Cytotoxicity of 3D-printed, milled, and conventional oral splint resins to L929 cells and human gingival fibroblasts

Abstract

Objectives: Evidence on the biocompatibility of three-dimensional (3D)-printed and milled resins for oral splints is limited. This in vitro study assessed the influence of the manufacturing method on the cytotoxicity of oral splint resins on L929 cells and human gingival fibroblasts (GF1).

Materials and methods: Standardized specimens of four 3D-printed, two-milled, one-thermoformed, and one-pressed splint resin were incubated with L929 and GF1 cells for 24 h. Immunofluorescence and WST-8 assay were performed to evaluate cytotoxic effects. One-way analysis of variance and Tukey's multiple comparison test were applied with the variables "splint resin" and "manufacturing method" (p < .05).

Results: Immunofluorescence showed attachment of L929 and GF1 cells to the splint resins. Irrespective of the manufacturing method, the WST-8 assay revealed significant differences between splint resins for the viability of L929 and GF1 cells. L929 cells generally showed lower viability rates than GF1 cells. The evaluation of cell viability by the manufacturing method showed no significant differences between 3D printing, milling, and conventional methods.

Conclusions: The cytotoxic effects of 3D-printed, milled, and conventional oral splint resins were similar, indicating minor influence of the manufacturing method on biocompatibility. Cytotoxicity of the resins was below a critical threshold in GF1 cells. The chemical composition might be more crucial than the manufacturing method for the biocompatibility of splint resins.

Keywords: 3D printing; cytotoxicity; milling; splint resins.

Figure 1

Fluorescence imaging of L929 and GF1 cells on the surface of dental splint resins after 24 h of incubation. Exemplary visualization shows adhesion of the cells to all of the tested resins. Morphology of GF1 cells includes more distinctive cell protrusions than L929 cells, which are generally rounder in shape. DAPI (blue) indicates cell nuclei, phalloidin (green), and vinculin (red) are associated with actin filaments of the cytoskeleton. DAPI, 4′,6‐diamidino‐2‐phenylindole.

Figure 2

Relative cell viability of L929 cells according to the Cell Counting Kit ‐8 assay after 24 h of incubation with the investigated oral splint resins. (a) Cell viability shows significant differences between resins indicated by asterisks (p < .05). (b) Arrangement of the data according to the manufacturing method shows no significant differences. Glass was used for normalization (=1.0).

Figure 3

Relative cell viability of GF1 cells according to the CCK‐8 assay after 24 h of incubation with the investigated oral splint resins. (a) Cell viability shows significant differences between resins. ^a p < .05 compared with Med610, ^b p < .01 compared with V‐print splint, ^c p < .05 compared with FREEPRINT ortho 385, ^d p < .01 compared with Dental LT clear, ^{d ′} p < .05 compared with Dental LT clear, ^e p < .0001 compared with M‐PM crystal, ^{e ′} p < .01 compared with M‐PM crystal, ^f p < .001 compared with Therapon Transpa, and ^g p < .05 compared with Erkodur. (b) Arrangement of the data according to the manufacturing method shows significant differences (*p < .05) between thermoforming and pressing. Glass was used for normalization (=1.0).