An In-Vitro Study of the Antibacterial Efficacy of Cavity Liners Against Streptococcus Mutans and Lactobacillus Casei

ARTICLE INFO Article Type Original Article


Introduction:
Secondary carious lesion is the main factor that influences the longevity of dental restorations. (1)Different bacterial species may be isolated from dental plaque.These bacteria are the main reasons of carious lesion and pulpal inflammation. (1,2) treptococcus mutans (S. mutans) and Lactobacillus casei (L.casei) are the two most frequently implicated bacteria in dental caries. (1)5) Caries removal is the first stage of tooth decay treatment.Many authors have reported that the bacterial flora is not completely removed from cavity walls during cavity preparation. (6,7) rthermore, microleakage may allow microorganisms to penetrate the gap between the filling material and dental tissue. (6,8) herefore, the antibacterial activity of dental materials is important from the clinical aspect, as it might inhibit recurrent caries.Glass Ionomers (GIs) have been used as cements, cavity liners and filling materials for over 20 years. (1)These materials have antibacterial activity, which is generally attributed to the low pH during setting time and also to fluoride release.Fluoride-releasing restorative materials can decrease bacterial metabolism and proliferation. (4)Release of strontium ions and fluoride may also create a synergistic antibacterial effect. (7)6)(7)(8) Compomers are resin composites which release fluoride. (5,9) he amount of fluoride released from these resin composites is lower than that released from GI cements (GICs), and it is not adequate for bacterial growth inhibition. (7)anufacturers not only improve the mechanical and aesthetic properties of GIs, but they also try to ease their use and increase their antibacterial efficacy. (6)Many studies have evaluated the antibacterial efficacy of different fluoride-releasing dental materials; (1)(2)(3)(4)(5)(6) however, new materials are introduced to the market every day in different forms including powder and liquid, capsulated, and one-component such as Ionoseal. (6)Therefore, it is necessary to compare these products with the dental materials that have been previously introduced to the market. (4,5) he aim of the present study was to compare the antibacterial activity of four fluoride-releasing dental cavity liners against S. mutans and L. casei.

Materials and methods:
In this in-vitro experimental study, the agar diffusion test was applied to compare the antibacterial activity of four dental cavity liners against S. mutans and L. casei. (1,2) he brands, categories and manufacturers of the dental materials used in this research are presented in Table 1.Indicator strains of S. mutans (ATCC35668) and L. casei (ATCC393) were obtained in the form of lyophilized culture (Biotechnology center of Islamic Azad University of Isfahan, Iran).They were grown separately in 15 ml of Brain Heart Infusion (BHI) agar (Merck, Germany) at 37 °C for 24 hours.The broth was placed in an anaerobic environment imitating the physiologic characteristic of L. casei.Suspensions were prepared according to 0.5 McFarland turbidity standard.Sterile saline 0.9% was used for 100-fold dilution to create a suspension corresponding to 10 6 colony-forming units according to McFarland scale.
The experimental specimens presented in Table 1 were made using a custom-built Teflon ring mold with the diameter of 7 mm and thickness of 2 mm, except for Chlorhexidine (CHX).The four dental materials were placed inside the molds and were sandwiched between two Mylar Downloaded from jrdms.dentaliau.ac.ir at 18:09 +0330 on Thursday December 27th 2018 strips placed over a glass slab, and subsequently were cured according to the manufacturer's recommendations presented in Table 2. Afterwards, all the specimens were autoclaved (Iran Tolid Medical Industries Co, Iran) at 121 °C and 15 psi pressure for 15 minutes.10 µl of aqueous 0.2% CHX was poured on a sterile filter paper disc with the diameter of 7 mm and served as positive control.
15 ml BHI Agar medium (Merck, Germany) was poured evenly in each of the sterile Petri dishes to a thickness of 5 mm.After the solidification of the culture medium at room temperature, 100 µl of each bacterial suspension was added with the use of a micropipette and was spread uniformly using a sterile cotton swab.In each Petri dish, four discs of specimens and one filter paper of CHX were placed.The culture plates of S. mutans were incubated at 37 °C for 7 days; whereas, L. casei culture plates were incubated in a 5% CO2 incubator.The antibacterial activity was evaluated at 24 and 48 hours, and at 7 days by measuring the diameter of the inhibition zone around the discs in millimeters (mm) using a digital caliper. (1,10,11) Te experiment was repeated 6 times for each dental cavity liner, and the mean diameter of the inhibition zone was calculated for each dental material.
The data are presented as mean ± standard deviation (SD).Statistical analysis was performed using the repeated measure ANOVA for intergroup differences and T-test.The level of significance was set at (P<0.05).

