Sensitivity analysis and quality indicators for an in vitro oral irritation assay
Article Sidebar
Main Article Content
Abstract
Biocompatibility testing using in vivo tests is often one of the final evaluations of new dental materials. To reduce the likelihood of failure at this late stage, predictive biocompatibility testing using in vitro methods is needed. In this study, we describe a sensitivity analysis of an oral irritation test by evaluating changes in the viability, using the MTT assay, of 3-D models with EpiOral constructs as a case study. Experiments that tested sources of variability in the assay led to recommendations regarding the storage of the constructs after arrival, pipetting procedure, use of MTT reagents from different vendors, use of transepithelial electrical resistance measurements, and statistical analyses. A statistical model was proposed to evaluate whether test substances yield a positive or negative result and the associated statistical confidence. Testing several test compounds such as the Y-4 polymer, that contains a known irritant, and dentally relevant substances such as sodium dodecyl sulfate (SDS) at varying concentrations revealed statistically significant results as expected. Lastly, a software app was designed to support a multiwell culture plate layout design. Overall, the findings and suggestions documented here will support the further development and potential standardization of this assay system and may be useful for the development of other assays using 3-D constructs.
Plain language summary
New in vitro methods can support the development of novel dental materials by yielding information about their biocompatibility. In this study, an in vitro oral irritation assay was investigated to better understand what aspects of the method contribute to its variability. There is a potential that such a method could yield similar information to in vivo experiments and reduce animal testing.
Article Details
This work is licensed under a Creative Commons Attribution 4.0 International License.
Articles are distributed under the terms of the Creative Commons Attribution 4.0 International license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is appropriately cited (CC-BY). Copyright on any article in ALTEX is retained by the author(s).
Aizawa, S., Yoshida, H., Umeshita, K. et al. (2023). Development of an oral mucosal irritation test using a three-dimensional human buccal oral mucosal model. Toxicol in Vitro 87, doi:10.1016/j.tiv.2022.105519
Barosova, H., Maione, A. G., Septiadi, D. et al. (2020). Use of EpiAlveolar Lung Model to Predict Fibrotic Potential of Multiwalled Carbon Nanotubes. ACS Nano 14, 3941-3956. doi:10.1021/acsnano.9b06860
Coleman, K. P., Grailer, T. P., McNamara, L. R. et al. (2018). Preparation of irritant polymer samples for an in vitro round robin study. Toxicol in Vitro 50, 401-406. doi:10.1016/j.tiv.2018.01.018
De Jong, W. H., Carraway, J. W., Liu, C. et al. (2020). The suitability of reconstructed human epidermis models for medical device irritation assessment: A comparison of In Vitro and In Vivo testing results. Toxicol in Vitro 69, 104995. doi:10.1016/j.tiv.2020.104995
Dhall, A., Tan, J. Y., Oh, M. J. et al. (2022). A dental implant-on-a-chip for 3D modeling of host-material-pathogen interactions and therapeutic testing platforms. Lab on a Chip 22, 4905-4916. doi:10.1039/d2lc00774f
Elliott, J. T., Rosslein, M., Song, N. W. et al. (2017). Toward Achieving Harmonization in a Nanocytotoxicity Assay Measurement Through an Interlaboratory Comparison Study. ALTEX 34, 201-218. doi:10.14573/altex.1605021
Farshidfar, N., Assar, S., Amiri, M. A. et al. (2023). The feasible application of microfluidic tissue/organ-on-a-chip as an impersonator of oral tissues and organs: a direction for future research. Bio-Design and Manufacturing doi:10.1007/s42242-023-00235-5
Franca, C. M., Balbinot, G. D., Cunha, D. et al. (2022). In-vitro models of biocompatibility testing for restorative dental materials: From 2D cultures to organs on-a-chip. Acta Biomaterialia 150, 58-66. doi:10.1016/j.actbio.2022.07.060
França, C. M., Tahayeri, A., Rodrigues, N. S. et al. (2020). The tooth on-a-chip: a microphysiologic model system mimicking the biologic interface of the tooth with biomaterials. Lab on a Chip 20, 405-413. doi:10.1039/C9LC00915A
Hargrove, M., Parr-Dobrzanski, B., Li, L. et al. (2021). Use of the MucilAir Airway Assay, a New Approach Methodology, for Evaluating the Safety and Inhalation Risk of Agrochemicals. App In Vitro Toxicol 7, 50-60. doi:10.1089/aivt.2021.0005
Ingber, D. E. (2022). Human organs-on-chips for disease modelling, drug development and personalized medicine. Nat Rev Genet 23, 467-491. doi:10.1038/s41576-022-00466-9
ISO (2021a). ISO 10993-12:2021 Biological evaluation of medical devices - Part 12: Sample preparation and reference materials.
ISO (2021b). ISO 10993-23:2021 Biological evaluation of medical devices - Part 23: Tests for irritation.
