Validation study for in vitro skin irritation test using reconstructed human skin equivalents constructed by layer-by-layer cell coating technology.

The aim of this study is to validate an in vitro skin irritation test (SIT) using three-dimensional reconstructed human epidermal (RhE) skin equivalents prepared by layer-by-layer (LbL) method (LbL-3D Skin) in a series of interlaboratory studies. The goal of these validation studies is to evaluate the ability of this in vitro test to reliably discriminate skin irritant from nonirritant chemicals, as defined by OECD and UN GHS. This me-too validation study is to assess the within- and between-laboratory reproducibility, as well as the predictive capacity, of the LbL-3D Skin SIT in accordance with performance standards for OECD TG 439. The developed skin model, LbL-3D Skin had a highly differentiated epidermis and dermis, similar to the validated reference methods (VRM) and native human skin. The quality parameters (cell survival in controls, tissue integrity, and barrier function) were similar to VRM and in accordance with OECD TG 439. The LbL-3D Skin SIT validation study was performed by three participating laboratories and consisted of three independent tests using 20 reference chemicals. The results obtained with the LbL-3D Skin demonstrated high within-laboratory and between-laboratory reproducibility, as well as high accuracy for use as a stand-alone assay to distinguish skin irritants from nonirritants. The predictive potency of LbL-3D Skin SIT using total 54 test chemicals were comparable to those in other RhE models in OECD TG 439. The validation study demonstrated that LbL-3D Skin has proven to be a robust and reliable method for predicting skin irritation.

Comparative assessment of 24-hr primary skin irritation test and human patch test data with in vitro skin irritation tests according to OECD Test Guideline 439 (for quasi-drugs in Japan).

The Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 439 is an in vitro test method of reconstructed human epidermis (RhE), which was developed for hazard identification of irritating chemicals in accordance with a primary skin irritation test using rabbits with 4-hr exposure. A regulation for quasi-drugs in Japan requires data from primary skin irritation tests using rabbits to undergo 24-hr exposure, and this is used as an evidence for 24-hr closed patch tests in humans. In this study with the same chemicals, primary skin irritation test data using rabbits undergoing 24-hr exposure and a 24-hr occlusive human patch test data were analyzed by comparing the results obtained with four test methods adopted in OECD TG 439. The performances of in vitro test methods showed a positive predictive value of 72.7-85.7% to predict the results of 24-hr primary rabbit skin irritation test knowing that its positive predictive value was 57.1% against humans only. The prediction factors of in vitro test methods were higher for the human patch test data with a sensitivity reaching 60 to 80%. Three surfactants gave false negatives in some of the RhE methods evaluated with the human patch test, but in each case, they were correctly classified as positive when evaluated at double concentration. Therefore, the approach of setting the margin to 2 was effective in eliminating false negatives. This suggests that in vitro test methods are useful for assessing skin irritation potential without animal testing for the application of quasi-drugs in Japan.

Keratinization induced by air exposure in the reconstructed human epidermal model: an in vitro model of a cultured epithelial autograft.

A reconstructed human epidermis, an in vitro model of a cultured epithelial autograft, was used to examine the formation of a stratum corneum induced by exposure to air. A prolonged wet condition and excess application of petrolatum on the dressing reduced efficient production of the stratum corneum.

A catch-up validation study of an in vitro skin irritation test method using reconstructed human epidermis LabCyte EPI-MODEL24.

Three validation studies were conducted by the Japanese Society for Alternatives to Animal Experiments in order to assess the performance of a skin irritation assay using reconstructed human epidermis (RhE) LabCyte EPI-MODEL24 (LabCyte EPI-MODEL24 SIT) developed by the Japan Tissue Engineering Co., Ltd. (J-TEC), and the results of these studies were submitted to the Organisation for Economic Co-operation and Development (OECD) for the creation of a Test Guideline (TG). In the summary review report from the OECD, the peer review panel indicated the need to resolve an issue regarding the misclassification of 1-bromohexane. To this end, a rinsing operation intended to remove exposed chemicals was reviewed and the standard operating procedure (SOP) revised by J-TEC. Thereafter, in order to confirm general versatility of the revised SOP, a new validation management team was organized by the Japanese Center for the Validation of Alternative Methods (JaCVAM) to undertake a catch-up validation study that would compare the revised assay with similar in vitro skin irritation assays, per OECD TG No. 439 (2010). The catch-up validation and supplementary studies for LabCyte EPI-MODEL24 SIT using the revised SOPs were conducted at three laboratories. These results showed that the revised SOP of LabCyte EPI-MODEL24 SIT conformed more accurately to the classifications for skin irritation under the United Nations Globally Harmonised System of Classification and Labelling of Chemicals (UN GHS), thereby highlighting the importance of an optimized rinsing operation for the removal of exposed chemicals in obtaining consistent results from in vitro skin irritation assays.

