Composite-derived monomers affect cell viability and cytokine expression in human leukocytes stimulated with Porphyromonas gingivalis

Abstract Objectives: Dental composites release unreacted resin monomers into the oral environment, even after polymerization. Periodontal cells are, therefore, exposed to substances that potentially elicit the immune inflammatory response. The underlying molecular mechanisms associated with the interaction between resin monomers and human immune cells found in the gingival crevicular fluid are not fully understood yet. This study investigated the ability of bisphenol A-glycidyl methacrylate (BISGMA), urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) to induce apoptosis and cytokine release by human leukocytes stimulated with a periodontal pathogen. Methodology: Peripheral blood mononuclear cells (PBMC) from 16 healthy individuals were included in this study. To determine the toxicity, the PBMC were incubated for 20 hours, with monomers, for the analysis of cell viability using MTT assay. To evaluate cell death in the populations of monocytes and lymphocytes, they were exposed to sub-lethal doses of each monomer and of heat-inactivated Porphyromonas gingivalis (P. gingivalis) for 5 hours. Secretions of IL-1β, IL-6, IL-10 and TNF-α were determined by ELISA after 20 hours. Results: UDMA and TEGDMA induced apoptosis after a short-time exposure. Bacterial challenge induced significant production of IL-1β and TNF-α (p<0.05). TEGDMA reduced the bacterial-induced release of IL-1β and TNF-α, whereas UDMA reduced IL-1β release (p<0.05). These monomers did not affect IL-10 and IL-6 secretion. BISGMA did not significantly interfere in cytokine release. Conclusions: These results show that resin monomers are toxic to PBMC in a dose-dependent manner, and may influence the local immune inflammatory response and tissue damage mechanisms via regulation of bacterial-induced IL-1β and TNF-α secretion by PBMC.


Introduction
The employment of resin-based materials in Dentistry is presently ubiquitous. Dental composite resins comprise a complex mixture of materials, usually consisting of an organic matrix, reinforcing inorganic filler and a silane-coupling agent that connects the filler and resin matrix. 1 Currently, the most common resin systems used in dentistry are methacrylates. 1 In these systems, the polymerizable organic matrix contains one or more base monomers, such as however, the complete reaction is still a challenge.
The literature suggests the best polymerization one can achieve in freshly light-cured dental composites is around 30-40%, which might increase up to 50-70% after 24 h. 3 As a consequence of this relatively poor polymerization, unreacted resin monomers may be released from dental composites, particularly in the first 24 hours after placement. 4,5 Then, monomers elute for many days after polymerization, both due to incomplete curing and as a consequence of natural degradation. 4 This fact undermines biocompatibility, and there is evidence that monomers act by disturbing the environment, ultimately influencing the function of various cell types. [6][7][8] In this regard, adverse biological reactions to resin components, such as local and systemic toxicity, pulp reactions, allergy, genotoxicity and cytotoxicity, have been reported. 6 Nonetheless, studies targeting PBMCderived immune cells stimulated with periodontal pathogens, commonly found in the gingival sulcus, are scarce. In addition, the underlying mechanism associated to the effect of sub-lethal doses of resin monomers in periodontal inflammation remains to be elucidated.
The presence of dental biofilms keeps a persistent sub-clinical inflammatory infiltrate in the gum tissue. 9,10 Porphyromonas gingivalis (P. gingivalis), a Gramnegative anaerobic bacteria that colonizes sub-gingival tissue sites, is an important pathogen associated with chronic periodontitis. 11 The immune system recognizes pathogenic invaders such as P. gingivalis and initiates an inflammatory response, launching the production of numerous inflammatory mediators such as pro-and anti-inflammatory cytokines. IL-1β, TNF-a and IL-6 are pro-inflammatory cytokines that enhance bone resorption and activate lymphocytes, 12 whereas IL-10 displays anti-inflammatory properties and inhibits bone resorption. 13 These molecules are regulated by tightly controlled pathways and orchestrate the intercellular communication, coordinating host immune response. 9 Mononuclear leukocytes play a critical role in this process. 9 Although the bacterial biofilm is important in periodontitis initiation and progression, it is primarily the host's inflammatory response that promotes considerable damage to periodontal tissues. 11 In fact, a delicate balance between health and disease maintains tissue integrity. 4 Any disability or excess of inflammatory response may result in tissue damage.
In this scenario, the contact of a composite resin restoration with gingival tissues, and the local release of monomers, may cause changes in periodontal cellular processes, acting as a local stressor.
In this study, we focused on evaluating the effects of resin monomers on leukocytes stimulated or not by P. gingivalis. To the best of our knowledge, this is the first report on the effect of monomers exposure in leukocytes stimulated with a periodontal pathogen.

