title: Determining the Depth of Injury in Bioengineered Tissue Models of Cornea and Conjunctiva for the Prediction of All Three Ocular GHS Categories.
abstract: The depth of injury (DOI) is a mechanistic correlate to the ocular irritation response. Attempts to quantitatively determine the DOI in alternative tests have been limited to ex vivo animal eyes by fluorescent staining for biomarkers of cell death and viability in histological cross sections. It was the purpose of this study to assess whether DOI could also be measured by means of cell viability detected by the MTT assay using 3-dimensional (3D) reconstructed models of cornea and conjunctiva. The formazan-free area of metabolically inactive cells in the tissue after topical substance application is used as the visible correlate of the DOI. Areas of metabolically active or inactive cells are quantitatively analyzed on cryosection images with ImageJ software analysis tools. By incorporating the total tissue thickness, the relative MTT-DOI (rMTT-DOI) was calculated. Using the rMTT-DOI and human reconstructed cornea equivalents, we developed a prediction model based on suitable viability cut-off values. We tested 25 chemicals that cover the whole range of eye irritation potential based on the globally harmonized system of classification and labelling of chemicals (GHS). Principally, the MTT-DOI test method allows distinguishing between the cytotoxic effects of the different chemicals in accordance with all 3 GHS categories for eye irritation. Although the prediction model is slightly over-predictive with respect to non-irritants, it promises to be highly valuable to discriminate between severe irritants (Cat. 1), and mild to moderate irritants (Cat. 2). We also tested 3D conjunctiva models with the aim to specifically address conjunctiva-damaging substances. Using the MTT-DOI method in this model delivers comparable results as the cornea model, but does not add additional information. However, the MTT-DOI method using reconstructed cornea models already provided good predictability that was superior to the already existing established in vitro/ex vivo methods.
authors.affiliation: University Medical Center Hamburg-Eppendorf, Department of Dermatology and Venerology, 20246 Hamburg, Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Zorn-Kruppa
authors.affiliation: University Medical Center Hamburg-Eppendorf, Department of Dermatology and Venerology, 20246 Hamburg, Germany.
authors.forename: Pia
authors.initials: P
authors.lastname: Houdek
authors.affiliation: University Medical Center Hamburg-Eppendorf, Department of Dermatology and Venerology, 20246 Hamburg, Germany.
authors.forename: Ewa
authors.initials: E
authors.lastname: Wladykowski
authors.affiliation: Jacobs University Bremen gGmbH, School of Engineering and Sciences, 28759 Bremen, Germany.
authors.forename: Maria
authors.initials: M
authors.lastname: Engelke
authors.affiliation: Jacobs University Bremen gGmbH, School of Engineering and Sciences, 28759 Bremen, Germany.
authors.forename: Melinda
authors.initials: M
authors.lastname: Bartok
authors.affiliation: Henkel AG & Co. KGaA, 40589 Düsseldorf, Germany.
authors.forename: Karsten R
authors.initials: KR
authors.lastname: Mewes
authors.affiliation: University Medical Center Hamburg-Eppendorf, Department of Dermatology and Venerology, 20246 Hamburg, Germany.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Moll
authors.affiliation: University Medical Center Hamburg-Eppendorf, Department of Dermatology and Venerology, 20246 Hamburg, Germany.
authors.forename: Johanna M
authors.initials: JM
authors.lastname: Brandner
mesh_terms: D000818:Animals; D002460:Cell Line; D003228:Conjunctiva / Q000187:drug effects* / Q000293:injuries; D003315:Cornea / Q000187:drug effects*; D005131:Eye Injuries; D008954:Models, Biological; D011817:Rabbits; D023822:Tissue Engineering