Establishment of a new in vitro test method for evaluation of eye irritancy using a reconstructed human corneal epithelial model, LabCyte CORNEA-MODEL.

Finding in vitro eye irritation testing alternatives to animal testing such as the Draize eye test, which uses rabbits, is essential from the standpoint of animal welfare. It has been developed a reconstructed human corneal epithelial model, the LabCyte CORNEA-MODEL, which has a representative corneal epithelium-like structure. Protocol optimization (pre-validation study) was examined in order to establish a new alternative method for eye irritancy evaluation with this model. From the results of the optimization experiments, the application periods for chemicals were set at 1min for liquid chemicals or 24h for solid chemicals, and the post-exposure incubation periods were set at 24h for liquids or zero for solids. If the viability was less than 50%, the chemical was judged to be an eye irritant. Sixty-one chemicals were applied in the optimized protocol using the LabCyte CORNEA-MODEL and these results were evaluated in correlation with in vivo results. The predictions of the optimized LabCyte CORNEA-MODEL eye irritation test methods were highly correlated with in vivo eye irritation (sensitivity 100%, specificity 80.0%, and accuracy 91.8%). These results suggest that the LabCyte CORNEA-MODEL eye irritation test could be useful as an alternative method to the Draize eye test.

Validation study of the in vitro skin irritation test with the LabCyte EPI-MODEL24.

A validation study on an in vitro skin irritation assay was performed with the reconstructed human epidermis (RhE) LabCyte EPI-MODEL24, developed by Japan Tissue Engineering Co. Ltd (Gamagori, Japan). The protocol that was followed in the current study was an optimised version of the EpiSkin protocol (LabCyte assay). According to the United Nations Globally Harmonised System (UN GHS) of classification for assessing the skin irritation potential of a chemical, 12 irritants and 13 non-irritants were validated by a minimum of six laboratories from the Japanese Society for Alternatives to Animal Experiments (JSAAE) skin irritation assay validation study management team (VMT). The 25 chemicals were listed in the European Centre for the Validation of Alternative Methods (ECVAM) performance standards. The reconstructed tissues were exposed to the chemicals for 15 minutes and incubated for 42 hours in fresh culture medium. Subsequently, the level of interleukin-1 alpha (IL-1 α) present in the conditioned medium was measured, and tissue viability was assessed by using the MTT assay. The results of the MTT assay obtained with the LabCyte EPI-MODEL24 (LabCyte MTT assay) demonstrated high within-laboratory and between-laboratory reproducibility, as well as high accuracy for use as a stand-alone assay to distinguish skin irritants from non-irritants. In addition, the IL-1α release measurements in the LabCyte assay were clearly unnecessary for the success of this model in the classification of chemicals for skin irritation potential.

[Development of alternative to animal experiment in evaluation of skin irritation caused by alcohol-based hand rubs].

Alcohol-based hand rubs are widely used for infection control in clinical practice. However, it is known that frequent use of the alcohol-based hand rubs may cause skin irritation. To predict the skin irritation in human, animal experiments are quite useful. Especially, the Draize Test using rabbits is suitable for this purpose because their skin is highly sensitive. On the other hand, the development of alternative to animal experiments is important not only from the viewpoint of ethical aspects but also from the efficient research and development. Reconstructed human epidermis (RhE) was developed as a human skin equivalent model in vitro, and has been applied to the evaluation of skin irritation. But the RhE has not been utilized for the evaluation of alcohol-based hand rubs because of the high skin permeability and cytotoxicity of alcohols. The aim of this study was to develop a new method using the RhE in evaluation of skin irritation caused by alcohol-based hand rubs. The authors propose an experimental technique named "Skin model blowing method (SMBM)" consisting of the sequential procedure as follows; applying small amount of testing sample on RhE, blow-dry, post incubation, and cell viability measurement. According to the SMBM, the skin irritation caused by alcohol-based hand rubs could be evaluated under the similar condition of their actual use. It was found that a high correlation existed between the cell viability obtained from SMBM and the skin irritation index in rabbit which had been reported previously.

