Oral mucosa: an alternative epidermic cell source to develop autologous dermal-epidermal substitutes from diabetic subjects

Abstract Oral mucosa has been highlighted as a suitable source of epidermal cells due to its intrinsic characteristics such as its higher proliferation rate and its obtainability. Diabetic ulcers have a worldwide prevalence that is variable (1%-11%), meanwhile treatment of this has been proven ineffective. Tissue-engineered skin plays an important role in wound care focusing on strategies such autologous dermal-epidermal substitutes. Objective The aim of this study was to obtain autologous dermal-epidermal skin substitutes from oral mucosa from diabetic subjects as a first step towards a possible clinical application for cases of diabetic foot. Material and Methods Oral mucosa was obtained from diabetic and healthy subjects (n=20 per group). Epidermal cells were isolated and cultured using autologous fibrin to develop dermal-epidermal in vitro substitutes by the air-liquid technique with autologous human serum as a supplement media. Substitutes were immunocharacterized with collagen IV and cytokeratin 5-14 as specific markers. A Student´s t- test was performed to assess the differences between both groups. Results It was possible to isolate epidermal cells from the oral mucosa of diabetic and healthy subjects and develop autologous dermal-epidermal skin substitutes using autologous serum as a supplement. Differences in the expression of specific markers were observed and the cytokeratin 5-14 expression was lower in the diabetic substitutes, and the collagen IV expression was higher in the diabetic substitutes when compared with the healthy group, showing a significant difference. Conclusion Cells from oral mucosa could be an alternative and less invasive source for skin substitutes and wound healing. A difference in collagen production of diabetic cells suggests diabetic substitutes could improve diabetic wound healing. More research is needed to determine the crosstalk between components of these skin substitutes and damaged tissues.

Effect of autologous platelet-rich plasma application on cutaneous wound healing in dogs

This study was conducted to identify the effectiveness of platelet-rich plasma (PRP) and efficacy of intralesional injection as a method of application to acute cutaneous wounds in dogs. Healthy adult beagles (n = 3) were used in this study. Autologous PRP was separated from anticoagulant treated whole blood in three dogs. Cutaneous wounds were created and then treated by intralesional injection of PRP in the experimental group, while they were treated with saline in the control group on days 0, 2 and 4. The healing process was evaluated by gross examination throughout the experimental period and histologic examination on day 7, 14 and 21. In PRP treated wounds, the mean diameter was smaller and the wound closure rate was higher than in the control. Histological study revealed that PRP treated wounds showed more granulation formation and angiogenesis on day 7, and faster epithelialization, more granulation formation and collagen deposition were observed on day 14 than in control wounds. On day 21, collagen deposition and epithelialization were enhanced in PRP treated groups. Overall, PRP application showed beneficial effects in wound healing, and intralesional injection was useful for application of PRP and could be a good therapeutic option for wound management in dogs.

Skin Tumours – Histopathological Review of 125 Cases.

The skin is the largest organ in the body. A wide variety of hyperplastic growths and tumours, both benign and malignant are encountered in the clinical practice. Any lesion, for which the diagnosis is uncertain, based on the history and clinical examination should be biopsied for histopathological examination to rule out malignancy. Objective: To analyze retrospectively tumours of skin with respect to age, sex, clinical features and histopathological features in a tertiary referral centre in Maharashtra, India. Material & Methods: The present study consisted of analysis of tumours of skin received in the histopathology section of department of pathology over a period of 5 years that is from August 2005 to July 2010. The material comprised of biopsies and excision specimens. The clinical and histopathological details were noted. The findings were compared with those reported by other authors. Results: One twenty five (125) tumours of skin were observed. The benign tumours were slightly more common (51.2%) than malignant tumours (48.8%). The maximum number of tumours was found in 7th decade (25.6%). Maximum number of tumours were found in third decade in benign tumours (20.3%) and seventh decade in malignant tumours (37.7%). Both benign and malignant tumours of skin were common in males than females. The equal numbers of skin tumours were seen in both the head and neck region (44.8%) and the extremities (44.8%). Face was the commonest site for skin tumours (35.2%). The keratinocytic tumours, both benign and malignant were common tumours of skin (62.4%) while neural tumours were rarely observed (1.6%). The Squamous Cell Carcinoma (SCC) was the commonest malignant tumour (45.9%) followed by Basal Cell Carcinoma (BCC) (34.4%). Verrucas (32.8%) were the commonest benign tumours followed by pyogenic granuloma (21.9%). Conclusion: SCC is the most common malignant skin tumour in India, unlike the Western countries. Histopathological study is a very important step in the diagnosis of skin tumours.

