanent correction of leptin-deficient ob/ob mice through oncortetrovirus-transduced human keratinocyte (HK) grafts resulted unsuccessful. When human keratinocytes were transduced with a leptin-ires GFP-encoding HIV- derived lentivirus, leptin levels in culture were 3-fold higher that those achieved with oncoretrovirus-transduced HK. Consistently, immunodeficient ob/ob mice orthotopically grafted with lentivirus- transduced HK as part of bioengineered skins, showed therapeutic serum leptin levels. Phenotypic correction of engrafted ob/ob (immunodeficient) mice included weight loss, decreased food intake and normalization of blood glucose. Permanent correction (longer than 22 weeks post-grafting) was achieved in two animals. Skin regenerated form leptin lentivirus-transduced HK displayed histological features similar to those of native human skin. This is the first report of the long-sought sustained correction of a systemic protein deficiency using orthotopic grafting of ex vivo genetically modified human keratinocytes acting as a bioreactor.

Microarray Analysis of Gene Expression in Cultured Skin Substitutes Compared with Native Human Skin

Cultured skin substitutes (CSS), prepared using keratinocytes, fibroblasts, and biopolymers, can facilitate closure of massive burn wounds by increasing the availability of autologous tissue for grafting. But because they contain only two cell types, skin substitutes cannot replace all of the functions of native human skin. To better understand the physiological and molecular differences between CSS and native skin, we undertook a comprehensive analysis of gene expression in native skin, cultured keratinocytes, cultured fibroblasts, and skin substitutes using Affymetrix gene chip microarrays. Hierarchical tree clustering identified six major clusters of coordinately regulated genes, using a list of 1030 genes that were the most differentially expressed between groups. These clusters correspond to biomarker pools representing expression signatures for native skin, fibroblasts, keratinocytes, and cultured skin. The expression analysis revealed that entire clusters of genes were either up- or downregulated upon combination of fibroblasts and keratinocytes in cultured skin grafts. Further, several categories of genes were overexpressed in CSS compared with native skin, including genes associated with hyperproliferative skin or activated keratinocytes. The observed pattern of expression indicates that CSS in vitro, which display a well-differentiated epidermal layer, exhibit a hyperproliferative phenotype similar to wounded native skin.

Skinomics

Skinomics

Reconstructed human epidermis: A model to study the barrier function

The use of in vitro reconstructed human epidermis (RHE) by the cosmetic and pharmaceutical industries is increasing because of its similar physiological mechanisms to native human skin. With the advent of ethic laws on animal experimentation, RHE provides an helpful alternative for the test of formulations. The aim of this study is to check that the RHE mineral status is comparable to that of human native skin by investigating the elemental distributions in the epidermis strata. In addition, possible deleterious effects of the transport on the epidermis ionic content were studied by nuclear microscopy.

MORE EFFORTS FOR TISSUE-ENGINEERING SKIN

A251 Aims: Submit a review of Tissue-engineering skin, and provide new sight for future efforts. Reviews: Requirements for functions of tissue-engieering skin are resemblance to native human skin including three aspects: protection, perception and regulation. Protective function consists of barrier, UV light absorption, immune surveillance and mechanical stability. There are some permeability differences between artificial skin and native skin which may be ascribed to the different composition and ultrastructure of stratum corneum(SC) lipids, and absence of hair. The UV light absorption ability has a direct ratio with the pigmentation of healed cultured skin substitutes, which is frequently irregular and unpredictable. Paracrine factors from skin cells and the extracellular matrix act as local environmental signals for the the pigmentation which is proportional to growth density of melanocytes. Immune surveillance requires the complete engraftment of native skin cells and a functional vascular plexus which the available artificial skin lacks. Many factors contribute to inevitable contraction of cultured skin substitutes, such as the effect of keratinocytes and fibroblasts, thickness of dermis, immaturity of the dermal-epidermal junction and materials. The perception and regulation functions have almost been studied only in experiments because of difficult transplantation of necessary elements like touch domes and skin appendages. Immune rejection is the main reason for limitting the popularity of allogeinic grafts. In the process immune cells, cytokines and blood supply work. CD80 on keratinocytes might bring the ultimate rejection of allogeneic keratinocytes. The host inflammatory response to some extents acts as a more significant role than immune reaction. Prospects: Tissue-engineering skin needs further developments. This may be achieved through improvement of culture conditions. crosslink agents, certain cytokines, phorbol ester and vitamine C can be added to culture medium to enhance mechanical stability of artificial skin and accelerate its formation. Perfection of cultured skin substitutes requires complete engraftment of the celluar part and a functional vascular plexus which may conflict with requirement of diminishing antigenicity. Here improvement of culture condition appears to be significant through which cells can move from the peripheral healthy region without transplantion. Stem cell is another attractive topic. Due to the difficult acquirement and culture, a stem cell bank can be built. Skin is perhaps the most accessible of all somatic tissues for therapeutic gene transfer. Gene therapy for cultured skin grafts develops quickly. The goal includes accelerating wound healing and curing diseases and the key is how to prolong expression of the transfected gene. As artificial skin makes a transition from research to clinical practice, the uniform criterions for qualitative and quantitative analysis of cultured skin substitutes must be established for evaluation of materials, composition, performance and safety.Figure

Induction of HSP27 nuclear immunoreactivity during stress is modulated by vitamin C.

