In Vitro Biocompatibility of Preheated Giomer and Microfilled-Hybrid Composite.

OBJECTIVE: The aim of this study was to evaluate cytotoxic potencies of two light cured composite materials after heating on different temperatures and cured directly and through CAD/CAM overlay.
MATERIALS AND METHODS: Composite materials (microfilled-hybrid Gradia Direct Posterior and Beautifil II) were heated in a Calset warming unit at three different temperatures (T1:37°C, T2:54°C, T3:68°C). A small amount of heated composite material was placed in a round mold (diameter 6mm; 0.65mm thick), covered with Mylar sheet, pressed and polymerized with Bluephase LED unit. One group of samples were polymerized directly, and the other group through 2mm thick CAD/CAM ceramic-reinforced polymer (CRP) and CAD/CAM lithium disilicate ceramic (LDC) overlay for 20 and 40 seconds. The polymerized samples were placed immediately after curing in a lymphocyte cell culture. The viability of peripheral blood lymphocytes was evaluated using a dye exclusion technique by simultaneous staining with ethidium bromide and acridine orange. Quantitative assessments were made by determination of the percentage of viable, apoptotic and necrotic cells. The Pearson chi-square test was used for statistical analysis.
RESULTS: In case of 20 seconds polymerization, the highest number of viable cells polymerization were recorded when materials were heated at 37°C (T1), while in case of 40 seconds polymerization, the highest number of viable cells were recorded when the materials were heated at 54°C (T2). The samples polymerized through CAD/CAM overlays showed less cytotoxicity than samples polymerized directly.
CONCLUSION: Apart from composite material composition, the cell viability was also influenced by curing time, temperature of pre-heating and polymerization pattern.

Introduction

Composite materials have been successfully used for many years as dental restorative materials due to their mechanical and excellent esthetic properties. They are used in everyday clinical practice not only as restorative materials but also as liners or as luting agents for cementation of inlays, onlays, crowns, veneers, and orthodontic brackets (). With CAD/CAM appearance on the market, the use of composite materials as a luting material becomes more popular. Besek et al. () first introduced the use of composite materials for CAD/CAM bonding at the time when CAD/CAM system was not so accurate. Due to mechanical properties of composite materials and extended curing time Besek et al. () introduced composite materials as a luting agent with the aim to prevent microleakage, postoperative sensitivity, recurrent caries and to improve overall esthetic appearance of a cemented restoration. When using composite materials as a luting agent, the bond strength is determined by achieving appropriate resin polymerization. Daronch et al. () reported in their study that increasing composite temperature up to 60° C might enhance the degree of conversion on the top of the composite material and in 2 mm of the bottom surfaces as well. Preheating of a composite material under an isothermal condition allows increasing monomer conversion which as a consequence has reduction of the free volume within polymer network, solvent uptake and material degradation, which can lead to less cytotoxicity of the polymerized material (-). However, some studies reported that pre-heating of a composite material may cause negative effects on the composite restoration margins due to the increased polymerization shrinkage in heated composite resin (). On the contrary, some studies showed that pre-heating of a composite material before light-curing did not alter mechanical properties and monomer conversion but did provide enhanced adaptation of composite materials to the cavity walls (). Deb et al. () confirmed in their study that a pre-heated composite material produced more shrinkage than non-heated composite, but still less than flowable composite materials. The same authors also concluded that there is no difference between cytotoxicity of heated and non-heated composite materials. Reactive components released from unpolymerized or underpolymerized composite resins may induce toxicity or inflammatory tissue responses depending on their aggressiveness and the thickness of the remaining dentin on the cavity floor (, ).

The aim of the present investigation has been to evaluate and compare cytotoxic potencies of one micro hybrid composite material (Gradia Direct Posterior) and one giomer composite material (Beautifill II) after heating at different temperatures and cured directly and through CAD/CAM overlay for 20 and 40 seconds. Isolated human peripheral lymphocytes were used as a model system. For the purpose of the study following null hypotheses were established:

The Pearson chi-square test was used.