Pluripotent lineage of CD133 stem cells isolated from human skin samples.

Skin stem cells are very important in cosmetics, pharmacological and regenerative medicine and burn cases. Foreskin samples surgically removed after circumcision from boys below 7 years were collected and primary epidermal cells were prepared by enzymatic and mechanical tituration method. Selecting CD133 (prominin-1) multipotent stem cell marker, enriched stem cells were analyzed by MACS using CD133 antibodies conjugated with magnetic beads. CD133 positive and negative cells with specific skin stem cells markers like - CD34 (Universal stem cells marker), CD29 (integrin beta-1) and CD49f (integrin alpha-6) immunophenotypes were screened and sorted in flowcytometer. Further the expression of four embryonic genes or transcription factors of pluripotent stem cells were analyzed for pluripotent character of sorted cells. It was found that skin stem cell markers associated with CD133 cells, differentially expressed CD34, CD29 and CD49f immunophenotyes on both positive and negative CD133 cells in FACS analysis. The embryonic stem cell markers (induced pluripotent stem cell markers) like Oct4, SOX2, Notch-2 and K19 genes were expressed in CD133 positive epidermal cells. It is therefore evident that foreskin derived epidermal stem cells showed pluripotent or multipotent nature. This finding opens up avenues for new uses of these stem cells for direct cell seeding in wound healing, surgical suturing and drug screening.

Delivery of epidermal neural crest stem cells [EPI-NCSC] to hippocamp in Alzheimer's disease rat model

Alzheimer's disease [AD] is characterized by progressive neuronal loss in hippocamp. Epidermal neural crest stem cells [EPI-NCSC] can differentiate into neurons, astrocytes and oligodendrocytes. The purpose of this study was to evaluate the effects of transplanting EPI-NCSC into AD rat model. Two weeks after induction of AD by injection of Amyloid-beta 1-40 into CA1 area of rat hippocamp, Y-maze and single-trial passive avoidance tests were used to show deficit of learning and memory abilities. EPI-NCSC were obtained from the vibrissa hair follicle of rat, cultured and labeled with bromodeoxyuridine. When Alzheimer was proved by behavioral tests, EPI-NCSC was transplanted into CA3 area of hippocamp in AD rat model. The staining of EPI-NCSC markers [nestin and SOX10] was done in vitro. Double-labeling immunofluorescence was performed to study survival and differentiation of the grafted cells. We showed that transplanted EPI-NCSC survive and produce many neurons and a few glial cells, presenting glial fibrillary acidic protein. Total number of granule cells in hippocamp was estimated to be more in the AD rat model with transplanted cells as compared to AD control group. We observed that rats with hippocampal damage made more errors than control rats on the Y-maze, when reward locations were reversed. Transplanted cells were migrated to all areas of hippocamp and the total number of granule cell in treatment group was equal compared to control group. Transplantation of EPI-NCSC into hippocamp might differentiate into cholinergic neurons and could cure impairment of memory in AD rat model

