Reconstructed Epidermal Skin Model And Its Features

Reconstructed epidermal skin model and its featuresIntroductionIn order to avoid the use of animal in cutanous studies, several experimental skin equivalents have been developed during the past decades. The skin equivalents were composed of epidermis reconstructed from cultured keratinocyte or skin explant grown on various substrates such as collagen matrix, de-epidermized dermis, or acetate cellulose-polycarbonate filter membrane. A stratified epidermis including the stratum corneum was produced using these models. Evidently, these models can provide an experimental platform to study epidermis in vitro for many purposes, for instance, biological and biochemical characteristics of reconstructed epidermis, as compared to those in vivo.In human skin wound healing, epithelial cells arise from epidermis at the wound edge. Proliferating and migrating, the new epithelia sheet eventually covers the wound bed. Studies of the process in reconstructed skin model in vitro could ideally illustrate native tissue behaviour. Beside that, the re-epithlization basically should include migration of epidermal keratinocytes, the proliferation of keratinocytes, and the stratification and differention of the neo-epithelium. It has been known that the migration of epithelial cells is very important event in wound healing. Since the neo-epithelia outgrowth morphologically reflect the migration of the epithelial cell, determination of its growing speed seems more important under various pathophysiological conditions. However, to our knowledge nearly all the assessments to this approach previously performed in some techniques were less suitable for quantitative evaluation of the neo-epithelia's out-growing.In this study we reconstructed neo-epithelia in a skin organ culture on DED. The characterization of epidermal proliferation and differentiation was observed and detected using light, electron microscope with a series of immunohistochemical techniques during the process of reepithelialization. The outgrowth rate of new epithelium can be maintained in culture to be visualised repeatedly by stained with fluorescence under fluorescence microscopy and measured by digital image analysis. Its possible application was assessed by applying different concentration of r-EGF and corticosteroid, with compared without those in culture.Materials and methods一,Preparation of dermis equivalent and skin organ culture1,Preparation of de-epidermized dermis(DED): Adult human skin sample was obtained from the removal breast of patients during plastic surgery operation. Pieces of skin were kept in Falcon tube with phosphate-buffered saline (PBS) at 4°C refrigerator. Before processed, it rinsed PBS for several times to clean the stained blood on the skin. Skin samples of 8-10-mm biopsy punch were taken from the pieces of skin. The samples were placed in the tube with PBS. The epidermis of 8-10mm biopsy skin samples was removed after heated at 56℃in warmed PBS for 30 min. The de-epidermized dermis samples were put into sterized cryovial. And then, the de-epidermized dermis was killed by ten successive freezing in nitrogen liquid and thawing. And then labelled and kept frozen at -70°C Freezer.2,Preparation of culture vessels: To keep the explant to grow at the air-liquid interface, we set up a modified cell strainer (Cell strainer with 70 um mesh, Falcon, DK) as support. The modified process as follows: The nylon membrane along the sides was cut away and the vertical polypropylene legs were divided at approximately 5 mm from the circular filter. The remaining membrane filter was placed upside-down on its legs in a 6-well plate.3,Preparation of cultured skin explant: Adult human skin was obtained from plastic surgery department. Skin from mammary and abdomen was generally used. 2mm explant was obtained from the donor skin, touched fibrin glue which is mixture of trombin, fibrinogen and cyclocapron at dermis side and placed on epidermal side of the DED, which was hold on a modified cell strainer. Culture medium was changed every other day and skin were incubated in 37℃in the atmosphere of 5% CO2 and 90% humidity.4,Preparation of culture medium: The tissue culture medium was composed of Dulbecco's modified Eagle's medium (DMEM), and Nutrient mixture F12, supplemented with non-essential amino acids, 5μg/ml insulin, 100μg/ml streptmycin, 100 units/ml penicilin, 0.18 mM adenin, 0.5μg/ml hydrocortisone, 10^-10 M cholera toxin and 10% fetal bovine calf sera. EGF was added (10ng/ml) for the comparative study of neo-epithelia outgrowth between the culture with EGF and without EGF. Betamethasome-17-valeratewas added (10^-3 M)for the comparative study of neo-epithelia outgrowth between the culture with corticosteroid and without corticosteroid.二,The examination of new-epithelium and specific marker of epidermal cells1,Preparation of light and electron microscope: For morphological observation, those staining were employed as following: Hematoxylin & Eosin stain, Periodic Acid-Schiff's reaction, Van Gieson stain, Weigart stain. Ultrastructural observation for DED and new epidermis was used by Transmission Electron Microscope(TEM), Scanning Electron Microscope(SEM).2,Preparation of Immunohistochemical Staining: Specific marker of epidermal cells, prolifertive and differential marker of epidermal development were detected by Immunohistochemicalstaining for Vimentin, collagenⅣ, CD1a, Mel-5, laminin, Involucrin, Transglutaminase-1(TGase 1), Keratin-1, Keratin-14, beta-1 intergrin, Ki67 antigen, 5-bromo-2—deoxyuridine(Brdu) and Hoechst33258 staining. 