Human dermal fibroblasts as a feeder layer promote the growth of human keratinocytes / 基础医学与临床

Objective To study the mechanism of dermal fibroblasts as a feeder layer to support the growth of human keratinocytes. Methods Human dermis fibroblasts were isolated and cultured and then treated with mitomycin-C. The expression of type Ⅰand type Ⅲ precollagen mRNA and relevant protein in feeder layer were examined by RT-PCR and Immunohistochemistry. KCs were cultured both on FB and NIH3T3 feed layer as control, the adhering numbers and the time of fusion were recorded. Results RT-PCR showed an increase of type Ⅰprecollagen mRNA in FB feeder layer as compared with that of normal fibroblasts (P

2D and 3D structural study of rete ridge in oral mucosa and skin paddle of various free flaps

OBJECTS: With the advancement of tissue engineering techniques, the effort to develop bioartificial mucosa have been actively delivered. The problem we met with this technique is the lack of mechanical strength between kerationocyte layer and dermal layer, where in the normal skin and mucosa, they are tightly bound with rete ridge structure. The purpose of this study is to understand the 2D and 3D structure of rete ridge of mucosa and skin paddle for rendering more biomimetic structure to the artificial mucosa. MATERIALS AND METHODS: Oral mucosa and skin from the patients who received the oral surgery and maxillofacial reconstruction were harvested. The epidermis was separated from the dermis after treating with dispase for 12-16 hours. H and E staining was performed for 2D(dimensional) structure study and confocal LASER and SEM study were performed for 3D structure. Mean height(Sc) and arithmetic mean deviation(Sa) of all surface height were calculated. RESULTS: The average height of rete ridge of skin flap was between 67.14micrometer and 194.55micrometer. That of oral mucosa was between 146.26micrometer and 167.51micrometer. Pressure bearing area and attached gingiva of oral mucosa showed deeper rete ridges. CONCLUSION: To obtain the adequate strength of artificially cultured keratinocyte skin and mucosa flap, it is necessary to imitate the original skin and mucosa structure, especially rete ridge. Through this study, 2D and 3D rete ridge structure of normal mucosa and skin was obtained. These results can be used as basis for substrate morphology for keratinocytes culture.

Antifungal Susceptibility Testing in Three-dimensional Keratinocyte Culture Model / 대한피부과학회지

BACKGROUND: Standardization of antifungal susceptibility testing for dermatophytes is important and several variables, such as inoculum size, length and temperature of incubation, media, and end point determination has recently been established. However, a more improved test model which can evaluate bioavailability and has clinical relevance is still needed. OBJECTIVE: We performed antifungal susceptibility testing by using three-dimensional keratinoctyte culture model, living skin equivalent (LSE), as an in vitro model. METHODS: LSE was prepared and various concentrations of antifungals were added to media. Microconidia of Trichophyton mentagrophytes was inoculated onto LSE and incubated for 6 days at 35 degrees C. RESULTS: Inhibition of fungal proliferation and invasion were observed at 0.1microgram/ml of terbinafine, 0.01 microgram/ml of itraconazole solution, 0.1 microgramg/ml of itraconazole powder, and 10 microgram/ml of fluconazole, respectively. CONCLUSION: Our culture model is similar to in vivo situation and the results were relatively well accordant to those of other previous studies. Our LSE model is considered as a promising in vitro system for evaluating the activity and safety of antifungal agents. However, further study using more various species of dermatophytes and more strains is still needed.

Study on the Composite Graft with an Acellular Xeno-dermis and Cultured Keratinocyte Sheets / 中国医师杂志

Objective To study a graft method of an acellular dermis composited with cultured human keratinocyte sheets,and investigate the outcomes of the composite graft on full-thickness wounds.Methods Using porcine skins,we prepared the acellular dermis and cultured human keratinocyte sheets in vitro.Athymic null mice were divided into the experimental group and the control group,the acellular xeno-dermis was implanted into the subcutaneous of the dorsal skin in experimental group,one week after the implantation the cultured keratinocyte sheets were grafted on the full-thickness defective area of the dorsal skin in the experimental group,in control group only implanted human keratinocyte sheet on the defective area wound,healing status was regular assessed,and biopsies for histological analysis and immunohistochemistry test were performed in post-operation.Results Compared with the control group,the appearance of wound coverage in the experimental group was better.Moreover,the histologic analysis revealed that fully differentiated epidermis,organized proliferated collagen fibers and significant reconstruction of epidermis-dermis junction were seen in the experimental group which lack of the acute immuno-rejection response.Conclusion As a kind of dermal substitutes,the acellular xeno-dermis can be compositely transplanted with cultured human keratinocyte sheet and can improve the quality of wound healing.

