Development and characterization of a novel bulk-fill elastomeric temporary restorative composite

Abstract Objectives: This study investigated the physical and mechanical properties, antibacterial effect and biocompatibility of novel elastomeric temporary resin-based filling materials (TFMs) containing zinc methacrylate (ZM). Material and Methods: Experimental TFMs were prepared by mixing the zinc methacrylate with monomer, co-monomer, photoinitiator and fillers. A ZM concentration of 0 (control), 0.5% (Z0.5); 1% (Z1), 2% (Z2), or 5% (ZM5) wt% was added to the TFMs. Fermit-N (F) was used for comparison with the experimental material. Microleakage, water sorption/solubility, degree of conversion, depth of cure, ultimate tensile strength, and hardness were determined and compared. A modified direct contact test (DCT) with Enterococcus faecalis and a Streptococcus mutans’ biofilm accumulation assay was carried out to evaluate the antimicrobial effect and cytotoxicity of the assay. Statistical comparisons were performed (α=5%). Results: The results showed that the physical and mechanical properties of the experimental TFMs with ZM are comparable with the properties of the commercial reference and some properties were improved, such as lower microleakage and water sorption, and higher ultimate tensile strength values. TFMs with ZM killed E. faecalis only after 1 h. Biofilm development of S. mutans was not affected by the inclusion of ZM in the experimental TFMs. Conclusions: The present findings suggest that the physical, mechanical and biological properties of the experimental TFMs with ZM are comparable with the properties of the commercial reference. However, some properties were improved, such as lower microleakage and water sorption, and higher ultimate tensile strength values.


Introduction
Temporary filling materials (TFMs) are commonly used for coronal sealing to prevent contamination and penetration of saliva, and for microorganism products during and after endodontic treatment.
Coronal microleakage appears to be of equal or greater clinical relevance as a factor in endodontic failure than apical leakage, due to risk of recontamination. 1,2 The literature has reported presence of residual flora after this use. [3][4][5] Therefore, dental materials are expected to have low cytotoxicity and to prevent bacterial colonization and biofilm formation on their surfaces. 6 Several TFMs are available commercially, such as: reinforced zinc-oxide eugenol-based; calcium sulfatebased; composite resin-based; resin reinforced glassionomer; and traditional glass-ionomer materials. 1,7 However, all these materials have been shown to adequate if placed in a thickness of 3 mm or greater. 8 New resin-based materials were introduced as temporary restorative materials. 9,10 These materials do not provide an immediately effective seal once they undergo polymerization shrinkage of 1 to 3%. 11,12 This contraction is compensated by the fact that they swell by absorbing water. Despite this, these materials provide good initial antimicrobial properties. 12 Zinc methacrylate, 13 a monomer that has demonstrated matrix metalloproteinase 2 (MMP-2) inhibition, 14 also contains a functional methacrylate group in its structure that is found in other monomers.
The use of a copolymerizable monomer with zinc in its constitution is very promising. Zinc is a metallic chemical element with an antibacterial effect, 13 however, by the time this study was conducted, in the author's knowledge it has not been added to temporary resin-based materials.

Microleakage
Sixty recently extracted bovine incisors were used.
Cavities were prepared on the buccal surface of each tooth. The saucer-shaped cavities, measuring 3 mm in diameter and 2 mm deep were located in the middlepart of the buccal surface. Teeth were randomly divided into seven groups (n=10). After the preparations, cavities were filled with the material; covered with polyester trip and polymerized for 20 s. All materials were used according to manufacturer's instructions.
The light polymerized materials were light activated. reaching pH 7 -at 23°C for 24 h, and subjected to thermal cycling in distilled water for 1000 cycles at temperatures from 5° to 55°C, with a dwell time of 30 s. The specimens were sectioned longitudinally in the buccal-lingual plane to obtain 2 slices (7 mm thick) that would be used to assess the dye infiltration.

Depth of cure
Depth of cure was analyzed by the scraping method.
The materials (n=3) were put into a cylindrical silicone mold (6 mm diameter, 20 mm height) and irradiated with LED light curing unit through a polyester strip for 20 s. At the end of the irradiation period, the composite sample was removed from the mold and the unpolymerized material was removed from the bottom of the sample by scraping it away manually with a spatula. 16 Using a digital caliper, three samples of each material were used to determine the material mean depth of cure.