三,Fluorescence imaging of reepithelialization1,Preparation of fluorescence: Two fluorescences were involved in this study. One is fluorescein diacetate (FDA) and another is CellTracker Green (CMFDA or CTG) It was diluted in PBS. Stock solution is 24 mM(FDA) and 10mM (CMFDA). Kept in -20℃Freezer. The work concentration is 2.4μM for both. It must be used fresh solution every time when changed medium with the fluorescence solution.2,Preparation of fluorescence microscopy and digital image analysis: Adding fresh fluorescein diacetate(FDA) or 5-chloromethyl derivative (CMFDA) solution onto one DED, then moved under a fluorescence microscopy. The fluorescent picture can be seen immediately under the microscopy, that taking about 15 seconds from the end of FDA or CMFDA dropping to be shown up of the fluorescent picture. The focus of fluorescent microscopy was adjusted as enough proper as possible (16x magnification) and took the photos. After that, replace the fluorescent solution with fresh medium. The image analysis was analysed by Olympus Dp Soft 8 Version 3.0 for Windows NT. Taking the function of Measure Image, we measure the whole fluorescence area and explant area. The outgrowth areas of neo-epithelia were obtained from the subtraction of whole fluorescence area and explant area.3,Measurement of neo-epithelial proliferative activity: Mean neoepidermial thickness (total area of viable neoepidermis/length of epidermal surface), number of viable cell layers, and papillomatosis index (length of basement membrane/length of epidermal surface) were measured in triplicate tissue sections of each sample, Proliferative cell density was defined as the number of Ki-67-reactive cells or Brdu positive cells per mm length of the basement membrane zone along the entire neoepidermal radius on the opposite sides of the explant using Leica's Q500 image analysis software.四,Statistical analysisThe data were analysed by GraphPad Prism version 4.00 for Windows and SPSS 11.5 software.. Quantitative data were showed by (?)±SD. Ap value of 0.05 or lower was considered statistically significant.Results一,The characteristics of reconstructed skin epidermis1,The biological characterization of DED: PAS staining was shown that there is a positive reaction line along the DED surface. Using Immunohistochemical Staining, the collagenⅣand laminin were also detected on DED at the same place where PAS is positive on DED. The principal component of artificial dermis is Collagen fibers and elastic fibers under light mrocoscopy after Staining with Van Gieson combined with Weigart stain Under electron microscope, the ultrastmctural appearance of rich and channel network of Collagen fibers were clearly illustrated in the dermis. However, with Vimentin Immunohistochemical Staining, the living fibroblast cell were not found DED after several times of freeze and thaw treatment. The fade ducts of piosebaceous and eccrine gland remain as original deposit, but they appeared no function.2,The morphological structure of neo-epidermis: After ten days' tissue culture, the new growing epidermis on DED has developed a full mature structures which bears similarities to human epidermis. The epidermis consists of basal layer, malpighian or prickle layer, granular layer, and horny layer. Electron microscopic studies have shown that keratinocytes accumulates within its cytoplasm intermediate filaments composed of tonofilaments intricately patterned into bundles. Between adjacent keratinocytes, the ultrastructural appearance of spicilized attachment plate called desmosome was identified as a characteristics of epidermis. It was also found that the partial basement membrane zone at the junction of epidermis and DED dermis, which composed of the hemidesmosomes. However, the neo-epithila sheet cannot form melanocyte and Langerhans'cell through immunohistochemical identification of MEL-5 and CD1a.3,Expression related markers of differentiation and plasticity in the neo-epithelial sheet during development:(1)Expression of Keratin 1 in the new epidermis: After 10 days'culture in this study, Keratin 1 was dominantly expressed in suprabasal keratinocytes.(2)Expression of Keratin 14 in the new epidermis: In this experiment, It was found in basal layer and innermost prickle layer in a ten days. It was also expressed in the basal and spinous layers after 20 days' culture.(3)Expression of involucrin in the new epidermis: Involucrin was found in all layer of neo-epithlia sheet except basal layers in 5 days'culture in this culture system. After 12 days, we examined involucrin expression in outermost spinous layers and granular layers.(4)Expression ofTgase 1 in the new epidermis: In ten days'culture, we detected TGases 1 mainly localized in spinous layers and granular layers.