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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 9  |  Issue : 1  |  Page : 33-37

In vitro antibacterial effectiveness of three different dentin bonding systems against Streptococcus mutans and Enterococcus faecalis


1 Department of Conservative Dentistry and Endodontics, C.S.I. College of Dental Sciences and Research, Madurai, Tamil Nadu, India
2 Department of Conservative Dentistry and Endodontics, Rajah Muthiah Dental College, Annamalai University, Chidambaram, Tamil Nadu, India

Date of Web Publication28-Feb-2017

Correspondence Address:
S Swathi Priyadharshini
Department of Conservative Dentistry and Endodontics, C.S.I. College of Dental Sciences and Research, Madurai, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jioh.jioh_2_16

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  Abstract 

Aims: This study aims to evaluate the antibacterial effect of three different bonding systems – G-Bond, Clearfil SE Bond and Prime and Bond – on Streptococcus mutans and Enterococcus faecalis in vitro. Materials and Methods: This study examined the antibacterial effectiveness of dentin bonding systems against S. mutans and E. faecalis. This study was carried out with the standardized inoculums with selective media and the materials assayed were straight away applied on the cultures. Discs of dentin bonding system (4 mm × 4 mm) were made, cured, washed and put on the bacterial inoculums for 24, 48, and 72 h and the growth inhibition was evaluated using a spectrophotometer. The dentin bonding agents evaluated in this study revealed different inhibitory effects. Statistical Analysis Used: Submitted to the analysis of variance. Results: Group A (G-Bond) has an inhibitory effect compared to Group B (Clearfil SE Bond) and Group C (Prime and Bond NT) at 24, 48, and 72 h against S. mutans. Group B (Clearfil SE Bond) has inhibitory effect at 24, 48, and 72 h compared to Group A (G-Bond) and Group C (Prime and Bond NT) against E. faecalis. Conclusions: This self-etching system G Bond and Clearfil SE Bond presented a greater inhibitory effect against S. mutans and E. faecalis than the conventional system, Prime and Bond did.

Keywords: Antibacterial effect, Enterococcus faecalis, laboratory research, self-etch adhesives, Streptococcus mutans


How to cite this article:
Priyadharshini S S, Ahmed A S, Savadamoorthi KS. In vitro antibacterial effectiveness of three different dentin bonding systems against Streptococcus mutans and Enterococcus faecalis. J Int Oral Health 2017;9:33-7

How to cite this URL:
Priyadharshini S S, Ahmed A S, Savadamoorthi KS. In vitro antibacterial effectiveness of three different dentin bonding systems against Streptococcus mutans and Enterococcus faecalis. J Int Oral Health [serial online] 2017 [cited 2019 Aug 24];9:33-7. Available from: http://www.jioh.org/text.asp?2017/9/1/33/201088


  Introduction Top


Dental caries development is a dynamic process between demineralization and remineralization which results in cavitation. While removing this carious lesion, bacteria may remain in dentinal tubules. Bacteria, living in the residual tissues and invading along the restoration-tooth interface, constitute the main cause for secondary caries. The presence of secondary caries is the most common reason for replacing dental restorations.[1] Two possible mechanisms are involved in the formation of secondary caries. First, resin polymerization shrinkage creates gaps between the restoration and the cavity walls that can be colonized by oral microorganisms from saliva. Second, removing caries incompletely can be another source of microbes.[2] The philosophy of surgical excision has greatly changed the concept of minimally invasive techniques due to the development of the functional and effective dentin bonding systems.

Minimal invasive dentistry focuses on less surgical intervention to preserve sound tooth structures as much as possible. Even after the bacterially contaminated, demineralized, and caries-affected dentin is removed, after minimally invasive caries intervention, some pioneer bacteria intrude in the remineralizable, caries-affected layer.[3] Composite restorations are effected to the tooth structure through adhesives. Ideally, the hybrid layer is characterized as a three-dimensional polymer/collagen network provides both a continuous and a stable link between the bulk adhesive and dentine substrate. The bonded interface is a weak link between the restoration and the tooth structure.[4] Residual bacteria, plaque accumulation on restorative materials, and microleakage have been taken as closely related to secondary caries.[5]

Recent dentin adhesives are classified according to the interaction with the dentin smear layer:[6]

  • The total etch technique
  • The self-etch technique.


In total, etch dentin bonding systems, phosphoric acid is used to etch dentin and enamel before applying primer or primer and adhesive together in one bottle. While etching dentin, the smear layer is removed, and dentinal tubules are opened.