Materials heated on lower temperature will cause less cytotoxicity regardless of the curing time and polymerization pattern (directly polymerized or through CAD/CAM overlay)

Longer curing time will cause less cytotoxicity regardless of the curing time and polymerization pattern (directly polymerized or through CAD/CAM overlay)

Samples polymerized directly will show less cytotoxicity than the samples polymerized through CAD/CAM overlay regardless of the temperature used.

Materials and methods

Preparation of CAD/CAM ceramic-reinforced polymer (CRP), CAD/CAM lithium disilicate ceramic (LDC), giomer and composite samples

Two blocks of the CRP CAD/CAM (shade A2, block size 14L, 3M ESPE, LAVA Ultimate, St. Paul, MN, USA), and LDC CAD/CAM (shade A2, block size 14L, e.max, Ivoclar Vivadent, Amherst, NY, USA) were used for preparing overlays for polymerization of composite materials.

CRP and LDC CAD/CAM blocks were cut using a low speed diamond saw in 2 mm thick slices. Thereafter, the samples were polished under water-cooling on both sides with a polishing machine (). For LDC CAD/CAM samples, each sample was coated with glaze (Crystall/Glaze Spray; Ivoclar/Vivadent, Schaan, Liechtenstein) and placed in in the oven Pro 100 (Whip-Mix; Louisville, KY) according to the manufacturer’s instructions.

Beautifill II (SHOFU Dental GmbH; Ratingen, Germany) is a nano-hybrid composite which belongs to the Giomer group. According to the manufacturer’s data, it contains Surface Pre-Reacted Glass (S-PRG) filler particles which are capable of releasing fluoride, sodium, strontium, aluminum, silicate and borate ions, bisphenol A-diglycidyl dimethacrylate (Bis-GMA) and tryethyleneglycol dimethacrylate (TEGDMA).

Gradia Direct Posterior (GC, Europe N.V.; Leuven, Belgium) is a micro-filled hybrid resin composite. According to the manufacturer’s data, it contains microfine pre-polymerized resin fillers (silica 19 wt % average particle size 0.85 µm; pre-polymerized filler 20 wt %). The matrix consists of a mixture of urethane dimethacrylate (UDMA) and dimethacrylate co-monomers (23 wt %). Fluoro-Alumino-Silicate glass is added to the posterior formulation to obtain radiopacity (38 wt %).

The Calset Composit Warmer (AdDent INc., Danbury, Connecticut, USA) was used for heating of tested composite materials. Manufacturer suggested temperature for warming composite materials in syringe is 68 oC. In this experiment three temperature levels were used: temperature T1: 37 °C, T2: 54 °C and T3: 68 °C. A tray placed on the top of the heating unit contains slots to place the composite compules. The desired temperature is reached in approximately 10 minutes and the composite is ready to use.

The samples for cytotoxicity testing were prepared as follows: small amount of tested material, pre-heated at T1, T2 or T3, was placed in a mold of 6 mm in diameter and 0.65 mm in thickness. The mold was positioned on stainless steel round 5 mm thick disc, which was covered with Mylar sheet. The mold was then filled with the composite material. The sample was covered with another Mylar sheet and pressed with another stainless steel round 5 mm thick disc to obtain homogenous and exact thickness of the sample (0.65 mm) (). The Mylar sheet was used in order to prevent the formation of an oxygen inhibited layer on the surface of polymerized composite material. After the sample had been pressed with the hand, the stainless steel was removed and Gradia Direct Posterior or Beautifil II samples were polymerized with Bluephase light curing unit (Vivadent, Schaan, Lichtenstein) using high intensity polymerization mode (1180 mW/cm2) for 20 and 40 seconds, respectively. Seven samples were made for each single tested group.

Three modes of sample polymerization were used:

Direct polymerization through the Mylar sheet,

Polymerization through the Mylar sheet overlaid with 2 mm CAD/CAM CRP overlay,

Polymerization through the Mylar sheet overlaid with 2 mm thick CAD/CAM LDC overlay.

So prepared samples were removed from the mold and the Mylar sheet and placed directly into the lymphocyte cell cultures.