Dimensional alterations
The TFMs were placed in cylindrical silicone molds For solubility (Figure 1B), the experimental groups Z1, Z2, Z5 presented less solubility than Group F ® (p<0.05), with statistic difference observed between them (p<0.001). Table 1        were similar for different depths of cure (p=0.080), and there was no statistically significant interaction between specimens and depth of cure (P=0.402).
Group F was excluded from calculations because of the pattern of absent data.
In the S. mutans UA159 biofilm accumulation test (Figure 2), Group F showed lower CFU/mg than all the other experimental TFMs (P<0.001). With regard to the direct contact test after 1 h (Figure 3A), Group F showed lower antibacterial effect than the other groups (p<0.001). Z0.5 and Z5 were similar to control and Z2, and Z1 was the group that showed the highest bacteria reduction after 1 h, not being, however, statistically significant (p>0.05). After the 24 h in direct contact test, all TFMs with ZM were statistically similar (p=0.058).
The results of the MTT assay are presented in Figure   4. According to statistical analysis, only Z5 were shown to present higher cytotoxicity after 24 h of exposre in DMEM (p=0.044).

Discussion
Temporary resin-based materials should have satisfactory mechanical properties to be accepted as a successful temporary material. The results obtained in this investigation indicated that TFMs with ZM had suitable mechanical properties that were at the same level as, or superior to, the properties of a commercial reference (F), depending on ZM concentration.
The incorporation of ZM improved some properties such as ultimate tensile strength, water sorption and microleakage. The null hypothesis was partially accepted, since the experimental material containing zinc methacrylate provided antibacterial effect similar to that of the control in the DCT assay.
Z5 was shown to have the least microleakage, and its properties may be effective for sealing the edges of the tooth. Furthermore, Z5 showed the largest amount of water sorption and least microleakage. This is explained because the TFMs undergo polymerization shrinkage and, after contact with water, they swell.
Moreover, the higher sorption of experimental TFMs with ZM was most likely due to the presence of Zn +2 , which form a network of Zn bonds and ZnOH in the polymer chains of the resin-based temporary material in the presence of water. In a more heterogeneous network, the space created between the polymer clusters (microporous) is larger and accommodates a higher amount of water. 15 In the DC test of the TFMs, no significant difference was observed between the control and the other groups containing ZM. The incorporation of ZM did not affect the DC, and these results are contrary to other studies, 13 probably due to the differences in composition of the materials assessed in these studies.
In the present study, the authors used exothane 8 ® . inhibiting the active transport and metabolism of sugars, as well as disrupting enzyme systems of dental biofilms by exchanging magnesium ions essential for the enzymatic activity of plaque. 26 Zinc can also reduce the acid production of S. mutans biofilms due to its ability to inhibit glucosyl transferase activity. 27,28 A point to consider is the fact that this is the first study that has evaluated zinc methacrylate in models of S. mutans biofilm. It has already been indicated in the literature that biofilms are organized in communities of microorganisms coated with extracellular polysaccharides and are more resistant than bacteria without that organization. This could explain the effect of (10-30 wt%) zinc methacrylate on a biofilm model with S. mutans UA159. 13 The DCT is a test for evaluating solid materials that have components with low solubility. In the DCT method, bacteria are under a previously controlled direct contact with the desired material, and after this, bacterial growth can be quantified. 21 In this study, the authors assessed two-timed intervals of TFMS exposure to E. faecalis: periods of 1 and 24 h.
The results provided on Figure 3A showed that TFMs can exert antibacterial effect against E. faecalis after 1 h of exposure at the concentrations of Z0.5, Z1 and Z5 tested. These results could not be compared with those of other studies because zinc was a functionality of methacrylate. After 24 h, all groups were similar, probably because of the antimicrobial effect of the zinc components of TFMs, or because there was a higher bacterial growth that resulted in zinc ion saturation in the media, and lack of nutrients causing the death of bacteria in the different groups tested.
The antibacterial effects of Zn are known, 25 however, attempting to explain the antimicrobial mechanism of ZM only by the leaching of Zn 2+ cannot be expected with this study, probably due to the difficulty of releasing the Zn ion, which can be supported by the data regarding the degree of conversion and solubility.
However, a considerable antibacterial activity of ZnO has also been attributed to their biological effect to the generation of reactive oxygen species on the surface of these oxides. The advantage of using this inorganic oxide as antimicrobial agent is that they contain mineral elements essential to humans and exhibit strong activity even when administered in small amounts. This activity is quantitatively evaluated by studying the growth medium caused by the bacterial metabolism. In this case, the cell wall rupture of bacteria must be due to the surface activity of ZnO in contact with the bacteria. 29 For the cytotoxicity test, the Z5 showed lower cell viability than all the other groups. This could be explained by the higher concentration of ZM. However, in this study, functionalized zinc was used. The results showed that the control material (ZM-free) had no cytotoxic effect on cells in the MTT test.

Conclusions
These novel composites based on zinc methacrylate exhibited promising elastomeric features and better sealing capacity, likely provided by increased water sorption as a compensatory feature with regard to composite shrinkage. In addition, in the direct contact test (DCT), the test TMF composites showed biocompatibility and an antibacterial effect against E. faecalis.