Construction of living skin equivalents using mixed autologous and allogeneic skin cells for repairing scar contracture of the hand in a patient with recessive dystrophic epidermolysis bullosa / 中华皮肤科杂志

Objective To evaluate the effect of living skin equivalents (LSE) constructed of mixed autologous and allogeneic skin cells and human amnion which served as a matrix on repairing scar contracture of the hand in a patient with recessive dystrophic epidermolysis bullosa (RDEB).Methods Skin tissues were obtained from a patient with RDEB and her mother,and epidermal keratinocytes and dermal fibroblasts were isolated from these tissues and cultured in vitro separately.Human amnion was obtained from the placenta of an unrelated healthy parturient undergoing cesarean delivery,and served as a matrix of the LSE.The autologous and allogeneic fibroblasts were mixed and seeded on the stromal side of the amnion,and then the autologous and allogeneic keratinocytes were mixed and seeded on the epithelial side of the amnion,so as to construct the human amnion-LSE (AM-LSE).Histological examination was performed to observe the structure of the skin tissues obtained from the patient and her mother,and immunofluorescence staining was conducted to detect the expression of type Ⅶ collagen in the skin tissues of the patient and her mother and in the AM-LSE.The AM-LSE was grafted on the skin defects of the patient after release of scar contracture of the hand.After grafting,the survival status of the AM-LSE graft and repairing effect on the wounds were evaluated.Results The constructed AM-LSE consisted of dermis,multilayered and fully differentiated epidermis and well-developed basement membrane.Immunofluorescence examination revealed that type Ⅶ collagen was linearly distributed along the basement membrane.Half a year after grafting,the AM-LSE survived well,and no obvious rejection reaction was observed.No blisters or ulcers occurred at the recipient sites,and the scar contracture was mild.The grafted area showed normal skin color with soft texture.The patient could grab and hold things,which had met self-care requirements of daily living.Conclusions The AM-LSE constructed of mixed autologous and allogeneic skin cells have good histological structures,and can be grafted on the wounds after resection of the scars in a RDEB patient.After grafting,no obvious rejection reaction was observed,and the skin was not liable to develop blisters,ulcers or scar contracture due to friction.

KHG26792 Inhibits Melanin Synthesis in Mel-Ab Cells and a Skin Equivalent Model

The purpose of this study is to characterize the effects of KHG26792 (3-(naphthalen-2-yl(propoxy) methyl)azetidine hydrochloride), a potential skin whitening agent, on melanin synthesis and identify the underlying mechanism of action. Our data showed that KHG26792 significantly reduced melanin synthesis in a dose-dependent manner. Additionally, KHG26792 downregulated microphthalmia-associated transcription factor (MITF) and tyrosinase, the rate-limiting enzyme in melanogenesis, although tyrosinase was not inhibited directly. KHG26792 activated extracellular signal-regulated kinase (ERK), whereas an ERK pathway inhibitor, PD98059, rescued KHG26792-induced hypopigmentation. These results suggest that KHG26792 decreases melanin production via ERK activation. Moreover, the hypopigmentary effects of KHG26792 were confirmed in a pigmented skin equivalent model using Cervi cornus Colla (deer antler glue), in which the color of the pigmented artificial skin became lighter after treatment with KHG26792. In summary, our findings suggest that KHG26792 is a novel skin whitening agent.

Fucoidan Promotes the Reconstruction of Skin Equivalents

In this study we investigated the effects of fucoidan on the proliferation of fibroblasts and the reconstruction of a skin equivalent (SE). Fucoidan significantly stimulated the proliferation of CCD-25Sk human fibroblasts and Western blot analysis demonstrated that fucoidan markedly increased the expression of cyclin D1 and decreased the expression of p27. Fucoidan was used to reconstruct SE. Immunohistochemical staining showed that the addition of fucoidan to dermal equivalents increased expression of proliferating cell nuclear antigen (PCNA) and p63. In addition, expression of alpha6-integrin was significantly increased by fucoidan, whereas expression of beta1-integrin, type 1 collagen, elastin, fibronectin did not markedly change. These results suggest that fucoidan has positive effects on epidermal reconstruction and will therefore be beneficial in the reconstruction of SE.

Solar ultraviolet radiation induces biological alterations in human skin in vitro: Relevance of a well-balanced UVA/UVB protection.

