Evaluation of physical-mechanical properties, antibacterial effect, and cytotoxicity of temporary restorative materials

Abstract The objective of this study was to compare selective physical-mechanical properties, antibacterial effects and cytotoxicity of seven temporary restorative materials (TRM): five resin-based materials [Bioplic (B), Fill Magic Tempo (FM), Fermit inlay (F), Luxatemp LC (L) and Revotek LC (R)], and zinc oxide-eugenol cement (IRM) and glass ionomer cement (GIC) as the controls. Material and methods The physical-mechanical properties were evaluated by determining microleakage (ML), ultimate tensile strength (UTS) and Shore D hardness (SDH). In addition, the polymerization rate (Pr-1), depth of cure (DC), water sorption and solubility (WS/SL) were evaluated. The antimicrobial effects of the materials were assessed by biofilm accumulation of Streptococcus mutans (BT) and the direct contact test (DCT) by exposure to Enterococcus faecalis for 1 and 24 h, and cytotoxicity by MTT assay. The data were analyzed by ANOVA or Kruskall-Wallis tests, and a complementary post-hoc method (p<0.05). Results Group B, followed by FM and GIC had significantly lower percentages of microleakage in comparison with the other groups; Groups FM and L showed the highest WS, while Groups R and FM showed the significantly lowest SL values (p<0.05). Group R showed the statistically highest UTS mean and the lowest DC mean among all groups. Group F showed the lowest S. mutans biofilm accumulation (p=0.023). Only the Group L showed continued effect against E. faecalis after 1 h and 24 h in DCT. The L showed statistically lower viability cell when compared to the other groups. Conclusions These findings suggest the antibacterial effect of the temporary materials Fill Magic and Bioplic against S. mutans, while Luxatemp showed in vitro inhibition of S. mutans biofilm accumulation and E. faecalis growth. Regarding the cell viability test, Luxatemp was the most cytotoxic and Fill Magic was shown to be the least cytotoxic.


Introduction
Temporary restorative materials are commonly used to seal the access cavity during the periods between visits and after the completion of endodontic therapy, their main function, both during and after the treatment, is sealing and preventing coronal microleakage. 1 Despite the use of intracanal dressing between endodontic therapy appointments, some studies have reported the presence of residual intracanal microorganisms after this procedure. [2][3][4] Temporary filling materials with good sealing ability and bactericidal properties may be advantageous to prevent bacterial invasions after an endodontic treatment. These materials can be divided into different groups according to their composition: reinforced zinc oxide-eugenol-based; calcium sulfatebased, resin-based composites; resin-modified glassionomer; and traditional glass-ionomer materials. 5 Generally, all these materials are adequate if placed in a thickness of 3 mm or greater. 6 Recently, new resin-based filling materials were introduced as temporary restorative materials. [6][7] These materials contain monomers, initiator systems, fillers and additives. Resin-based temporary materials must be bonded to provide an effective seal, because they undergo polymerization shrinkage of 1 to 3%. [8][9][10][11] This contraction is compensated by the fact that they swell by absorbing water. These materials provide the best initial seal usually, 7 but they lack antimicrobial properties. 8 The antibacterial properties of restorative materials have been evaluated in vitro using various methods, and the agar diffusion test (ADT) was the standard assay in most of these studies, despite its limitations.
Weiss, et al. 12 (1996) introduced a direct contact test (DCT) that quantitatively measures the effect of direct and close contact between the test microorganism and the tested materials, regardless of the solubility and diffusivity of their components. [13][14][15][16][17][18] The goal of this study was to investigate the physical-mechanical properties, antibacterial effects and cytotoxicity of seven different temporary fillings, as these may decrease the risk of caries development and failure of endodontic therapies.

Materials and methods
The materials tested in this study are described in Figure 1.   Ltda., São Paulo, SP, Brazil). Two coats of nail varnish were applied on the tooth surfaces, except for the restoration, and a distance of 2 mm around their margins. The specimens were immersed in methylene blue solution at 23°C once more, for 10 min, and then washed in tap water for the same time and dried. The specimens were serially sectioned longitudinally in the buccal-lingual plane to obtain two (7 mm thick) slices that would be used to assess dye infiltration.

Depth of cure (DC)
Depth of cure was analyzed by the scraping method.
The materials were put into a cylindrical silicone mold

Hardness measurements
The measurements were made in accordance with ASTM D2240 using the Shore D hardness (SDH)

Biofilm accumulation test
Streptococcus mutans UA159 is one of the major bacterial species responsible for dental caries, 20  The CFU were counted and CFU/mL was calculated. 23 The experiments were performed in duplicate.

Microbiological effect
The development of S. mutans in biofilm was significantly affected by the materials (Figure 3), except for group F (p=0.023). The results of the DCT test after 1 h showed that groups L and B were significantly more potent bacterial growth inhibitors than the other materials (p<0.001).

Discussion
In vitro tests remain an indispensable method for initial screenings of dental materials and setting a theoretical maximal amount of leakage that could be present in vivo. 25 Dye leakage is the cheapest and fastest method to test the sealing ability of restorative materials. 26 26 In this study, immersion in methylene blue solution 0.5% for 24 h was adopted.
These parameters were reported in a systematic review about microleakage tests as the most commonly found Regarding polymerization kinetics, group L was not considered in the results because it was not possible identify a peak (1610 cm −1 ) for analysis.
The polymerization rate of the other materials was evaluated and kept stable after 20 s of light polymerization. In Figure 2D, the highest rate of polymerization can be observed to occur in the first 5 s, probably indicating that more than one co-initiator may be used in their composition. 29 These materials have advantages, such as polymerization occurring in an increment exceeding 4 mm. This property was evaluated by the depth of cure, and we must highlight that in all materials, more than 4 mm was polymerized, except for group R. was the standard assay in most studies. 23 The direct contact test (DCT) is a reproducible method that simulates the contact of the tested microorganisms with the material. This test provides information on bacterial viability and growth rate, 32 by allowing the number of the viable bacteria to be estimated after incubation periods in direct contact with the material.
In endodontics, the difficulties to eliminate E. faecalis from the root canal system may be related to its ability to penetrate into dentinal tubules and organize itself into biofilms. The antibacterial activity of materials may help to eliminate microorganisms present in the root canal, thus, improving the success of endodontic treatments.
After 24 h of DCT, group L showed the strongest antibacterial activity against E. faecalis, followed by Evaluation of physical-mechanical properties, antibacterial effect, and cytotoxicity of temporary restorative materials J Appl Oral Sci. 2018;26:e20170562 9/10 group FM. The lowest antimicrobial activity among all samples was from groups R and F. The materials R and F were also assessed in a previous study regarding their antibacterial activity. 2 Similarly, to our findings, material R revealed no antimicrobial effect against E. faecalis; whereas, the results of material F differed from those found in this study, because in the referred study, F also showed no antibacterial effect. 3