For the investigation of the skin irritancy potential of chemicals in an in vitro model it is necessary to have sensitive endpoints that predict the effects of those compounds on native human skin. Recently, we have identified that 27-kDa heat shock protein (HSP27) can serve as a sensitive marker of skin irritation, as exposure of human skin to sodium lauryl sulfate (SLS) both in vitro and in vivo induced relocalization of HSP27 from the cytoplasm to the cell nucleus. The aim of the present study was to determine whether nuclear localization of HSP27 could be used as a parameter for evaluation of potential skin irritants in screening assays in vitro. For this purpose, human skin equivalent consisting of epidermis reconstructed on de-epidermized dermis was exposed to SLS or UV light. Stress-induced nuclear relocalization of HSP27 was observed in excised skin exposed to SLS or UV light and in reconstructed epidermis only when the latter was generated in the absence of vitamin C. The omission of vitamin C results in an impaired barrier function. In the presence of vitamin C, however, the barrier function was comparable with excised skin, suggesting that vitamin C may control the response to stress in the reconstructed epidermis. Besides the presence of vitamin C, the response of skin equivalents may strongly depend on other conditions under which they are generated, because the stress-induced HSP27 relocalization was not detected in the commercially available epidermal kit EpiDerm. The results of the present study show that HSP27 nuclear staining can serve as a sensitive marker for skin irritation or cellular stress in excised skin as well as in certain well-characterized human skin equivalents in vitro.

Barrier Function in Reconstructed Epidermis and Its Resemblance to Native Human Skin

One of the prerequisites for the use of human skin equivalents for scientific and screening purposes is that their barrier function is similar to that of native skin. Using human epidermis reconstructed on de-epidermized dermis we demonstrated that the formation of the stratum corneum (SC) barrier in vitro proceeds similarly as in vivo as judged from the extensive production of lamellar bodies, their complete extrusion at the stratum granulosum/SC interface, and the formation of multiple broad lamellar structures in the intercorneocyte space. The presence of well-ordered lipid lamellar phases was confirmed by small-angle X-ray diffraction. Although the long periodicity lamellar phase was present in both the native and the reconstructed epidermis, the short periodicity lamellar phase was present only in native tissue. In addition, the SC lipids predominantly formed the hexagonal sublattice. Analysis of lipid composition revealed that all SC lipids are synthesized in vitro. Differences in SC lipid organization in reconstructed epidermis may be ascribed to the differences in fatty acid content and profile indicating that further improvement in culture conditions is required for generation of in vitro reconstructed epidermis with stratum barrier properties of the native tissue.

Assessment with the dermal torque meter of skin pliability after treatment of burns with cultured skin substitutes.

The assessment of visco-elastic (V-E) properties in cutaneous scars is critical to reduction of impairment and restoration of function after grafting of excised burns. Cultured skin substitutes (CSS) that consist of autologous keratinocytes and fibroblasts attached to biopolymer substrates are alternatives for permanent closure of excised, full-thickness burns, but assessment of scarring has been subjective. V-E properties of CSS were measured with a Dia-Stron Dermal Torque Meter (DTM 310, Dia-Stron, Ltd, Broomall, Pa), which applies a constant torque (10 mNm) for a fixed interval (10 seconds) and measures rotational deformation and recovery. Parameters of skin deformation were measured in patients (n = 10) after grafting of CSS or meshed skin autograft. Native human skin (NHS) of healthy volunteers (n = 13) served as the control. Skin healed after treatment with CSS or autograft was evaluated for 1 year or longer after grafting. Elastic stretch (Ue), viscous stretch (Uv), total extensibility (Uf), elastic recovery (Ur), total recovery (Ua), and residual plasticity (R) were measured as degrees of rotation, were tested for significance (P < .05) by Student t test comparisons between treatment groups and controls, and were subjected to regression analysis. Assessment of burn scar with the Dermal Torque Meter detected time-dependent increases of all individual parameters of V-E properties for both CSS and autograft during the first year after grafting. At 1 year or later, no statistical differences were found between CSS and autograft for individual parameters, but Ue and Ur for autograft were significantly lower than for NHS. At 1 year or longer, autograft was significantly different from CSS or NHS, with a greater ratio of Uv to Ue, and both graft types had a lower ratio of Ur to Uf than NHS had. These results suggest that instrumental measurement of scar pliability may increase objectivity in assessment of patient recovery and establish an absolute scale for quantitative analysis of V-E properties in skin after grafting of conventional or alternative skin substitutes.