Assessment of the human epidermal model LabCyte EPI-MODEL for In vitro skin corrosion testing according to the OECD test guideline 431.

A new OECD test guideline 431 (TG431) for in vitro skin corrosion tests using human reconstructed skin models was adopted by OECD in 2004. TG431 defines the criteria for the general function and performance of applicable skin models. In order to confirm that the new reconstructed human epidermal model, LabCyte EPI-MODEL is applicable for the skin corrosion test according to TG431, the predictability and repeatability of the model for the skin corrosion test was evaluated. The test was performed according to the test protocol described in TG431. Based on the knowledge that LabCyte EPI-MODEL is an epidermal model as well as EpiDerm, we decided to adopt the the Epiderm prediction model of skin corrosion for the LabCyte EPI-MODEL, using twenty test chemicals (10 corrosive chemicals and 10 non-corrosive chemicals) in the 1(st) stage. The prediction model results showed that the distinction of non-corrosion to corrosion corresponded perfectly. Therefore, it was judged that the prediction model of EpiDerm could be applied to the LabCyte EPI-MODEL. In the 2(nd) stage, the repeatability of this test protocol with the LabCyte EPI-MODEL was examined using twelve chemicals (6 corrosive chemicals and 6 non-corrosive chemicals) that are described in TG431, and these results recognized a high repeatability and accurate predictability. It was concluded that LabCyte EPI-MODEL is applicable for the skin corrosive test protocol according to TG431.

Assessment of human epidermal model LabCyte EPI-MODEL for in vitro skin irritation testing according to European Centre for the Validation of Alternative Methods (ECVAM)-validated protocol.

A validation study of an in vitro skin irritation testing method using a reconstructed human skin model has been conducted by the European Centre for the Validation of Alternative Methods (ECVAM), and a protocol using EpiSkin (SkinEthic, France) has been approved. The structural and performance criteria of skin models for testing are defined in the ECVAM Performance Standards announced along with the approval. We have performed several evaluations of the new reconstructed human epidermal model LabCyte EPI-MODEL, and confirmed that it is applicable to skin irritation testing as defined in the ECVAM Performance Standards. We selected 19 materials (nine irritants and ten non-irritants) available in Japan as test chemicals among the 20 reference chemicals described in the ECVAM Performance Standard. A test chemical was applied to the surface of the LabCyte EPI-MODEL for 15 min, after which it was completely removed and the model then post-incubated for 42 hr. Cell v iability was measured by MTT assay and skin irritancy of the test chemical evaluated. In addition, interleukin-1 alpha (IL-1alpha) concentration in the culture supernatant after post-incubation was measured to provide a complementary evaluation of skin irritation. Evaluation of the 19 test chemicals resulted in 79% accuracy, 78% sensitivity and 80% specificity, confirming that the in vitro skin irritancy of the LabCyte EPI-MODEL correlates highly with in vivo skin irritation. These results suggest that LabCyte EPI-MODEL is applicable to the skin irritation testing protocol set out in the ECVAM Performance Standards.

Fabrication of cultured oral gingiva by tissue engineering techniques without materials of animal origin.

BACKGROUND: Cultured gingival substitute has been found to be a useful graft material for treatment of gingival recession. However, such substitutes include xenograft derivative materials that involve concomitant risk of viral contamination. To eliminate this risk, we designed new gingival substitutes made of recombinant human collagen types I and III sponges and cultured these substitutes in animal-free media (HFDM-1). METHODS: Gingival fibroblasts were seeded onto sponges of type I or III recombinant collagen. These sponges were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), HFDM-1 with 2% human serum (HS), or HFDM-1. Fibroblast proliferation in these samples was compared using the cell-counting kit assay. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released into the cultured media were examined by enzyme-linked immunosorbent assay. RESULTS: The fibroblasts proliferated significantly in all six combinations of collagen and medium types. The fibroblast growth rate after 9 days of culture was equal between HFDM-1 with 2% HS and DMEM with 10% FBS. The type III collagen sponge showed a higher fibroblast growth rate than the type I sponge. VEGF concentrations in HFDM-1 with 2% HS were higher than those in other media. The highest HGF levels were detected in DMEM with 10% FBS. CONCLUSIONS: The new cultured gingival substitute containing no animal-derived materials produced good cell proliferation and VEGF release. The results suggested that the substitute may provide a new tool for the treatment of gingival recession.