Primary lymphocyte cultures

This study was approved by the Ethical Committee, School of Dental Medicine, University of Zagreb, Croatia. To overcome possible inter-individual differences in response to the treatment, a blood sample was obtained from one healthy male donor (age 39 years, non-smoker), with no medical records of chronic or acute adverse health conditions. Prior to blood sampling, the donor was acquainted with the procedure, purpose of blood donation, and the aim of blood testing. He signed an informed consent. Venous blood (40 ml) was collected under sterile conditions in heparinized vacutainer tubes (Becton Dickinson, UK) containing lithium heparin as anticoagulant. Lymphocytes were freshly isolated using the Histopaque-1077 reagent (Sigma Chemical Co., St. Louis, MO, USA) according to the manufacturer’s instructions. Following the isolation, 50,000 lymphocytes were seeded in sterile tubes (Nange Nunc Int, Naperville, IL, USA) in RPMI 1640 culture medium with penicillin and streptomycin (Gibco Invitrogen, Paisley, UK). The final culture volume was 7 ml. No newborn calf serum or mitogen was added. Each culture was treated for 24 h with 0.06 g of unpolymerized or polymerized tested material at 37 oC in a 5% CO2 atmosphere (Heraeus Hera Cell 240 incubator, Langenselbold, Germany). The same study design has been proved in our previous investigation (-).

Quantitative fluorescent assay for the assessment of cell viability, apoptosis and necrosis

After 24 hours of treatment, the cultures were centrifuged at 600 rpm for 10 minutes, supernatant was removed and the remaining pellet was gently re-suspended. Aliquots of lymphocyte suspension (V=20 μl) were pipetted, put on the microscope slide and mixed with the same volume of ethidium bromide and acridine orange dyes (Sigma-Aldrich, USA), prepared in final concentrations of 100 µg/ml (1:1; v/v). After covering the preparation with a coverslip, lymphocyte viability was immediately evaluated under a fluorescence microscope (Olympus BX 51; 400 x magnification; Olympus, Tokyo, Japan), by applying a dye exclusion method (). Quantitative assessments were made by determination of the percentage of viable, apoptotic and necrotic cells. Viable cells with intact plasma membrane excluded ethidium bromide and the appearance of their nuclei with an intact structure was bright green. Non-viable necrotic cells had orange to red colored chromatin with organized structure, while apoptotic cells were bright green with highly condensed or fragmented nuclei. Three tests with aliquots of the same sample were performed and a total of 300 cells per sample were counted. The untreated lymphocyte culture was studied in parallel as a control group.

Comparisons between values obtained for the cell viability treated and control samples were made by the Pearson’s χ2 test for two-by-two contingency tables. Statistical decisions were made at a significance level of p < 0.05.

Results

The results of the quantitative fluorescent assay for simultaneous identification of apoptotic and necrotic cells in lymphocyte samples incubated with non-polymerized and polymerized Beautifil II are shown in Table 1 and 2.

Table 1

Results of the quantitative fluorescent assay for simultaneous identification of apoptotic and necrotic cells. Lymphocytes were treated in vitro for 24 hours with unpolymerized and polymerized composite material Beautifill II. Control, non-treated cells were studied in parallel.