Klausner, M., Ayehunie, S., Breyfogle, B. A. et al. (2007). Organotypic human oral tissue models for toxicological studies. Toxicol in Vitro 21, 938-949. doi:10.1016/j.tiv.2007.01.024
Klausner, M., Handa, Y. and Aizawa, S. (2021). In vitro three-dimensional organotypic culture models of the oral mucosa. In Vitro Cellular & Developmental Biology-Animal 57, 148-159. doi:10.1007/s11626-020-00539-1
Koning, J. J., Rodrigues Neves, C. T., Schimek, K. et al. (2022). A Multi-Organ-on-Chip Approach to Investigate How Oral Exposure to Metals Can Cause Systemic Toxicity Leading to Langerhans Cell Activation in Skin. Front in Toxicol 3, doi:10.3389/ftox.2021.824825
Lafarge, T. and Possolo, A. (2015). The NIST Uncertainty Machine. NCSLI Measure 10, 20-27. doi:10.1080/19315775.2015.11721732
Ly, K. L., Rooholghodos, S. A., Rahimi, C. et al. (2021). An Oral-mucosa-on-a-chip sensitively evaluates cell responses to dental monomers. Biomedical Microdevices 23, doi:10.1007/s10544-021-00543-6
Makkar, H., Zhou, Y., Tan, K. S. et al. (2023). Modeling Crevicular Fluid Flow and Host-Oral Microbiome Interactions in a Gingival Crevice-on-Chip. Adv Healthc Mater 12, e2202376. doi:10.1002/adhm.202202376
Miyauchi, M., Ishikawa, S., Kurachi, T. et al. (2022). Oral Absorption across Organotypic Culture Models of the Human Buccal Epithelium after E-cigarette Aerosol Exposure. ACS Omega 7, doi:10.1021/acsomega.2c06304
Muniraj, G., Tan, R. H. S., Dai, Y. et al. (2023). Microphysiological Modeling of Gingival Tissues and Host-Material Interactions Using Gingiva-on-Chip. Adv Healthc Mater 12, e2301472. doi:10.1002/adhm.202301472
Padovani, G. C., Feitosa, V. P., Sauro, S. et al. (2015). Advances in Dental Materials through Nanotechnology: Facts, Perspectives and Toxicological Aspects. Trend Biotechnol 33, 621-636. doi:10.1016/j.tibtech.2015.09.005
Petersen, E. J., Hirsch, C., Elliott, J. T. et al. (2020). Cause-and-Effect Analysis as a Tool To Improve the Reproducibility of Nanobioassays: Four Case Studies. Chem Res Toxicol 33, 1039-1054. doi:10.1021/acs.chemrestox.9b00165
Petersen, E. J., Nguyen, A., Brown, J. et al. (2021a). Characteristics to consider when selecting a positive control material for an in vitro assay. ALTEX 38, 365-376. doi:10.14573/altex.2102111
Petersen, E. J., Sharma, M., Clippinger, A. J. et al. (2021b). Use of Cause-and-Effect Analysis to Optimize the Reliability of In Vitro Inhalation Toxicity Measurements Using an Air–Liquid Interface. Chem Res Toxicol 34, 1370-1385. doi:10.1021/acs.chemrestox.1c00080
Petersen, E. J., Elliott, J. T., Bancos, S. et al. (2022a). Predictive Alternative Methods for Assessing Biocompatibility of Dental Materials: A NIST-NIDCR Workshop Report. ALTEX 39, 522-524. doi:10.14573/altex.2206241
Petersen, E. J., Uhl, R., Toman, B. et al. (2022b). Development of a 96-Well Electrophilic Allergen Screening Assay for Skin Sensitization Using a Measurement Science Approach. Toxics 10, 257. doi:10.3390/toxics10050257
Petersen, E. J., Elliott, J. T., Gordon, J. et al. (2023). Technical framework for enabling high-quality measurements in new approach methodologies (NAMs). ALTEX 40, 174-186. doi:10.14573/altex.2205081
Rösslein, M., Elliott, J. T., Salit, M. et al. (2015). Use of Cause-and-Effect Analysis to Design a High-Quality Nanocytotoxicology Assay. Chem Res Toxicol 28, 21-30. doi:10.1021/tx500327y
Sae, H., Arakawa, S., Takahashi, M. et al. (2014). Effects of ozone nano-bubble water on periodontopathic bacteria and oral cells - In vitro studies. Sci Technol Adv Mater 15, 055003. doi:10.1088/1468-6996/15/5/055003
Searle S., C. G., McCulloch C. (1992). In (eds.), Variance Components.
Simon, C. G., Borgos, S. E., Calzolai, L. et al. (2023). Orthogonal and complementary measurements of properties of drug products containing nanomaterials. Journal of Controlled Release 354, 120-127. doi:10.1016/j.jconrel.2022.12.049
Song, G., Banov, D., Bassani, A. et al. (2016). Evaluation of the Safety, Cell Migration, and Mucoadhesive Properties of a Mucoadhesive Polymer Blend in Human Oral Mucosa. AAPS PharmSciTech 18, 1-7. doi:10.1208/s12249-016-0630-z
Sun, J., Petersen, E. J., Watson, S. S. et al. (2017). Biophysical characterization of functionalized titania nanoparticles and their application in dental adhesives. Acta Biomaterialia 53, 585-597. doi:10.1016/j.actbio.2017.01.084
Welch, J., Wallace, J., Lansley, A. B. et al. (2021). Evaluation of the toxicity of sodium dodecyl sulphate (SDS) in the MucilAir™ human airway model in vitro. Regul Toxicol Pharmacol 125, 105022. doi:10.1016/j.yrtph.2021.105022
Zhang, J. H., Chung, T. D. Y. and Oldenburg, K. R. (1999). A simple statistical parameter for use in evaluation and validation of high throughput screening assays. Journal of Biomolecular Screening 4, 67-73. doi:10.1177/108705719900400206