Self-etch adhesives, without a separate acid-etch step; do not remove the smear layer completely. Self-etch adhesives have been classified into three categories:[7]

  • Mild (pH > 2)
  • Moderate (pH = 1.5)
  • Aggressive (pH < 1).


Mutans streptococci are a group of seven bacterial species highly related to the development of caries in humans, where Streptococcus mutans is considered the main etiological agent of this disease. Enterococcus faecalis is a resistant microorganism, but it is found only in a small fraction of untreatedroot canals, it has a big role in periradicularlesions after completing root canal treatment.[8] Till date, a very limited number of studies have been carried outto evaluate the effect of dental adhesives on this bacterialspecies.[8]

Several studies describe the antibacterial effects of the 12-methacryloyloxydodecylpyriminium bromide (MDPB) monomer, still little is known about the antimicrobial effects of other adhesive systems without MDPB.

Few studies have evaluated whether acetone/ethanol based self-etch adhesive systems inhibit bacterial growth compared to fluoride containing Prime and Bond NT a total etch adhesive agent.

Hence, the purpose of this study is to know the efficiency of self-etch adhesive agents namely G-Bond and Clearfil SE Bond respectively in inhibiting S. mutans and E. faecalis compared to Prime and Bond NT a total etch adhesive agent incorporated with fluoride as an antibacterial agent.


  Materials and Methods Top


The present study is a laboratory investigation to determine the antibacterial efficacy of 5th, 6th, and 7th generation adhesive systems against S. mutans and E. faecalis.

A low viscosity polyvinyl siloxane mold (Aquasil, Dentsply DeTrey, Konstanz, Germany) with 4 mm diameter and 4 mm high was used to make ten dentin bonding discs in each. Group A (G-Bond), Group B (Clearfil SE Bond), and Group C (Prime and Bond NT) as described in [Table 1]. Discs of G-Bond, Clearfil SE Bond (CB) and Prime and Bond NT were prepared as per the manufacturer's recommendation. Using the software program G*Power, version 3.1.7 (Franz Faul, Kiel University, Germany), we calculated that 60 samples would be required for this study.
Table 1: Materials used in the study

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In Group A, one drop of bonding agent (G-Bond) was used in the mold, and light cured for 20 s with light curing unit. In Group B, one drop of self-etch primer (Clearfil SE Bond) was used in the mold, and after 20 s one drop of bonding system was added to the primer and light cured for 20 s with light curing unit. In Group C, one drop of bonding agent (Prime and Bond NT) was used in the mold, and light cured for 20 s with light curing unit.

After 48 h curing period, following method of Prati et al.[9] All material samples were shaken in a sterile solution (0.85% w/v of distilled water) before exposing to the test microorganisms. This procedure is done to simulate the effects of saliva in extracting any unreacted components. This practice was carried out by transferring the specimens of each material aseptically to a 100 ml Erlenmeyer flask with 25 ml of previously sterilized distilled water. The flasks were incubated for 15 min at 37°C on a rotary shaker at 150 rpm. The specimens of washed materials were then picked up out of the flasks and aseptically transferred into the Eppendorf tubes.

The bacteria used in the study were S. mutans and E. faecalis. Overnight bacterial precultures were taken with the tested bonding discs in the Eppendorf tubes. The tubes were incubated at 24, 48, and 72 h. The bacterial growth was monitored in a spectrophotometer at 640 nm after 24, 48, and 72 h respectively as shown in [Table 2].
Table 2: Absorbance mean (nm)±standard deviation at different time intervals

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Statistical analysis

The antibacterial activity of each dentin-bonding agent was analyzed statistically using the analysis of variance (ANOVA) test, with a level of significance set up at P < 0.05. The software program was SPSS software, version 16.0 (SPSS Inc., Chicago, IL, USA).


  Results Top


The values were statistically analyzed by one-way ANOVA. The result of the present study showed that there was a statistically significant difference in the antibacterial property among Group A, Group B, and Group C against S. mutans and E. faecalis.

Results of this study against S. mutans, Group A showed a mean bacterial inhibition value of 0.193 (P < 0.01) which is statistically significant at 24 h interval [Table 3]. At 48 h of interval, there was no statistical difference among Group A, Group B, and Group C [Table 4]. At 72 h of interval Group A was with a mean bacterial inhibition value of 0.142 (P < 0.01) and Group C with a mean of bacterial inhibition 0.151 (P < 0.01) which is statistically significant [Table 5].
Table 3: Comparative evaluation of antibacterial effectiveness against Streptococcus mutans at 24 h interval

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Table 4: Comparative evaluation of antibacterial effectiveness against Streptococcus mutans at 48 h interval