Material – Beautifil II	Viable cells (%)	Non-viable cells (%)
		Σ	Apoptosis	Necrosis
Control	97.3±1.5	2.7±1.5	2.0±1.0	0.7±1.2
Unpolymerized	16.0±6.6	84.0±6.6	24.3±3.5	59.7±8.7
Polymerized for 20 seconds
D–T1	87.7±2.1	12.3±2.1	8.0±1.0	4.3±1.2
D–T2	56.7±10.7	43.0±11.1	22.3±11.1	20.7±0.6
D–T3	78.7±4.2	21.3±4.2	17.0±4.6	4.3±2.5
CRP–T1	92.0±1.0	8.0±1.0	5.7±2.1	2.3±1.2
CRP–T2	82.7±8.6	17.3±8.6	14.0±6.2	3.3±2.5
CRP–T3	68.3±8.0	31.8±8.0	15.0±9.5	16.7±3.8
LDC–T1	90.3±1.5	9.7±1.5	4.3±3.2	5.3±2.1
LDC–T2	64.3±3.2	39.0±8.2	22.7±1.5	13.0±2.0
LDC–T3	81.0±7.5	19.0±7.5	13.3±7.5	5.7±1.2
Polymerized for 40 seconds
D–T1	83.3±2.1	16.7±2.1	9.7±2.3	7.0±1.7
D–T2	67.0±6.6	33.0±6.6	11.7±4.7	21.3±8.4
D–T3	72.7±5.0	27.3±5.0	17.3±6.5	10.0±6.2
CRP–T1	75.7±3.8	24.3±3.8	14.3±1.5	10.0±3.0
CRP–T2	81.0±1.0	19.0±1.0	11.3±3.5	7.7±4.2
CRP–T3	74.0±7.8	26.0±7.8	11.0±3.6	15.0±4.4
LDC–T1	75.7±2.5	24.3±2.5	12.3±0.6	12.0±2.0
LDC–T2	84.3±1.5	15.7±1.5	11.3±0.6	4.3±1.5
LDC–T3	77.0±4.6	23.0±4.6	12.0±1.7	11.0±4.6

Note.

D-directly polymerized; T1, T2, T3 – polymerization temperatures; CRP-CRP CAD/CAM overlay; LDC-LDC CAD/CAM e.max overlay

300 cells per sample per each experimental point were analysed.

Statistical significance of data was evaluated using Pearson χ2 test.

Table 2

Results of the quantitative fluorescent assay for simultaneous identification of apoptotic and necrotic cells. Lymphocytes were treated in vitro for 24 hours with unpolymerized and polymerized composite material Gradia Direct Posterior. Control, non-treated cells were studied in parallel.

Material – Gradia Direct Posterior	Viable cells (%)	Non-viable cells (%)
		Σ	Apoptosis	Necrosis
Control	97.3±1.5	2.7±1.5	2.0±1.0	0.7±1.2
Unpolymerized	74.0±11.1	26.0±11.1	15.7±11.1	10.3±0.6
Polymerized for 20 seconds
D–T1	83.0±1.0	17.0±1.0	8.7±1.5	8.3±0.6
D–T2	74.3±6.1	25.7±6.1	15.7±5.1	10.0±1.0
D–T3	67.0±9.5	33.0±9.5	17.7±9.6	15.3±4.0
CRP–T1	73.0±2.6	27.0±2.6	16.3±3.5	10.7±5.7
CRP–T2	70.7±3.8	29.3±3.8	11.3±2.3	18.0±3.0
CRP–T3	75.3±5.5	24.7±5.5	13.7±1.2	11.0±4.4
LDC–T1	83.3±2.3	16.7±2.3	14.7±2.9	2.0±2.6
LDC–T2	75.3±5.5	24.7±5.5	11.7±3.1	13.0±4.4
LDC–T3	79.0±3.6	21.0±3.6	11.7±3.8	9.3±0.6
Polymerized for 40 seconds
D–T1	68.3±1.5	31.7±1.5	12.0±2.6	19.7±3.1
D–T2	78.7±7.8	21.3±7.8	13.3±2.3	8.0±8.7
D–T3	75.7±5.1	24.3±5.1	11.7±4.2	12.7±4.9
CRP–T1	81.0±2.6	19.0±2.6	10.7±1.5	8.3±1.5
CRP–T2	90.0±2.0	10.3±1.5	7.0±1.0	3.0±2.6
CRP–T3	79.7±5.0	20.3±5.0	10.3±4.0	10.0±1.0
LDC–T1	83.0±1.7	17.0±1.7	12.7±2.1	4.3±0.6
LDC–T2	84.0±2.0	16.0±2.0	9.0±2.0	7.0±0.0
LDC–T3	81.0±6.9	19.0±6.9	13.0±4.4	6.0±2.6

Note.

D-directly polymerized; T1, T2, T3 – polymerization temperatures; CRP-CRP CAD/CAM overlay; LDC-LDC CAD/CAM e.max overlay

300 cells per sample per each experimental point were analysed.