(5) Expression ofβ1 integrins in the new epidermis: About 10 days'culture, we found it mainly distributed in the basal and adjacent spinous layers.4,Expression proliferative markers in the development of neo-epithelial sheet:(1)The Expression of Ki67 associated with the neo-epithelial proliferation: Ki67 is expressed by proliferating cells. It was easily detected in the basal layer on DED, so that we can understand the growing rate of keratinocytes in the reconstructed epidermis.(2)BrdU-labelled keratinocytes regeneration in the reepithiliazation: BrdU is usually used in the detection of proliferating cells in living tissues. It is also clearly detected in the proliferating keratinocytes at the basal layers in the growing neo-epithelial sheet, taking as understanding the growing rate of the epithelial cells.二,Illustration of neo-epithelia outgrowth area on DED with fluorescence.1,Neo-epithelia outgrowth can be recorded by fluorescence with FDA and CMFDA: During the period of culture, resurfacing of the DED with neoepidermis from the skin explant was monitored daily by fluorescencce imaging of reepithelialization technique using the conventional viability probe FDA or CMFDA.2,Neoepidermal surface area measurements and assay variation:(1)Intra variation: To obtain the intra assay variation in one experiment, measurement of outgrowth area illustrated with CMFDA fluorescence in 36 explants, which came from one piece of skin sample of a health donor (abdomen, 61 years old, woman) at day 2, day 5, day 7, and day 9 was carried out using a flurescence microscopy and digital image analysis. The coefficients of variation (CV) in the series of date observations were 41.2%, 18.8%, 12.87 %, and 9.47%, respectively.Further more, we took another sample from a health donor (breast skin, woman, 34 years old) to confirm intra variation in the experiment. Stained with FDA, the neo-epithelia outgrowth area of 24 explants has been illustrated and measured under the fluorescence microscope and digital image analysis at day3, day5, day7, day 10. The coefficients of variation (CV) was recorded 34.1%, 22.3%, 13.79%, 10.99% at day 3, day 5, day 7 and day 10, respectively.(2)Inter variation: An indication of the inter assay variation has been obtained from four- separate experiments. The samples of explants came from four healthy individuals, whose age and donor sites varies and the experiments was done in different dates, but all was prepared under similar culture conditions. The differences of neo-epithelia outgrowth area (FDA staining) measurement between the four subjects at day3, day5, day7, day10 have been analysed. It was found that there was significant difference between the skin from the four healthy donors at day 3, day 5, day 7, day 10 (P＜0.01, P＜0.01, P＜0.01, P＜0.01, respectively. One-way Anova).3,The effects of fluoroprobe exposure on neoepidermis: In order to get a impression about whether the stained fluorescence can influence the neo-epithelia outgrowth, we set up a experiment, in which 36 biopsy explants from a piece of skin sample of a donor were shared out equally on random into three 6 well culture cluster. The culturing conditions were same in the three all, but those in cluster one were stained with FDA, those in cluster two were stained with CMFDA when took photographs at day 3, day 5, day 7, and day10, while the samples observed in cluster three were just add a certain amount of DMSO in PBS when processed illustration at the same days as those of another two clusters for a group of control, until day 10, at that time we took it as final day to cease the observation and stained with FDA for pictures.Difference of outgrowth area recordings between cluster one(FDA) and cluster two(CMFDA) is not statistically significant at day 3, day5, day7, and day 10(P＞0.05, P＞0.05, P＞0.05, P＞0.05, respectively, unpaired student's t-test), and the difference of outgrowth area measurement at day 10 between the three different treated groups is not statistically significant, too. (P＞0.05, one-way Anova).四,The effect of cytokine and glucorticosteroid on neo-epithelial growth1,EGF promoting the speed of neo-epithelial growth: In ten days'culture, the EGF-treated samples had larger area of epithelial outgrowth(P＜0.01), greater thickness(P＜0.01), more layers of cell (P＜0.05), higher keratinocytes proliferation index(P＜0.05), higher cell density(P＜0.05), as compared to control culture.2,High concentration of potent glucorticosteroid reducing growing of neo-epithiliazation: Betamethasone-17-valerate treated samples had reducing the area of epithelial outgrowth as compared to control culture (P＜0.05) after nine days culture. Conclusions1,The artificial de-epidermized dermis(DED) remaining some biological characteristics of dermis on which can induce epidermal regeneration, development and differentiation in vitro.2,Architecture of new regenerated epidermis including basal layer, prickle layer, granular layer, and stratum corneum which share similarities to human epidermis.3,The main proliferating and differential markers of keratinocytes found in the epidermis.4,With fluorescence, the outgrowth area of epithcial sheet can be recorded and enables non-invasive assay of reepithelialization without interrupting cultures progress.5,The potential of application with this skin model should be advantageous in biological studies of normal and pathological epidermalization.