Stem cells in embryonic skin development

The skin is a complex stratified organ which acts not only as a permeability barrier and defense against external agents, but also has essential thermoregulatory, sensory and metabolic functions. Due to its high versatility and activity, the skin undergoes continuous self-renewal to repair damaged tissue and replace old cells. Consequently, the skin is a reservoir for adult stem cells of different embryonic origins. Skin stem cell populations reside in the adult hair follicle, sebaceous gland, dermis and epidermis. However, the origin of most of the stem cell populations found in the adult epidermis is still unknown. Far more unknown is the embryonic origin of other stem cells that populate the other layers of this tissue. In this review we attempt to clarify the emergence, structure, markers and embryonic development of diverse populations of stem cells from the epidermis, dermis and related appendages such as the sebaceous gland and hair follicle.

DNA release and uptake associated with the development of pleomorphic cells in mammalian skin autotransplants / Liberación y absorción de ADN asociada con el desarrollo de células pleomórficas en autotrasplantes de piel en mamíferos

OBJECTIVE: Although several in vitro studies have demonstrated active release of DNA by living cells, there is still doubt. There are no such in vivo studies (1). The following experiment is an in vivo study to determine whether DNA release and uptake by cells and tissues occur and can be related to normal growth and differentiation, abnormal growth and cancer. METHODS: Epidermal and full-thickness ear-skin grafts were separately autotransplanted into two groups of mice. In a second group, host mice were labelled with tritiated thymidine and autografted separately, with unlabelled epidermal and full-thickness ear-skin grafts. Animals were sacrificed regularly in both cases. RESULTS: Full thickness grafts revealed cysts in 15 out of 16 grafts, with well-differentiated squamous epidermis, DNA labelling ofdermal fibroblasts and no DNA labelling ofepidermal cells. Epidermal grafts revealed cysts in six out of 20 grafts, with epidermal cells variable in shape and arrangement; some appeared normal but others were two to four times larger, forming solid nests ofcells. In some grafts, there were spindle-shaped pleomorphic cells loosely interconnected. DNA labelling was ob served in occasional epidermal cell. Two lung adenocarcinomas were found. CONCLUSION: These results suggest active release of DNA by host cells and DNA uptake by grafted cells. This phenomenon and the differential uptake of DNA labelling ofepidermal and dermal cells in the epidermal and full-thickness grafts suggest an association with abnormal, even pleomorphic epidermal cell behaviour due to the interference of dermal/epidermal interacting factors.

OBJETIVO: Aunque varios estudios in vitro han demostrado la liberación activa de DNA por las células vivas, todavía persisten las dudas. No existen tales estudios in vitro (1). El siguiente experimento constituye un estudio in vitro para determinar si hay liberación y absorción de ADN por parte de las células y los tejidos, y si estos procesos guardan relación con el crecimiento normal y la diferenciación, así como con el crecimiento anormal y el cáncer. MÉTODOS: Injertos de piel de la oreja, tanto de espesor total como epidérmicos fueron auto trasplantados por separado a dos grupos de ratones. En el segundo grupo, ratones huéspedes fueron etiquetados con timidina tritiada, y autoinjertados, por separado, con injertos de piel de la oreja no etiquetados, tanto de espesor total como epidérmicos. En ambos casos, fue necesario sacrificar animales de manera regular. RESULTADOS: Los injertos de espesor total revelaron quistes en 15 de cada 16 injertos, con epidermis escamosa bien diferenciada, etiquetado ADN de fibroblastos dérmicos, y no etiquetado ADN de células epidérmicas. Los injertos epidérmicos revelaron quistes en seis de 20 injertos, siendo las células epidérmicas variables en forma y ordenamiento. Algunas parecían normales, pero otras eran de dos a cuatro veces mayores, y formaban anidamientos celulares sólidos. En algunos de los injertos, se presentaron células pleomórficas en forma de huso, interconectadas con laxitud. Se observó etiquetado de DNA en células epidérmicas ocasionalmente. Se hallaron dos adenocarcinomas pulmonares. CONCLUSIÓN: Estos resultados sugieren la liberación activa de ADN por las células huéspedes y la absorción de ADN por las células injertadas. Este fenómeno y la absorción diferencial de etiquetado de ADN de células dérmicas y epidérmicas en los injertos epidérmicos y de espesor total, sugieren una asociación con el comportamiento celular anormal, e incluso pleomórfico epidérmico, debido a la interferencia de los factores dérmicos/epidérmicos interactuantes.