Statistical significance of data was evaluated using Pearson χ2 test.

Polymerization time 20 seconds

Cytotoxicity profiles of both tested materials are shown in Figure 1. Unpolymerized Beautifil II had a significantly higher cytotoxicity than Gradia Direct Posterior (P<0.0001). After direct polymerization at T1 and T3, Beautifil II had lower cytotoxicity than Gradia Direct Posterior, but the difference was statistically significant only at T3 (P=0.0013). At this polymerization temperature, Gradia Direct Posterior caused a significantly higher frequency of lymphocyte necrosis than Beautifil II (P<0.0001). Quite opposite results were obtained at T2, when Gradia Direct Posterior showed a significantly lower cytotoxicity than Beautifil II (P<0.0001, due to lower frequency of necrotic cells, P=0.0003). After polymerization through CRP CAD/CAM overlay at T1, Gradia Direct Posterior was more cytotoxic than Beautifil II (P<0.0001) due to increased frequencies of both apoptotic and necrotic cells P<0.0001). Similar results were obtained at T2: Gradia Direct Posterior was more cytotoxic than Beautifil II (P=0.0005) due to higher frequency of necrotic cells, P=0.0001). At T3, Beautifil II was more cytotoxic than Gradia Direct Posterior, but the difference was not statistically significant. After polymerization through LDC CAD/CAM overlay at T1, Gradia Direct Posterior was more cytotoxic than Beautifil II (P=0.0112) due to increased frequencies of apoptotic cells, P<0.0001 and necrotic cells P=0.0298). Quite opposite was found at T2, when Gradia Direct Posterior showed a significantly lower cytotoxicity than Beautifil II (P=0.0112). At T3, Gradia Direct Posterior was slightly more cytotoxic than Beautifil II, but the difference was not statistically significant.

Figure 1

Comparison of cytotoxicity between Beautifil II and Gradia Direct Posterior polymerized for 20 seconds. Percentages of viable, apoptotic and necrotic cells were determined using the quantitative fluorescent assay after simultaneous staining with ethidium bromide and acridine orange. DP-directly polymerized; P-polymerized at temperatures T1-T3; CRP-CRP CAD/CAM overlay; LDC-LDC CAD/CAM overlay

Polymerization time 40 seconds

Cytotoxicity profiles of both tested materials are shown in Figure 2. Unpolymerized Beautifil II had significantly higher cytotoxicity than Gradia Direct Posterior (P<0.0001). After direct polymerization at T1, Beautifil II had a significantly lower cytotoxicity than Gradia Direct Posterior (P<0.0001). This was mostly influenced by an increased frequency of lymphocyte necrosis. At two higher polymerization temperatures, Gradia Direct Posterior showed lower cytotoxicity than Beautifil II, which was statistically significant at T2 (P=0.0013) due to an increased frequency of necrotic cells, P<0.0001). Polymerization through both overlays contributed to lowering of cytotoxicity, and better results in both cases were obtained for Gradia Direct Posterior. Polymerization of Beautifil II and Gradia Direct Posterior through CRP CAD/CAM overlay at T1 and T2 resulted with higher lymphocyte viability, as compared with T3. A statistically significant difference was recorded only between two tested materials at T2 (P=0.0017). In this case, Beautifil II caused more lymphocyte necrosis as compared to Gradia Direct Posterior (P=0.0082). After polymerization through LDC CAD/CAM overlay at T1, Beautifil II was more cytotoxic than Gradia Direct Posterior (P=0.0265) due to increased frequency of necrotic cells P=0.0006). At T2 their cytotoxicity was similar. At T3 Beautifil II was again more cytotoxic than Gradia Direct Posterior, but the difference was not statistically significant.