Cutaneous damages such as sunburn, pigmentation, and photoaging are known to be induced by acute as well as repetitive sun exposure. Not only for basic research, but also for the design of the most efficient photoprotection, it is crucial to understand and identify the early biological events occurring after ultraviolet (UV) exposure. Reconstructed human skin models provide excellent and reliable in vitro tools to study the UV-induced alterations of the different skin cell types, keratinocytes, fibroblasts, and melanocytes in a dose- and time-dependent manner. Using different in vitro human skin models, the effects of UV light (UVB and UVA) were investigated. UVB-induced damages are essentially epidermal, with the typical sunburn cells and DNA lesions, whereas UVA radiation-induced damages are mostly located within the dermal compartment. Pigmentation can also be obtained after solar simulated radiation exposure of pigmented reconstructed skin model. Those models are also highly adequate to assess the potential of sunscreens to protect the skin from UV-associated damage, sunburn reaction, photoaging, and pigmentation. The results showed that an effective photoprotection is provided by broad-spectrum sunscreens with a potent absorption in both UVB and UVA ranges.

Tratamiento de las úlceras crónicas en los miembros inferiores con un equivalente cutáneo autólogo y desbridación con larvas de Lucilia sp. (Diptera: Calliphoridae). Reporte de un caso / Chronic lower limb ulcers treatment with autologous skin equivalent and larvae debriding (Diptera Calliphoridae)

Introducción: las úlceras en los miembros inferiores constituyen una causa importante de hospitalización y deterioro en la calidad de vida de los pacientes, al interferir con sus actividades laborales y sociales. Estas lesiones obedecen a diferentes enfermedades, la más frecuente de las cuales en Medellín es la insuficiencia venosa; una vez diagnosticadas, existen muchas alternativas de tratamiento, que buscan crear un lecho apropiado para el cierre de la herida. El uso de larvas para el desbridamiento es una alternativa rápida y de bajo costo, con la cual se logra un tejido de granulación adecuado para la aplicación de equivalentes cutáneos autólogos, que constituyen una opción para pacientes refractarios a otros tratamientos. Materiales y métodos: se seleccionó y evaluó a una paciente de acuerdo con los criterios de inclusión establecidos para el estudio. Para desbridar la úlcera se recurrió a la aplicación de larvas de Lucilia sp. (Diptera: Calliphoridae), btenidas a partir de huevos previamente desinfectados con hipoclorito de sodio y sembrados en un medio de cultivo con antibióticos. Se aplicaron las larvas directamente en la lesión y posteriormente se procedió a tapar esta con muselina, permitiendo la obtención de oxígeno y facilitando el drenaje del tejido necrótico. Las larvas se dejaron por 48 horas, al cabo de las cuales se retiraron con una pinza estéril y se descartaron en alcohol al 70%; luego se procedió a evaluar la limpieza de la lesión. Este procedimiento se llevó a cabo en el Laboratorio de Entomología (GIEM) de la Universidad Antioquia. Se procesó una biopsia de piel de la paciente, obtenida siguiendo un protocolo previamente establecido, para obtener queratinocitos y fibroblastos. Para el cultivo primario de queratinocitos el 90% de las células obtenidas de la biopsia se sembró en cajas de cultivo de 75 cm2, en presencia de 7 x 106 células de la línea 3T3-Swiss tratadas con mitomicina C (10 μg/mL) como capa alimentadora; para el cultivo primario de fibroblastos, se sembró el 10% de las células en cajas de cultivo de 75 cm2 con el medio DMEM suplementado. En la producción del equivalente cutáneo se utilizó un gel obtenido con una mezcla de plasma humano de sangre AB de banco de sangre, 6-7,5 x 104 fibroblastos, CaCl2 y ácido tranexámico. Sobre dicho gel se sembraron luego los queratinocitos provenientes del cultivo primario y se hizo seguimiento del cultivo en microscopio invertido. Cuando se obtuvo una confluencia celular cercana al 100%, se desprendió el equivalente cutáneo con pinzas estériles para aplicarlo inmediatamente sobre la úlcera. Se reportan los resultados del tratamiento de una úlcera en el miembro inferior izquierdo de una paciente de 66 años con la terapia combinada de desbridamiento con larvas y cultivo de equivalentes cutáneos autólogos. Después de 15 meses de iniciado el tratamiento, la úlcera continúa cerrada y la paciente no tiene dolor ni impedimentos funcionales en la extremidad.

Effects of Ascorbic Acid on Keratinocyte and Epidermalization of Skin

BACKGROUND: There are different models of skin substitutes, but no skin substitutes have the characteristics of native skin. It was reported that the incubation of skin substitutes in medium containing ascorbic acid extends cellular viability and promotes formation of an epidermal barrier in vitro. OBJECTIVE: The purpose of this study is to observe the effects of ascorbic acid on the proliferation of keratinocytes and on the reconstruction of epidermis. MATERIALS AND METHODS: Normal human keratinocytes and fibroblasts were isolated and used for culturing living skin equivalent (LSE). RESULTS: When ascorbic acid was added, the expression of p63 and a6 integrin was definitely increased compared to control models. In addition, ascorbic acid increased the proliferation of normal human keratinocytes at a dose dependent manner. Especially, ascorbic acid induced the phosphorylation of ERK and up-regulation of EGF-R CONCLUSION: Results suggest that ascorbic acid is essential in the control of keratinocyte proliferation and basement membrane formation. Ascorbic acid-related keratinocytes proliferation is seemed to be mediated by ERK phosphorylation and EGF-R up-regulation.