Nkx-2.3 gene in mouse epidemial maturation

In higher vertebrates, from amphibians to humans, epidemial maturation is a conserved developmental process. Using adult epidemial tissue and an established keratinocyte cell line, the mouse Nkx-2.3 homeobox gene was demonstrated, for the first time, to be expressed in mouse epidermal keratinocytes. Under the normal culture condition, the spontaneous aggregation phenomenon, a common initiation step of ES cell differentiation, and the induction of mouse adult K1 keratin, a marker of mature epidermal keratinocytes, were both observed in vitro when the Xenopus Nkx-2.3 gene was stably transfected into a mouse pluripotent P19 EC cell line. The induction of mouse K1 keratin by using its Xenopus orthologous gene in the mouse P19 cell implies that Nkx-2.3 may play a conserved role in the epidermal maturation of the mouse, as it does in that of the frog (Ma, 2004). However, the CAT assay study on frog adult keratin promoter could not find the induction of adult keratin. This implies there might not be a direct activation of its promoter.

[Culturing of kerationocytes in serum-free medium]

To establish a simple method for preparation of pure epidermal cell in serum-free medium, without co culturing with lethally irradiated 3T3 cells. A piece of skin biopsy was taken from a rat. After trypsinazation to separate epidermal layer, the epidermis was cut into explants 1x1 mm and were laid at 50 mL on tissue cultured flasks. Then, the explants were covered with serum-free keratinocyte growth medium, while the culture medium was changed every two days. The keratinocyte colonies were expanded in flasks and proliferated to 70% confluency on day 21. After two subcultures, the cells were frozen. Pure epidermal cells are established by this technique in a serum-free keratinocyte growth medium, under feeder cell-free condition. Culturing of keratinocytes in serum-free medium is proved to be a useful and simple method for keratinocyte isolation. The successful culturing of keratinocytes from a small skin biopsy can be useful in the treatment of major burn wounds

Expression of N-terminal truncated desmoglein 3 (Delta NDg3) in epidermis and its role in keratinocyte differentiation

During a search for keratinocyte differentiation-related genes, we obtained a cDNA fragment from the 5'-untranslated region of a previously identified splicing variant of desmoglein 3 (Dg3). This transcript encodes a protein of 282 amino acids, which corresponds to the N-terminal truncated intracellular domain of Dg3 (Delta NDg3). Northern blot analysis detected a 4.6-kb transcript matching the predicted size of Delta NDg3 mRNA, and Western blot analysis with an antibody raised against the Dg3 C-terminus (H-145) detected a 31-kDa protein. Increased Delta NDg3 expression was observed in differentiating keratinocytes by RT-PCR and Western blot analysis, suggesting that Delta NDg3 is indeed a differentiation-related gene product. In immunohistochemical studies of normal and pathologic tissues, H-145 antibody detected the protein in the cytoplasm of suprabasal layer cells, whereas an antibody directed against the N-terminal region of Dg3 (AF1720) reacted with a membrane protein in the basal layer. In addition, Delta NDg3 transcript and protein were upregulated in psoriatic epidermis, and protein expression appeared to increase in epidermal tumors including Bowen's disease and squamous cell carcinoma. Moreover, overexpression of Delta NDg3 led to increased migration and weakening of cell adhesion. These results suggest that Delta NDg3 have a role in keratinocyte differentiation, and that may be related with tumorigenesis of epithelial origin.