Figure 2

Comparison of cytotoxicity between Beautifil II and Gradia Direct Posterior polymerized for 40 seconds. Percentages of viable, apoptotic and necrotic cells were determined using the quantitative fluorescent assay after simultaneous staining with ethidium bromide and acridine orange. DP-directly polymerized; P-polymerized at temperatures T1-T3; CRP-CRP CAD/CAM overlay; LDC-LDC CAD/CAM overlay

Discussion

The present study reports results regarding the in vitro assessment of cytotoxic potencies of one micro hybrid composite material (Gradia Direct Posterior) and one giomer composite material (Beautifill II) polymerized at three temperatures for two time periods. Peripheral blood lymphocytes were used as a model system in the present study. This test system is well-established in in vitro toxicology. Lymphocytes are easily available and proven to be good surrogate cells in different testing conditions. The most important fact is that lymphocytes are primary cells. Since an in vivo situation is generally better-simulated by primary cultures (, , ), such experimental design seems to be appropriate for the assessment of biocompatibility of composite materials as it was performed in the present study.

Although both studied materials possessed certain degrees of cytotoxicity, our results suggest that in the experimental conditions as applied here, Gradia Direct Posterior was more biocompatible material than Beautifill II. The obtained results have also shown that, apart from material composition, cell viability was also influenced by curing time, temperature of pre-heating and the polymerization pattern. It has to be stressed that greater cell viability was observed after polymerization of both materials through CAD/CAM overlays, as compared to direct polymerization.

To assess the lymphocyte viability in this study, we applied a rapid viability assay with acridine orange and ethidium bromide, which allowed for counting the fractions of viable, apoptotic and necrotic cells based on the cell morphology, nuclear and chromatin disintegration. While control lymphocytes showed intact morphology, in late apoptotic cells we observed membrane blebbing, fragmentation of nuclei and formation of apoptotic bodies. In necrotic cells, on the other hand, more irregular chromatin destruction was noticed, along with vacuole formation in the cytoplasm. Due to breakdown of the plasma membrane, necrotic cells accumulated ethidium bromide and their chromatin thus was stained bright red, as reported in the literature (). On the whole, our fluorescent microscopic findings suggest that the cytotoxic effects of both tested materials in their polymerized forms have been predominantly mediated by apoptosis. Such results are important from the clinical point of view as apoptosis represents a well-controlled, and tightly-regulated physiological process, which does not result in inflammation around the dying cell, in contrast to necrosis (-). The highest percentage of necrotic cells (about 60%) was found in the sample incubated with unpolymerized Beautifill II, which resulted in 84% of dead cells after 24 hours of in vitro exposure. The most important are induction of lipid peroxidation, glutathione depletion, downregulation of glutathione peroxidase levels and increase of intracellular calcium levels.

From the dentists’ point of view, using heated composite material as a luting agents for inlay and onlay restorations has advantage regarding prolonged handling time, easier removal of excess of the material, better sealing of unideal fitting of the restoration as it is documented in some clinical studies (, -). Dab et al. () measured shrinkage in pre-heated and non-heated composite and concluded that despite a shrinkage increase, this increase may not be significant in clinical scenarios. This may also occur due to drop of the material temperature, while the material is taken from the heating unit and placed in the cavity. Daronch et al. () observed the behavior of composite materials heated in Calset heating unit at different temperatures and noticed a rapid decrease in composite temperature after the removal from the heating unit. They reported a drop of 50% in material temperature 2 minutes after the removal from the heating unit (). Regardless of whether the pre-heated or non-heated composite materials are used, it is essential to cure composite material properly. A previous study () demonstrated that pre-heated composite allows for a shorter time of light exposure with a similar degree of conversion rate than when the composite is irradiated for a longer exposure time at a room-temperature.

The first working hypothesis of this study stating that materials heated on lower temperature will cause less cytotoxicity regardless of the curing time and polymerization pattern (directly polymerized or through CAD/CAM overlay) was accepted in all cases except in the case of polymerization of Gradia Direct Posterior composite through CRP overlay for 20 seconds. For Beautifil II composite, the highest number of viable cells was recorded in the case of 20 seconds polymerization and heating temperature T1 for direct polymerization and polymerization through both CAD/CAM overlays and for 40 seconds direct polymerization. For 40 seconds, Beautifil II polymerization through CAD/CAM overlays samples heated at temperature T2 showed the highest number of viable cells. For Gradia Direct Posterior composite material, all samples polymerized for 40 seconds showed the highest number of viable cells when the material was heated at temperature T2, while for 20 seconds polymerization, the highest number of viable cells were recorded when the material was heated at temperature T1, apart from polymerization through CRP CAD/CAM overlay as stated before.