Chemicals
Three monomers (BISGMA, TEGDMA and UDMA) were chosen, as they are constituents of a variety of dental resin systems. All monomers were obtained

Peripheral blood mononuclear cells (PBMC) preparation
The study protocol was approved by the Research Ethics Committee of the Pontifical Catholic University of Minas Gerais, Minas Gerais, Brazil.
Sixteen healthy individuals were recruited. All of them gave informal consent to participate. Inclusion criteria were: individuals between 25 and 50 years old, who did not report any systemic or oral disease, were not immunocompromised and did not use drugs were challenged with heat-inactivated P. gingivalis at moi: 100 (1.0x10 8 CFU/mL). All samples were cultured for 1 h, at 37°C in an atmosphere with 5% CO 2 .
Next, resin monomers were added to the test group samples to a final concentration of their TC 20

Measurement of inflammatory cytokines
To analyze the production of cytokines, PBMC (0.5x10 6 cells/well) from 16 healthy individuals (9 females, 7 males) were seeded in 24-well plates and incubated under the above-mentioned conditions. Samples were cultured for 1 h, at 37°C in an atmosphere with 5% CO 2

Statistical analysis
The Shapiro-Wilk test showed whether data came from a normal distribution. All samples were submitted to a rout test to identify outliers. Data that met the criteria for parametric tests were analyzed by a Student's paired t-test or repeated-measurements one-way ANOVA, followed by Tukey's test. Data groups that failed normality tests were analyzed by Wilcoxon or Friedman test, followed by Dunn's test. Correlations between cytokine levels and cell death data were   Figure 5F). Apart from that, no other changes were observed in lymphocytes and monocytes death following incubation with this monomer, regardless of P. gingivalis stimulus. TEGDMA did not interfere with lymphocyte death ( Figure 5A).
On the other hand, TEGDMA incubation produced a statistically significant increase in monocyte death when compared with media control ( Figure 5D).
Likewise, TEGDMA associated to P. gingivalis produced a higher percentage of dead monocytes compared to P. gingivalis alone ( Figure 5D). When analyzing the percent of apoptotic monocytes, one can observe an  Cytokine release analysis PBMC were exposed to heat-inactivated P. gingivalis stimulation alone (Figures 6 and 7). Analysis of correlation between cytokine levels and cell death did not show any negative correlation that could link the great cell death to a decreased production of these cytokines. However, UDMA exposure statistically decreased the IL-1β secretion by PBMC stimulated with P. gingivalis (Figure 6A), and a negative correlation between IL-1β levels and the percentage of apoptotic lymphocytes was observed (rho= -0.9, p=0.002), indicating the decreased IL-1β production could be linked to a decreased number of cells. No influence was observed in TNF-α secretion ( Figure 6B).
In all groups, in the absence of P. gingivalis, no detectable quantities of IL-10 were observed after 5 and 20 h. While IL-6 was detected in the supernatants from cultures in all groups, no statistical differences among groups was observed. The release of IL-10 and IL-6 was not influenced neither by the treatment with P. gingivalis nor by the monomers incubation (data not shown).
* represents a significant difference (p<0.05) regarding the control group, and connecting lines represent significant differences (p<0.05) between the experimental groups   The PBMC TC 50 to TEDGMA was 3161 μM. Using primary human gingival fibroblast cells, the TEGDMA TC 50 value was 3460 μM. 16 In contrast, human THP-1 monocytes displayed TC 50 of 1500 μM. 18 Furthermore, the PBMC TC 50 to BISGMA was 69 μM, whereas other authors found 31 μM for immortal human endothelial cells. 14 UDMA exhibited a TC 50 value of 505 μM in this study, while 106 μM for primary human gingival fibroblasts was previously described. 16 Therefore, our results confirmed that cytotoxicity varied considerably depending on the cell type and on the resin monomer tested. The TEGDMA TC 50 was considerably higher than that observed for UDMA, and BISGMA presented the lowest value. This result is corroborated by other studies that observed a cytotoxicity ranking as follows: BISGMA>UDMA>TEGDMA. 6,16 However, most articles on this subject report the use of TC 50 , and the authors of this study decided to use the TC 20 because a 50% toxic concentration toxic in the cell population compromises too many cells and seems to be above the level one may expect to find in the periodontal environment exposed to resin composites. 4 This research provides experimental evidence that UDMA induces apoptosis in lymphocytes, as described by other authors. 19 It has been suggested this resin monomer induces apoptosis through DNA lesion or mitochondrial dysfunction. 19 Heat-inactivated P.
gingivalis did not induce apoptosis in lymphocytes after 5 hours of culture. This finding is similar to a previous study that tested PBMC stimulated with the same bacteria. 20 Interestingly, co-incubation of UDMA and P. gingivalis led to a statistically significant decrease in the number of apoptotic lymphocytes when compared to UDMA alone. As apoptosis can be triggered by the imbalance between pro-and anti-apoptotic intracellular molecules, it is tempting to hypothesize that P. gingivalis may interfere with the signaling pathways associated with the expression of these molecules. In fact, previous studies demonstrated that activated lymphocytes express molecules that antagonize apoptosis. 21 The pathway involved in the protective effect of heat-killed P. gingivalis on UDMAexposed PBMC is an intriguing issue to be explored.
It is worth mentioning that UDMA did not significantly affect monocytes viability, inducing lymphocyte death primarily via apoptosis.
After a short-time exposure to TEGDMA, we observed an increased monocyte death mainly via apoptosis. Such result is similar to that detected by other authors in human pulp-derived cells after 6 h of incubation. 22 Additionally, we showed that BISGMA did not significantly affect cell death via apoptosis or necrosis in lymphocytes and monocytes. As a matter of fact, the frequency of annexing V-positive cells in our experimental groups was low. Some studies demonstrated that apoptosis caused by resin monomers is time-and dose-dependent. 22,23 For instance, BISGMA exhibited a dose-dependent apoptotic effect in dental pulp cells after 24 hours of incubation. 23 Thus, an experiment with longer exposure periods might render higher rates of apoptosis related to TEGDMA and UDMA exposure and may help determine whether sub-lethal doses of BISGMA induce apoptosis in human mononuclear