Model for human skin reconstructed in vitro composed of associated dermis and epidermis

CONTEXTO E OBJETIVO: A técnica para obtenção de pele humana que apresente derme e epiderme, reconstruída a partir de células isoladas de pacientes, pode possibilitar a realização de enxertos autólogos de pele reconstruída em laboratório em pacientes com áreas doadoras escassas, além de permitir ensaios com substâncias químicas e drogas in vitro e não mais in vivo. O objetivo do trabalho é demonstrar um método de obtenção de pele humana reconstruída in vitro composta de derme e epiderme associadas. TIPO DE ESTUDO E LOCAL: Estudo experimental laboratorial realizado no Laboratório de Cultura de Células da Pele da Faculdade de Ciências Médicas da Universidade Estadual de Campinas, Campinas, São Paulo, Brasil. MÉTODOS: A partir da cultura de fibroblastos humanos, é possível obter um número suficiente de células que podem ser injetadas em uma matriz de colágeno bovino tipo I que, mantida imersa em meio de cultura específico para fibroblastos, permite a formação de uma derme humana reconstruída in vitro. Sobre essa derme, por meio de cultura de queratinócitos e melanócitos humanos, forma-se epiderme diferenciada, levando à formação de pele humana reconstruída in vitro, composta de derme e epiderme associadas. RESULTADOS: Demonstramos que é possível reproduzir pele humana reconstruída in vitro, composta de derme e epiderme associadas. Essa pele humana formada é, histologicamente, semelhante à pele humana in vivo. Na derme, identifica-se o tecido colágeno, com suas células, e a matriz extracelular organizados paralelamente à epiderme. Esta se desenvolve em várias camadas. CONCLUSÃO: É possível obter pele humana reconstruída in vitro, completamente diferenciada, composta de derme e epiderme, associadas, a partir da injeção de fibroblastos humanos em uma matriz de colágeno bovino tipo I e da cultura seqüencial de queratinócitos e melanócitose humanos sobre essa matriz contendo fibroblastos em seu interior.

Model of human epidermis reconstructed in vitro with keratinocytes and melanocytes on dead de-epidermized human dermis

CONTEXTO: Recentes progressos no campo das técnicas de cultura epitelial têm levado ao desenvolvimento de sistemas de cultura nos quais a epiderme reconstruída obtida exibe características de diferenciação morfológica semelhantes àquelas vistas in vivo. Uma epiderme humana reconstruída in vitro pode ser utilizada como melhor alternativa para testes toxicológicos e de eficácia de produtos de uso tópico in vitro e ainda no tratamento de queimaduras e úlceras crônicas de pele. OBJETIVO: Demonstrar um método de obtenção de epiderme humana reconstruída in vitro, utilizando queratinócitos e melanócitos cultivados sobre uma derme humana morta desepidermizada. TIPO DE ESTUDO: Experimental Laboratorial. LOCAL: Laboratório de Cultura de Células da Pele da Faculdade de Ciências Médicas da Universidade Estadual de Campinas, Campinas, São Paulo, Brasil. PROCEDIMENTOS: Queratinócitos e melanócitos humanos cultivados in vitro foram semeados sobre uma matriz biológica (derme humana morta desepidermizada) e o sistema foi mantido em interface ar-líquido, em meio de cultura adequado, até haver a formação de uma epiderme humana estratificada, mantendo as características histológicas da epiderme in vivo. RESULTADOS: Demonstramos, histologicamente, que é possível reproduzir uma epiderme diferenciada, a partir da cultura de queratinócitos e melanócitos sobre uma derme humana morta desepidermizada, obtendo uma epiderme humana reconstruída in vitro, com queratinócitos e melanócitos funcionais, corretamente posicionados, equivalente à epiderme in vivo. CONCLUSÕES: É possível obter uma epiderme humana reconstruída in vitro completamente diferenciada a partir da cultura de queratinócitos e melanócitos sobre uma derme humana morta desepidermizada.

