Influence of dual-cure resin-cement curing modes on gingival cytotoxicity and inflammatory responses

Abstract

Background/purpose: Dual-cure resin-cements are used for various dental restorations. However, whether the curing modes of these resin-cements influence gingival inflammation remains unclear. Hence, herein, we evaluated the effects of dual-cure resin-cement curing modes on gingival cytotoxicity and inflammatory responses.

Materials and methods: Specimens were prepared using two dual-cure resin-cements-RelyX Unicem 2 (RU) and G-CEM ONE (GO)-by light-cure or self-cure modes. Degree of conversion (DC) and monomer elution of the resin-cements were measured using Fourier-transform infrared spectroscopy and high-performance liquid chromatography, respectively. Human gingival fibroblasts (GFs) and macrophages were cultured on resin-cements, and inflammatory cytokine levels, intracellular reactive oxygen species (ROS) generation, and mitogen-activated protein (MAP) kinase activation were evaluated.

Results: Light-cured (LC) resin-cements exhibited significantly higher DC and lower monomer elution than did self-cured (SC) resin-cements. Triethyleneglycol dimethacrylate (TEGDMA) and urethane dimethacrylate (UDMA) were substantially eluted from RU and GO, respectively. Neither LC resin-cement exhibited cytotoxicity and enhanced pro-inflammatory cytokine expression in GFs and macrophages. However, both SC resin-cements significantly decreased cell numbers and promoted cellular inflammatory responses. SC generated higher intracellular ROS levels compared to that seen with LC, and different patterns of MAP kinase activation were observed between SC-RU and SC-GO.

Conclusion: Compared with LC dual-cure resin-cements, SC dual-cure resin-cements show stronger cytotoxicity and elicit greater inflammatory responses in gingival cells owing to residual monomers (e.g., TEGDMA and UDMA) by activating MAP kinases in GFs and macrophages. Clinicians should ensure adequate light irradiation during prosthesis cementation and make efforts to remove the excess cement.

Keywords: Curing modes; Cytotoxicity; Dual-cure resin-cements; Gingival tissue; Inflammatory responses.

Figure 1

Effects of dual-cure resin-cement [RelyX Unicem 2 (RU) and G-CEM ONE (GO)] curing mode on the degree of conversion (DC) and mechanical properties of dual-cure resin-cements. (A) DC of light-cured (LC) or self-cured (SC) resin-cements determined using Fourier-transform infrared spectroscopy with a universal attenuated total reflectance (n = 6). (B) Vickers hardness of LC or SC resin-cements measured with a microhardness tester (n = 10). Data were analyzed using analysis of variance (ANOVA) and Tukey's multiple comparison test, and P < 0.05 was considered significant. Data are presented as the mean ± standard deviation (SD), with different letters indicating statistically significant differences between multiple groups. D1, day 1; D2, day 2; D3, day 3; D7, day 7.

Figure 2

Effects of dual-cure resin-cement [RelyX Unicem 2 (RU) and G-CEM ONE (GO)] curing mode on resin monomer release from dual-cure resin-cements. (A) Representative chromatograms of triethylene glycol dimethacrylate (TEGDMA) and urethane dimethacrylate (UDMA) standard solutions and the supernatant of self-cured RU (SC–RU) immersed in water on day 1. (B) Released TEGDMA concentrations in the supernatant of light-cured (LC) or SC–RU immersed in water (n = 7). (C) Representative chromatograms of TEGDMA and UDMA standard solutions and the supernatant of SC–GO immersed in water on day 1. (D) Released UDMA concentrations in the supernatant of LC or SC–GO immersed in water (n = 7). Red arrows indicate detected monomer peaks. Data were analyzed using analysis of variance (ANOVA) and Tukey's multiple comparison test, and P < 0.05 was considered significant. Data are presented as the mean ± standard deviation (SD), with different letters indicating statistically significant differences between multiple groups. 1h, 1 h; D1, day 1; D2, day 2; D3, day 3; D7, day 7.

Figure 3

Effects of dual-cure resin-cement [RelyX Unicem 2 (RU) and G-CEM ONE (GO)] curing mode on gingival fibroblasts (GFs). Gene expression of (A) pro-inflammatory cytokines interleukin (IL)-1β, IL-6, prostaglandin E synthase 2 (PTGES2), tumor necrosis factor α (TNFα), and (B) matrix metalloproteinase 2 (MMP2) and MMP9 relative to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in GFs cultured on light-cured (LC) or self-cured (SC) resin-cements on day 1 (n = 3). (C) Water-soluble tetrazolium 1 (WST-1)-based evaluation of GF cell proliferation cultured on LC or SC resin-cements (n = 3). Data were analyzed using analysis of variance (ANOVA) and Tukey's multiple comparison test, and P < 0.05 was considered significant. Data are presented as the mean ± standard deviation (SD), with different letters indicating statistically significant differences between multiple groups. D0, day 0; D1, day 1; D2, day 2; D3, day 3.

Figure 4

Effects of dual-cure resin-cement [RelyX Unicem 2 (RU) and G-CEM ONE (GO)] curing mode on macrophages. Gene expression of (A) pro-inflammatory cytokines tumor necrosis factor α (TNFα) and nitric oxide synthase 2 (NOS2) relative to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in macrophages cultured on light-cured (LC) or self-cured (SC) resin-cements on day 1 (n = 3). (B) Water-soluble tetrazolium 1 (WST-1)-based evaluation of cell proliferation of macrophages cultured on LC or SC resin-cements (n = 3). Data were analyzed using analysis of variance (ANOVA) and Tukey's multiple comparison test, and P < 0.05 was considered significant. Data are presented as the mean ± standard deviation (SD), with different letters indicating statistically significant differences between multiple groups. D0, day 0; D1, day 1; D2, day 2; D3, day 3.

Figure 5

Effects of dual-cure resin-cement [RelyX Unicem 2 (RU) and G-CEM ONE (GO)] curing mode on intracellular reactive oxygen species (ROS) generation and mitogen-activated protein (MAP) kinase activation of gingival fibroblasts (GFs) and macrophages. Intracellular ROS levels of (A) GFs and (B) macrophages cultured on light-cured (LC) or self-cured (SC) resin-cements measured and normalized to DNA concentrations at 1 h (n = 3). Activation of MAP kinases in (C) GFs and (D) macrophages cultured on SC resin-cements. Data were analyzed using analysis of variance (ANOVA) and Tukey's multiple comparison test, and P < 0.05 was considered significant. Data are presented as the mean ± standard deviation (SD), with different letters indicating statistically significant differences between multiple groups. hrs, hours; P-ERK1/2, phosphorylated extracellular signal-regulated kinase 1 and 2; P-p38, phosphorylated p38; P-JNK, phosphorylated c-Jun N-terminal kinase (JNK).