Measurement of the Cytotoxicity of Several Solvents Using a Normal Human Keratinocytes Culture Model / 대한피부과학회지

BACKGROUND: As in vivo models such as animal and human tests have many potential problems the keratinocyte culture model has previously been used as an in vitro model for testing skin irritancy for common skin irritants. OBJECTIVE: To determine the skin irritant potency of several solvents, we employed cultured human keratinocytes as an in vitro model. METHODS: To evaluate the cytotoxicity of solvents, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) test, lactic dehydrogenase(LDH) release test, and tritiated thymidine incorporation test were used. RESULTS: Dose dependent decrease of cell viability and DNA synthesis, and dose dependent increase in leakage of LDH liberation were observed in normal cultured human keratinocytes after exposure to several solvents. The cytotoxicity potency of several solvents measured by each method were slightly different. CONCLUSION: These observations suggest that the cultured human keratinocyte model would be useful in evaluating the cytotoxicity of solvents.

Measurement of the Cytotoxicity of Several Solvents Using a Normal Human Keratinocytes Culture Model / 대한피부과학회지

BACKGROUND: As in vivo models such as animal and human tests have many potential problems the keratinocyte culture model has previously been used as an in vitro model for testing skin irritancy for common skin irritants. OBJECTIVE: To determine the skin irritant potency of several solvents, we employed cultured human keratinocytes as an in vitro model. METHODS: To evaluate the cytotoxicity of solvents, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) test, lactic dehydrogenase(LDH) release test, and tritiated thymidine incorporation test were used. RESULTS: Dose dependent decrease of cell viability and DNA synthesis, and dose dependent increase in leakage of LDH liberation were observed in normal cultured human keratinocytes after exposure to several solvents. The cytotoxicity potency of several solvents measured by each method were slightly different. CONCLUSION: These observations suggest that the cultured human keratinocyte model would be useful in evaluating the cytotoxicity of solvents.

In Vitro Effects of Several Irritants Using Human Keratinocyte Culture Model / 대한피부과학회지

Primary irritant dermatitis is one of the most common skin disease caused by various hazardous chemicals produced from the environment. For the detection of skin irritant potency, in vivo models such as human and animal patch test have been used, Keratinocyte culture method which has been set up very recently is another alternative in vivo method of detecting skin irritarlcy. LVe have investigated the effects of three skin irritants, phenol, benzoyl peroxide (BP), and sodium lauryl sulfate(SLS) on the keratinocyte culture system. Prostaglandin E(PGE) measurement, cell count and electron microscopic observation were performed after adding three irritants of different concentrations to the cultured keranocyte cells. The main results of this study were as follows : 1. There were statistically significant decreased cell number in concentration of 10 M phenol, 10 4M BP and SLS. The order of cytotoxic potency was SLS>BP >phenol. 2. In case of PGE production, decreased PGE production was observed 6 hours after addition of the irritants, except 10 M phenol and 10M BP groups. Decrea sing tendency sustained until 24 hours, however all were statistically nonsignificant comparing with control group. 3. Electron microscopic finding showed that dilatation of endoplasmic reticulums in 10 M phenol group, condensation and dilatation of mitochondrias in 10 4M BP group, and most of the cells were swollen in 10 4M SLS group. These results suggest that cell count is a useful model for performing cytotoxi city test in keratinocyte culture decreased PGE production represents cytotoxic effect in high concentration of primary irritants and ultrastructural changes may reflect the different pathomechanisms in cytotoxicity.

Ultrastructural Study of Human Epidermal Keratinocytes Cultured in Low Calcium Medium / 第二军医大学学报

A low calcium medium developed for epidermal keratinocytes were prepared according to the MCDB 153 modified formula and used in human epidermal keratinocyte culture compared with DMEM culture system. The observation by contrast microscopy and electron microscopy showed that in the low calcium medium keratinocytes grew as a monolayer of high proliferation and had many characteristics of basal cells, with a more rounded shape and large intercellular spaces. Increasing the calcium ion concentration in the medium or changing the other culture conditions the cells in these cultures could be induced stratification and terminal differentiation. The results suggest that the growth, proliferation and differentiation of cultured human epidermal keratinocytes can be controlled and regulated someway.