Results:
The mean diameters of the growth inhibition zones of the studied dental materials are shown in Table 3.The data related to the antibacterial activity of the tested dental materials were col lected and analyzed using the repeated meas-

Discussion:
The results of the present study indicated that the antibacterial efficacy of the studied dental cavity liners varied at different time intervals (p<0.001).Vitrebond cavity liner showed the largest mean diameter of the inhibition zone; however, there were no statistically significant differences between the four assessed cavity liners in terms of antibacterial potential (p=0.07).Moreover, there were no statistically significant differences in the growth inhibition zones of the cavity liners against the two bacterial species (p=0.342).
The primary purpose of restorative dentistry is to maintain or restore the function of tooth by arresting caries or preventing their recurrence.
Caries removal is the first stage of tooth decay treatment.Many authors have declared that the bacterial flora is not completely removed from cavity walls during cavity preparation. (6,7)Furthermore, microleakage may allow microorganisms to penetrate the gap between the filling material and dental tissue. (6,8) herefore, it is important to use restorative materials that can provide a long-term seal against cariogenic bacteria. (6)he antibacterial activity of GICs is mostly attributed to fluoride release. (4,7) owever, some authors have stated that the antibacterial properties of GICs are more related to the low pH during setting time rather than fluoride release. (7)Fluoride-releasing restorative materi-als can decrease bacterial metabolism and proliferation in the agar culture medium but may be incapable of preventing the rapid and spontaneous initial adhesion of bacteria.RMGICs also exhibit antimicrobial activity, which is due to the release of chemical components such as fluoride and metallic ions and also due to the low initial pH. (7)Release of strontium ions and fluoride may also create a synergistic antibacterial effect. (7)Compomers are resin composites which release fluoride. (5,9) he amount of fluoride released from these resin composites is lower than that released from GICs, and it is not adequate for bacterial growth inhibition. (7)(11) These results further highlight the contrasting views on the role of fluoride in the antibacterial activities of dental restorative materials.
A systematic review in 2015, which included 14 (500 treated lesions) of 113 identified studies, reported a disagreement between those clinical studies stating that the use of GIC or no lining is highly successful, and the other studies incorporated in this review which found MTA or antibacterial liners to be more suitable for exerting antibacterial effects.Eventually, it has been stated that the current evidence is inadequate to support certain cavity treatments before restoration. (8)ntibacterial activity of GICs has attracted the attention of scientists since the 19th century until recent years. (12,13) n spite of the fairly abundant literature regarding the issue, evaluation and comparison of the results is not simple. (6)The methodology of these studies varies greatly, that in turn hampers the comparison of the results of these studies.Most authors, including us, have performed their experiments using the agar diffusion method; (6) however, some recommended other techniques such as Direct Contact Test (DCT). (6)The duration of studies also varied.The majority of them were short-term studies of 24 or 48 hours, and only a small number of them were conducted over a longer period of 7 days (similar to the present study). (6)Also, there are some inconsistencies regarding the antibacterial activity of GICs observed in our study and studies of other authors, which can be attributed to the evaluated cements, bacterial strains and period of evaluation.
difference in terms of antibacterial activity according to the mean diameter of the inhibition zones (p=0.07).However, the p-value was close to the signif-Downloaded from jrdms.dentaliau.ac.ir at 18:09 +0330 on Thursday December 27th 2018 bacterial activity in the first 24 hours, which decreased during 7 days.This can be related to the low pH level of GICs during the first 24 hours.Vitrebond and Ionobond maintained their antibacterial activity after 7 days.
It has been well demonstrated that the release of fluoride from restorative materials is a very complex process, and can be affected by intrinsic variables such as fillers and formulations, and also by experimental factors such as storage media and the frequency of changing the storage medium. (15)urther investigations are needed to compare various dental cavity liners with antibacterial properties, and to determine the antibacterial activity of fluoride-releasing dental cavity liners over a longer period of time, and also to evaluate these dental cavity liners in vivo.

Conclusion:
The four evaluated dental cavity liners (Ionobond, Ionoseal, Ionosit and Vitrebond) demonstrated antibacterial properties; however, none of them was statistically superior.These findings indicate that fluoride-releasing dental materials may play an important role in decreasing the number of bacteria in the prepared dental cavity walls.