Tissue-engineered human living skin substitutes: development and clinical application.

The skin acts as a barrier to exogenous substances, pathogens, and trauma. Skin defects caused by burns, venous ulcer, diabetic ulcer, or acute injury occasionally induce life-threatening situations. Tissue engineering provides an alternative for autologous or allogeneic tissue transplantation, which is required because of donor site limitations and the risks of transmitting infection. Currently, skin substitutes are made of only extracellular matrix, mainly cells, or combination of cells and matrices. New biotechnological approaches have led to the development of the skin equivalent, the closest match yet to native human skin in terms of histological and functional properties. This review article focuses upon the development of the in vitro and in vivo epidermis and dermis and their clinical applications.

Incubation of cultured skin substitutes in reduced humidity promotes cornification in vitro and stable engraftment in athymic mice

Cultured skin substitutes have been used successfully for adjunctive treatment of excised burns and chronic skin wounds. However, limitations inherent to all models of cultured skin include deficient barrier function in vitro, and delayed keratinization after grafting in comparison to native skin autografts. Experimental conditions for incubation of skin substitutes were tested to stimulate barrier development before grafting, and measure responses in function and stability after grafting. Cultured skin substitutes consisted of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan biopolymer substrates. Parallel cultured skin substitutes were incubated at the air-liquid interface in ambient (48-61%) or saturated (79-91%) relative humidity, and grafted to athymic mice on culture day 14. Additional cultured skin substitutes were incubated in the experimental conditions for a total of 28 days. Cadaveric human skin and acellular biopolymer substrates served as controls. Epidermal barrier was evaluated as the change in surface hydration by surface electrical capacitance with the NOVA Dermal Phase Meter. Cultured skin substitutes and cadaveric skin incubated in ambient humidity had lower baseline surface electrical capacitance and less change in surface electrical capacitance than parallel samples incubated in saturated humidity at all time points in vitro. Data from healing cultured skin substitutes at 2, 4, 8 and 12 weeks after grafting showed an earlier return to hydration levels comparable to native human skin, and more stable engraftment for skin substitutes from ambient humidity. The data indicate that cultured skin substitutes in ambient humidity have lower surface electrical capacitance and greater stability in vitro, and that they reform epidermal barrier more rapidly after grafting than cultured skin substitutes in saturated humidity. These results suggest that restoration of functional epidermis by cultured skin substitutes is stimulated by incubation in reduced humidity in vitro.

Skin substitutes from cultured cells and collagen-GAG polymers.

Engineering skin substitutes provides a potential source of advanced therapies for the treatment of acute and chronic wounds. Cultured skin substitutes (CSS) consisting of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates have been designed and tested in preclinical and clinical studies. Cell culture techniques follow general principles of primary culture and cryopreservation in liquid nitrogen for long-term storage. Biopolymer substrates are fabricated from xenogeneic (bovine) collagen and glycosaminoglycan that are lyophilised for storage until use. At maturity in air-exposed culture, CSS develop an epidermal barrier that is not statistically different from native human skin, as measured by surface electrical capacitance. Preclinical studies in athymic mice show rapid healing, expression of cytokines and regulation of pigmentation. Clinical studies in burn patients demonstrate a qualitative outcome with autologous skin that is not different from 1:4 meshed, split-thickness autograft skin, and with a quantitative advantage over autograft skin in the ratio of healed skin to biopsy areas. Chronic wounds resulting from diabetes or venous stasis have been closed successfully with allogeneic CSS prepared from cryopreserved skin cells. These results define the therapeutic benefits of cultured skin substitutes prepared with skin cells from the patient or from cadaver donors. Future directions include genetic modification of transplanted cells to improve wound healing transiently or to deliver gene products systemically.

Expression of skin-derived antileukoproteinase (SKALP) in reconstructed human epidermis and its value as a marker for skin irritation.

For the investigation of the skin irritancy potential of chemicals in an in vitro model, it is necessary to have sensitive end-points that predict the effects on native human skin. Our aim was to investigate whether the induction of the proteinase inhibitor SKALP in reconstructed epidermis can be used as a marker. The influence of culture conditions and the effect of topical application of sodium lauryl sulfate and oleic acid on SKALP expression were evaluated using immunohistochemistry and Northern blotting. SKALP expression was induced by serum, epidermal growth factor and fibroblasts. In the presence of retinoic acid and 1,25-dihydroxyvitamin D3 SKALP expression was inhibited, whereas supplementation with ascorbic acid and a-tocopherol had no effect. Tape-stripping of excised skin and topical treatment with sodium lauryl sulfate induced SKALP protein expression. Application of sodium lauryl sulfate and oleic acid on reconstructed epidermis also induced SKALP at the protein level but no significant effects could be demonstrated at mRNA levels. In conclusion, SKALP expression, which was increased upon application of sodium lauryl sulfate and oleic acid, can be used as an in vitro end-point for skin irritancy, irrespective of the modifying effects of culture conditions.

