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.

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.

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.