The Investigation of the Melanocytes in a Cultured Skin Equivalent Model / 대한해부학회지

Melanocytes grown in the pure monolayer culture lack the three-dimentional organization and the cellular interactions that exist in vivo. These can be partially overcome by growing melanocytes together with other epidermal cells in cultured skin equivalent models. In this study, skin equivalents were prepared by seeding mixtures of cultured human keratinocytes and melanocytes in ratio 10 : 1 onto artificially constructed dermis. They were cultured in DMEM/F12 (1 : 1) for 4 days and then lifted to the air-liquid interface and maintained in DMEM/F12 (3 : 1) for 10 days. Histological and electronmicroscopic examinations of the cultured skin equivalants revealed a structure that closely resembled human interfollicular epidermis; 1. Melanocytes, were identified by positive staining with melanoma-specific antibodies (NKI/C3 and S-100 protein) and prominent cytoplasm with rich cell organelles, were located in the basal layer. 2. Melanocytes contained predominently early stage melanosomes and prominent Golgi apparatus. Mature melanins, were usually abundant in normal skin, were hardly seen both in melanocytes and in neighbouring keratinocytes. 3. Melanocytes were surrounded by keratinocytes but did not form intercellular junctions with them. 4. Keratinocytes were charaterized by microfilament bundles and intercellular junctions such as desmosomes and hemidesmosomes with neighbouring keratinocytes and artificial dermis. The melanocyte in the above skin equivalents had a strong resemblance to the one of normal human skin and therefore this model can be used as artificial skin for the transplantation and in the investigation of melanocytes' role to the UV stimuli.

Influences of Human Cytokines and Mixed Lymphocyte Culture-derived Cytokines on the Proliferation and Differentiation of the Living Skin Equivalent / 대한피부과학회지

BACKGROUND: A living skin equivalent(LSE) is useful as a skin replacement and as a model system for basic studies such as skin physiology and pathology. This system attempts to reproduce in vitro the cell-cell and cell-matrix interactions responsible for cell differentiation. Recently, there have been many studies that cytokines(IL-1, IL-6, IL-8, TGF-beta, TNF-alpha) play an important role in proliferating skin disease, especially psoriasis. Hesides, many cytokines can influence the proliferation and differentiation of normal human keratinocytes. OBJECTIVE AND METHODS: In the present study, the author investigated the proliferation and differentiation of cytokines in LSE as compared with control skin using histopathological and immunohistochemical staining methods. The author also investigated the proper concentration of cytokines on the proliferation of cultured human keratinocytes. RESULTS: Morphological analysis of LSE showed reorganization of epidermal layers with the appearance of a distinct basal laer and of a hyperkeratotic horny layer. We demonstrated that the fine granules resembling keratchyaline granules were observed in the control and cytokine treated groups(IL-l alpha, IL-2, IL-6, TNF-alpha). We observed significant epidermal proliferation in the PBMC (25%), IL 6(10ng), TGF-beta(20ng) treated groups than in the control group(p<0.01). Immunohistochemical analysis demonstrated that the terminal differentiation marker involucrin was expressed at the level of the prickle cell layer in the cytokine treated groups. Also the cellular proliferation marker PCNA was expressed at the level of the basal layer in the PBMC(25%), IL-2(100ng) and IL-6(10ng) treated groups. CONCLUSION: The results indicate that cytokine IL-6 and TGF-beta can induce a proliferation and differentiation of the epidermis in this in vitro model.

Effects of Normal Fibroblasts and Peripheral Blood Mononuclear Cells on Squamous Cell Carcinoma Cell Line ( SCL - 1 ) / 대한피부과학회지

Many carcinomas have an active mononuclear cell infiltrates surrounding tumor. Various in vitro assays have shown that cellular constituents of peripheral blood mononuclear cells(PBMC) can alter growth of carcinoma cell line. Author compared the effects of normal fibroblasts on squamous cell carcinoma cell line(SCL-1) along with those of sctivated and/or nonactivated PBMC on SCI 1 using a skin equivalent system. This system prevents direct cellular contact by growing SCL-1 on an overlying Millicell-HA membrane and normal fibroblast or supernatants of PBMC in a lower chamber. Normal fibroblasts enhanced the outgrowth of SCL-1 and induced a more organized phenotype of SCL-1. Supernatants from nonstimulated PBMC suppressed outgrowth of SCL 1, and concanavalin A stimulated PBMC supernatants alterd rnorphology of cultured SCL-1 from a disorganized phenotype to a more organized phenotype. It is concluded that fibroblasts and PBMC may affect the growth and differentiation of SCL-1 via their mediators(cytokines)