leukocytes.
As demonstrated, TC 20 BISGMA, TEGDMA and UDMA did not alter cytokine production by unstimulated human mononuclear leukocytes. These findings are similar to data obtained using fibroblasts and THP-1 macrophages. 8,15 This speaks in benefit of likely favorable properties of resin monomers in low concentrations, which would be an important assurance when using resin composites in dental treatments.
After co-stimulation with bacteria, the methacrylate monomer UDMA decreased IL-1β release by PBMC.
Moreover, TEGDMA inhibited the secretion of both IL-1β and TNF-a. Previous studies using murine or immortalized cells described similar results. 7, 8,18,22 However, it should be pointed out the lower secretion of cytokines could be due to a decrease on cells able to produce then. In fact, we observed a negative correlation between IL-1β levels and the percentage of apoptotic lymphocytes incubated with UDMA. However, we did not find similar correlation between levels of TNF-a or IL-1β and cells incubated with TEGDMA, suggesting that other mechanisms could be linked to the decreased production of cytokines by PBMC cells. not expected to be released from dental fillings. 4,24 Lastly, we used heat-inactivated P. gingivalis, a major periodontal pathogen, 13 whereas other studies used LPS from E. coli. As different bacteria act differently in the same cell, triggering different signal pathways, 25 a study such as this one -analyzing bacteria with oral relevance -is important to verify their possible effects in the periodontal tissue.
IL-1β and TNF are pro-inflammatory cytokines that regulate host response. 12 In particular, IL-1β is a cytokine whose bioactivity is controlled by

Conclusion
The effect of resin monomers on human leukocytes stimulated with a periodontal pathogen, shown for the first time in this paper, indicates that sub-lethal doses of TEGDMA and UDMA induce cell death via apoptosis and/or necrosis depending on the cell type, in addition to influencing PBMC cytokine production. Our results suggest that resin monomers interfere with the local immune inflammatory response and with the tissue damage mechanisms associated with IL-1β and TNF secretion.