A reliable and cost-effective in vitro assay of skin viability for skin banks and burn centers.

The tetrazolium salt, MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), was used to evaluate the viability of fresh native human skin and cryopreserved human skin under a wide variety of conditions. Viability of fresh and cryopreserved skin was determined with our modification of the MTT assay, and compared with the well-described tetrazolium reductase assay. The MTT assay provides a precise and reproducible index of viability for both fresh and cryopreserved skin. In comparison, the tetrazolium reductase assay (1) is subject to higher levels of variability and, (2) underestimates the viability of fresh native skin. The precision, simplicity, and economy of the MTT assay support its utility in the routine assessment of skin viability in skin banks, burn centers, and skin biology research units.

Surface Electrical Capacitance as a Noninvasive Index of Epidermal Barrier in Cultured Skin Substitutes in Athymic Mice

Restoration of an epidermal barrier is a definitive requirement for wound closure. To determine formation of an epidermal barrier as a function of hydration of the stratum corneum, we measured surface electrical capacitance (SEC) of the epidermis in cultured skin substitutes (CSS) in vitro and after grafting to athymic mice. CSS were prepared from human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan substrates. On culture days 3, 7, 14, 17, and 21, SEC was measured in situ. CSS (n = 18; mean +/- SEM) showed a time-dependent decrease of SEC (picoFarads, "pF") from 4721 +/- 28 pF on day 3 to 394 +/- 117 pF on day 14, and subsequent increase to 1677 +/- 325 pF on day 21. After 14-d incubation, parallel CSS samples (n = 5) or murine autografts (n = 5) were grafted orthotopically to athymic mice. After grafting, CSS showed decreases in SEC from 910 +/- 315 pF at 2 wk to 40 +/- 10 pF at 4 wk with no significant decreases thereafter. Control values for murine autograft were 870 +/- 245 pF at 2 wk, and 87 +/- 30 pF at 4 wk. SEC values for native murine skin (n = 10) were 91 +/- 18 pF, and for native human skin (n = 10) were 32 +/- 5 pF. The data demonstrate that SEC decreases with time in culture and that healed or intact skin has approximately 10- to 100-fold lower SEC than CSS in vitro. This noninvasive technique provides a quantitative index of epidermal barrier in CSS in vitro and demonstrates the development of functional epidermal barrier during healing of wounds treated with cultured skin substitutes.

Influence of intercellular agents on proliferation and gene activity of cultured human skin epithelium cells (NCTC 2544)

"Human skin epithelium cells" (NCTC strain 2544; HSEpicell) are established cells and grow to a monolayer the same way as epithelial cells. Addition of proliferating or antiproliferating substances results in a typical dose-dependent influence on the cell growth: steroids inhibit mitosis, polyamines stimulate proliferation, while prostaglandin E2, theophylline and papaverine reduce cell growth. Since the pattern of chromosomal nonhistone proteins indicates alterations of gene activity, DNA-binding proteins of HSE picells are analysed. Compared to native human skin fibroblasts (La Col 1115) there are only slight differences, in contrast to cancer cells. Therefore HSE picells may represent undifferentiated non-cancer cells. Hydrocortisone and theophylline inhibit cell proliferation by different mechanisms. As indicated by the pattern of DNA-binding proteins, both substances also act on HSEpicells in two different ways. As HSEpicells can be used for studying cell regulation, water-soluble extract and DNA-binding proteins of psoriatic scales as well as sera of psoriasis patients are tested in respect to any proliferating component. However, no influence on cell proliferation could be found.

Collagen-and collagen peptide-induced chemotaxis of human blood monocytes.

The ability of collagen and collagen-derived peptides to act as chemotactic stimuli was investigated by in vitro chemotaxis assays. Native human and chick skin collagen (type I) and alpha-chains obtained from purified chick skin collagen were each chemotactic for human peripheral blood monocytes. In addition, smaller peptides obtained either by digesting native collagen with bacterial collagenase or by degrading purified alpha-chains with cyanogen bromide or pepsin were also chemotactic for monocytes. In contrast, native collagen, alpha-chains, and smaller collagen-derived peptides were not chemotactic for human neutrophils. Since collagen is degraded at sites of tissue damage and inflammation, our findings suggest the possibility that such collagen-derived degradation products might directly serve as chemotactic stimuli for human peripheral blood monocytes in vivo.