Influence of Test Specimen Geometry and Water Soaking on the In Vitro Cytotoxicity of Orthocryl®, Orthocryl® LC, Loctite® EA 9483 and Polypropylene

Richard Behnke; Franka Stahl; Kathrin Duske; Mareike Warkentin; Margit Schwartz; Burkhard Hinz; Udo Walther

doi:10.3390/molecules27123949

Influence of Test Specimen Geometry and Water Soaking on the In Vitro Cytotoxicity of Orthocryl^®, Orthocryl^® LC, Loctite^® EA 9483 and Polypropylene

Molecules. 2022 Jun 20;27(12):3949. doi: 10.3390/molecules27123949.

Authors

Richard Behnke¹, Franka Stahl¹, Kathrin Duske¹, Mareike Warkentin², Margit Schwartz³, Burkhard Hinz³, Udo Walther³

Affiliations

¹ Department of Orthodontics, University Dental School, Rostock University Medical Center, 18057 Rostock, Germany.
² Department of Materials Science and Medical Engineering, University of Rostock, 18119 Rostock, Germany.
³ Institute of Pharmacology and Toxicology, Rostock University Medical Center, 18057 Rostock, Germany.

Abstract

Depending on their composition, plastics have a cytotoxic potential that needs to be evaluated before they are used in dentistry, e.g., as orthodontic removable appliances. Relevant guidelines set out requirements that a potential new resin in the medical field must meet, with a wide scope for experimental design. In the present study, test specimens of different geometries consisting of varying polymers (Orthocryl^®, Orthocryl^® LC, Loctite^® EA 9483, Polypropylene) were soaked for different periods of time, then transferred to cell culture medium for 24 h, which was subsequently used for 24-h cultivation of A549 cells, followed by cytotoxicity assays (WST-1, Annexin V-FITC-propidium iodide (PI) flow cytometry). In this context, a reduction in the cytotoxic effect of the eluates of test specimens prepared from Orthocryl^® LC and Loctite^® EA 9483 was particularly evident in the Annexin V-FITC-PI assay when the soaking time was extended to 48 h and 168 h, respectively. Consistent with this, a reduced release of potentially toxic monomers into the cell culture medium, as measured by gas chromatography-mass spectrometry, was observed when the prior soaking time of test specimens of all geometries was extended. Remarkably, a significant increase in cytotoxic effect was observed in the WST-1 assay, which was accompanied by a higher release of monomers when the thickness of the test sample was increased from 0.5 to 1.0 mm, although an elution volume adapted to the surface area was used. However, further increasing the thickness to 3.0 mm did not lead to an increase in the observed cytotoxicity or monomer release. Test specimens made of polypropylene showed no toxicity under all test specimen sizes and soaking time conditions. Overall, it is recommended to perform toxicity studies of test specimens using different geometries and soaking times. Thereby, the influence of the different specimen thicknesses should also be considered. Finally, an extension of the test protocols proposed in ISO 10993-5:2009 should be considered, e.g., by flow cytometry or monomer analysis as well as fixed soaking times.

Keywords: cytotoxicity; monomer release; orthodontics; polymers; removable appliances; resins.

MeSH terms

Materials Testing / methods
Methacrylates
Methylmethacrylates / chemistry
Polypropylenes*
Water*

Substances

Methacrylates
Methylmethacrylates
Orthocryl
Polypropylenes
Water
Loctite

Grants and funding

This work was supported by Mecklenburg-Vorpommersche Gesellschaft für Zahn-, Mund- und Kieferheilkunde an den Universitäten Greifswald und Rostock e. V., Germany.