Microscopic analysis of epidermal melanocytes in human abdominal skin

Microscopic analysis of epidermal melanocytes in human abdominal skin with respect to age and sex. Design: Cross sectional study. Place and Duration of Study: Department of Anatomy. BMSI, JPMC, Karachi. About one year in 1998. Subjects and Demonstration of epidermal melanocytes in 5mm thick vertical paraffin embedded sections of thirty-eight skin samples from different age and sex groups, using dihydroxyphenyl alanine [Dopa] reagent. The melanocytes count per unit area of skin was significantly higher in the younger than older age groups. No significant difference was noticed between males and females epidermal melanocytes counts. Distribution of epidermal melanocytes was inversely proportional to the advancing age. However, there was no significant gender differences in the distribution of epidermal melanocytes

Growth and differentiation of adult Mongolian gerbil epidermal cells in culture

A simple protocol for the growth and differentiation of adult Mongolian gerbil epidermal cells is reported. Insulin (8 micrograms/ml) and reduced levels of serum supplementation (2 percent) were sufficient for the maintenance of these cells in culture. Primary cultures were maintained as a proliferative monolayer in a medium with low calcium concentration (< 0.3 mM). Terminal differentiation of cultures was induced by raising the calcium concentration (1.6 mM) in the medium. These results support the concept derived from mouse epidermal cell culture that calcium is an important regulator of mammalian epidermal cell growth and differentiation. The present protocol also represents a useful tool for studies of mechanisms involved in epidermal cell growth and differentiation in a laboratory animal

Adult gerbil epidermal cells resistant to terminal differentiation associated with initiation of carcinogenesis

In order to identify the cellular basis of the gerbil skin unresponsiveness to two-stage carcinogenesis, it was studied the effect of an initiating dose of carcinogen on the biological behaviour of gerbil skin. Treatment of adult gerbil epidermal cells either in vivo or in vitro with 3-methylcholanthrene yielded cells which were resistant to terminal differentiation induced by calcium. These results support the concept derived from the mouse model system of skin carcinogenesis in which initiation is associated with an altered program of epidermal differentiation. The results also suggest that relative resistance of gerbil skin to two-stage carcinogenesis is related to promotion stage

Moléculas de adhesión celular y piel / Adhesion molecules and skin

Las moléculas de adhesión celular tienen un papel importante en múltiples fenómenos biológicos, tanto normales (generación de la respuesta inmune, coagulación, cicatrización, órgano-génesis) como patológicos (inflamación, trombosis, metástasis de células tumorales). En el presente trabajo se revisan los aspectos básicos de las moléculas de adhesión celular y su papel en diversas condiciones patológicas que afectan a la piel

Células dendríticas epidérmicas Thy-1 positivas un nuevo grupo celular asociado con inmunovigilancia cutánea / Dendritic epidermal cells thy-1 positives. A new cell group asociated with cutaneous immune surveillance

Las células dendríticas epidérmicas Thy-1 positivas (CDE Thy-1) son un tipo celular recientemente descubierto en la epidermis de ratones. Estás células se caracterizan por presentar en su superficie el antígeno Thy-1, que hasta 1975 se había encontrado sólo en timocitos y células nerviosas. Las CDE Thy-1 expresan las glicoproteínas CD3 y gamma delta (* *) del receptor antígenico de linfocitos T, lo cual las relaciona consistentemente con este grupo celular. Hasta el presente se desconoce la función específica de este nuevo tipo celular. Sin embargo, experimentos in vitro han demostrado que estas células presentan citotoxicidad frente a los grupos de células tumorales. Por otra parte, pruebas in vivo demuestran que las CDE Thy-1 ejercen una función derreguladora en las repuestas de hipersensibilidad por contacto. Además su ubicación en el epitelio y la expresión del receptor antigénico **, han permitido asociarlas a mecanismos de vigilancia inmunológica de la integridad cutánea a través del reconocimiento antigénico de proteínas de stress