The second hypothesis stating that longer curing time causes less cytotoxicity regardless of the curing time and polymerization pattern (directly polymerized or through CAD/CAM overlay) was accepted for Gradia Direct Posterior composite. For Beautifil II, this hypothesis was accepted in the case of material heating at the highest temperature, T3 (68 oC), but it was rejected at two lower temperatures where higher numbers of viable cells were recorded when the samples were polymerized for 20 seconds.

The third working hypothesis was that samples polymerized directly would show less cytotoxicity than the samples polymerized through CAD/CAM overlay regardless of the temperature used was rejected for both materials and both curing times. It would be expected that the material which is cured directly will possess better curing quality and less unreacted components left after curing. Our results, however, did not speak in favor of this assumption. Obviously, there is another reason that might influence the number of viable cells in cultures that were incubated with directly polymerized material, and that is most likely the temperature. If this was the case, a higher temperature produced from the curing unit in direct contact with the Mylar sheet which was covering the material could contribute to an increase in lymphocyte cytotoxicity. Findings of our previous study about the influence of curing mode intensity on cytotoxicity of composite materials () are in agreement with the results of the present study. Nevertheless, the reasons behind lymphocyte death following incubation with samples that were polymerized using different modes should be investigated more thoroughly in future studies.

The results also showed that polymerization through both CAD/CAM overlays contributed to lowering of cytotoxicity of the tested material, and better results in both cases were obtained for Gradia Direct Posterior composite material in case of 40 seconds polymerization. Explanation for this may be in the different organic structure of each material: Gradia Direct Posterior has UDMA in its composition which should lead to less cytotoxicity, while Bis-GMA, which is present in Beautifil II material according to the literature, causes more cytotoxicity (). Tadin et al. () tested genotoxicity of Gradia Direct Posterior and found that Gradia Direct Posterior show higher cytotoxicity after five days than after first day which was explained by gradual release and biodegradation of UDMA.

Beautifil II contains a surface pre-reacted glass (S-PRG) filler that has been shown to possess acid neutralization capabilities and inhibition of plaque formation according to the manufacturer data. Glass-ionomers and compomers require water absorption after light curing in order to release fluoride ions. Conversely, giomers contain a multifunctional glass core which undergoes an acid-base reaction during manufacturing procedure and is protected by surface modified layer. Restorative materials with potential fluoride release when placed in a cavity may serve as a fluoride reservoir and lead to low fluoride release, thus increasing the fluoride level in oral fluids and preventing the dental caries. Madhyastha et al. () tested the fluoride release at different temperatures and concluded that it is highest at the temperature of 55 °C. It is interesting that Beautifil II in this study showed the highest number of viable cells at temperature T1 (37 °C) and T2 (54 °C). The same authors also concluded that the highest amount of fluoride release occurs the first day after application followed by the days 7 and 14 with least release after 28 days. Some other studies also showed that the cytotoxicity level drops with time in the same manner (). The findings of Madahystha et al. () suggest that pre-heating of this material prior to placement in the cavity will accelerate and facilitate the fluoride release. Potentially, the use of this material for luting of CAD/CAM may be beneficial due to fluoride release of this material. However, although the results of this study have clinical implications, further clinical research is needed to implement this material as a potential luting agent for CAD/CAM restorations.

Conclusion

Bearing in mind the fact that similar studies on this topic have been rare, our findings provide a preliminary insight into the cytotoxicity of micro hybrid composite material (Gradia Direct Posterior) and giomer composite material (Beautifill II) toward human non-target cells. Since these findings are observed on a cell culture system, they cannot be directly extrapolated to in vivo situations. However, our results make a solid frame for designing future studies with the same materials aiming to further clarify mechanisms involved in their cytotoxic action.