Cytotoxicity testing of dental materials: Establishment of a 3D-printed dentin barrier culture system

Cytotoxicity testing is critical in assessing the biocompatibility of dental materials with regard to the pulp, however, conventional methods such as the extract test (ISO 10993-5) do not reflect the protective effects of dentin in the dentin-pulp complex. Traditional dentin barrier testing techniques (ISO 7405) address this problem, but face challenges due to their complex and expensive production and the difficult and error-prone handling. Therefore, the aim of this study was to develop a novel, user-friendly and easily accessible dentin barrier culture system (DBCS) to improve cytotoxicity testing of dental materials.

The DBCS was designed and manufactured using stereolithography 3D printing. The components were assembled with dentin discs to create a barrier between an upper and lower compartment (pulp side/restoration side). Dentin discs were pretreated (EDTA or citric acid) and primary human pulp cells (HPC) or mouse fibroblasts (L-929) were cultured in the upper chamber, while a self-adhesive composite (Vertise Flow, Kerr; Scafati, Italy) was applied to the opposite side of the disc after 48 h. Scanning electron microscopy (SEM) was used to visualize dentin structure and cell attachment. Analyses included assessment of cell metabolism (MTT test), membrane integrity (LDH assay), and performing live/dead staining. To validate the model, various dentin disc parameters were evaluated, including species origin (human or bovine), location relative to the pulp (proximal or distal), thickness, and the effects of autoclaving. Statistical evaluation was performed using the Kruskal-Wallis or the Mann-Whitney test (P ≤ 0.05).

Both HPC and L-929 formed confluent cell layers in the DBCS setup. Pretreatment of dentin discs with EDTA or citric acid reliably removed the smear layer, with the latter having a positive effect on cell viability (P ≤ 0.03). The self-adhesive composite had a similar effect on the viability of both cell types, and SEM revealed apoptotic cell morphology. Dentin provided a protection only at a thickness of greater than 400 µm. There were no significant differences in cellular responses to the material depending on dentin origin or autoclaving (P > 0.9999).

This 3D-printable culture system offers a readily available, user-friendly dentin barrier model for evaluating the cytotoxicity of dental materials and can be easily reproduced by anyone with access to a standard 3D printer.