Novel biodegradable cholesterol-modified polyrotaxane hydrogels for cartilage regeneration.

Cholesterol was introduced to a hydrolyzable polyrotaxane (PRx), not only to improve cell proliferation and glycosaminoglycan (GAG) production, but also to control the degradation rate of the hydrogels. The cholesterol was introduced to hydrolyzable PRx species by threading many alpha-cyclodextrins (alpha-CDs) on a poly(ethylene glycol) (PEG) chain having hydrolyzable ester linkages at the terminals; the PRx species were then cross-linked with other PEGs to prepare cholesterol-modified PRx hydrogels. The degree of cholesterol substitution was varied in the range of 1-25%. These hydrogels were examined to clarify the effect of cholesterol groups on mechanical properties, erosion time and chondrocyte proliferation. Highly porous biodegradable cholesterol-modified PRx hydrogels were fabricated using a combination of potassium hydrogen carbonate (as an effervescent salt) and citric acid. This fabrication process enabled the homogeneous expansion of pores within the polymer matrices, leading to well-interconnected macroporous hydrogels with a mean pore size of around 200-400 microm, ideal for high-density chondrocyte seeding. Time to complete degradation of the hydrogels was shortened by increasing the degree of substitution due to the aggregation of alpha-CDs through hydrophobic interaction of cholesterol groups. The presence of approx. 10% cholesterol improved the chondrocyte proliferation and GAG production. The modification of cholesterols to PRx is a good approach for creating new biodegradable hydrogels in terms of chondrocyte culture and controlling degradation time of the hydrogels.

Novel poly(ethylene glycol) scaffolds crosslinked by hydrolyzable polyrotaxane for cartilage tissue engineering.

Highly porous poly(ethylene glycol) (PEG) hydrogel scaffolds crosslinked with hydrolyzable polyrotaxane for cartilage tissue engineering were prepared by a solvent casting/salt leaching technique. The resultant scaffolds have well interconnected microporous structures ranging from 87 to 90%. Pore sizes ranging from 115.5-220.9 microm appeared to be dependent on the size of the sieved sodium chloride particulates. Moreover, a dense surface skin layer was not found on either side of the scaffold surfaces. Using microscopic Alcian blue staining of the chondrocyte-seeded scaffolds, well adhered cells and newly produced glycosaminoglycans (GAG) were confirmed. Following the initial chondrocyte seeding onto the hydrogel scaffolds, the cell number was significantly increased, reaching 149, 877, and 1228 cells/mg of tissue at 8, 15, and 21 days in culture, respectively. The micrograph shows well adhered and spread chondrocytes in the interior pores and a cartilaginous extracellular matrix with a GAG fraction produced from the chondrocytes. Results suggest that the PEG hydrogel scaffolds crosslinked with the hydrolyzable polyrotaxane are a promising candidate for chondrocyte culture.

Effect of glycosaminoglycan production on hardness of cultured cartilage fabricated by the collagen-gel embedding method.

To assess applicability of the tactile sensor in hardness measurement of cultured cartilage and to clarify the relationship between hardness and tissue structure of cultured cartilage fabricated by the collagen-gel embedding method, we studied the effect of glycosaminoglycans on hardness of such cultured cartilage using a tactile sensor and electron probe x-ray microanalyzer (EPMA). Hardness measured by the tactile sensor, that is, change in frequency of naturally oscillating piezoelectric elements caused by contact with a testing material, increased with the number of days of culture or seeding cell density. Analysis of the sulfur component in EPMA results mainly reflected glycosaminoglycans produced by chondrocytes. Sulfur mapping indicated that tissue of the cultured cartilage differed between its surface and the inside; layers rich in glycosaminoglycans and cells had formed in the surface. Changes in frequency showed close correlation with the amount of glycosaminoglycans in the surface and the inside (r = 0.98 and 0.85, respectively) of cultured cartilage measured by EPMA. Thus, the tactile sensor is capable of measuring hardness of cultured cartilage, reflecting the change in tissue structure between the surface and the inside of the cartilage.