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Table 5: Comparative evaluation of antibacterial effectiveness against Streptococcus mutans at 72 h interval

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Results of this study against E. faecalis, as shown in [Table 6],[Table 7],[Table 8] Group B showed a mean of bacterial inhibition values of 0.15, 0.14, and 0.14 (P < 0.01) which is statistically significant at 24, 48, and 72 h of intervals.
Table 6: Comparative evaluation of antibacterial effectiveness against Enterococcus faecalis at 24 h interval

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Table 7: Comparative evaluation of antibacterial effectiveness against Enterococcus faecalis at 48 h interval

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Table 8: Comparative evaluation of antibacterial effectiveness against Enterococcus faecalis at 72 h interval

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  Discussion Top


Composite resin restoration has been widely employed in dental practice for more than two decades. The clinical performance of composites has been improved considerably through modifications in its formulation. Irrespective of good esthetic and mechanical properties, it has major disadvantages of polymerization shrinkage and susceptibility to degradation in the oral environment.

Dentin bonding system involves an unfilled, liquid acrylic monomer mixture which is placed onto an acid conditioned and primed dentin surface. The bonding primer is based on hydrophilic monomers such as hydroxyethyl methacrylate to drench dentin surfaces that may contain moisture.

Sixth generation bonding systems consist of two-step self-etch adhesives and one-step, two-component self-etch adhesives. The self-etch is derived from monomers to which one or more carboxylic or phosphate groups are added. Strong self-etch adhesives usually have a pH of 1 or less. This acidity results in rather deep demineralization effects. In enamel, the resulting etching pattern of self-etch adhesives resemble that of an etch and rinse approach. In dentin, collagen is exposed, and all hydroxyapatite is dissolved. Consequently, the underlying bonding mechanism of strong self-etch adhesives is primarily based on diffusion, similar to that of etch and rinse approach.[10]

To simplify the clinical procedure, seventh generation dentin bonding systems was introduced as “all in one” adhesives that combine etch, prime, and bond in a single solution. They do not need a separate acid-etch and subsequent rinsing procedure.

No dental resin composite material currently available is free of shrinkage during polymerization. Because of polymerization shrinkage, the gap formed between restoration and cavity walls are colonized by oral microorganisms, which may result in secondary caries. The bacteria that invade the gaps and permeate the dentin tubules, sometimes causing pulp inflammation.[11]

Data from numerous studies have shown that the longevity of dental restorations is shorter than generally expected and secondary caries is a main cause for the replacement of restorations.[12] Restorations must be hermetically sealed to prevent bacterial infection. Although dentin bonding systems have been developed to seal cavity preparations and prevent marginal microleakage, even the currently bonding systems are incapable of providing a complete seal in clinical conditions. Therefore, bonding systems that show antibacterial activity may help in preventing pulpal and periapical infections.[13]

An effective bactericidal effect from adhesives systems can be an alternative to suppress bacterial invasion and microleakage, which leads to the improvement of the longevity of restorations.[14] A few studies have evaluated the antibacterial effectiveness of dentin bonding systems.

In this study, the antibacterial effects of 5th, 6th, and 7th generation bonding agents were evaluated against S. mutans and E. faecalis.

S. mutans have been chosen because of early colonization in oral cavity and their major role in the development of dental caries.

E. faecalis have been chosen because of their presence in the persistent periapical infection. Coronal seal after root canal therapy plays a vital role in the success of endodontic treatment. In the majority of cases, the failure of endodontic treatment is due to periapical lesions as a result of microleakage and penetration of microorganisms.[8]

The introduction of self-etch adhesive systems has led to the elimination of separate etching and rinsing steps; bacteria may still remain at the tooth-restoration interface. The longevity of the restorations can be improved, if the primer or the adhesive in the bonding system exhibits antibacterial activity so that the bacteria remaining at the interface and also the bacteria penetrating due to microleakage might be eliminated.[8]

The dentin bonding agents used in the present study was Group A (G-Bond), Group B (Clearfil SE Bond), and Group C (Prime and Bond NT). The antibacterial effect of the tested materials used in this study took place after curing which resembled to those encountered in the restorative treatment in dental caries.

The resin monomer MDPB has been incorporated into the composition of some bonding systems to enhance the antibacterial effect of these materials.[15] Although all the tested bonding agents used in this study do not contain MDPB, they exhibit significant antibacterial effect against the two examined bacteria.

In this study, direct contact test was performed. The test allows water-insoluble materials to be tested. It relies on a direct and close contact between the test microorganism and the tested materials. It is virtually free from the diffusion properties of both the tested material and the medium. With its reproducible and quantitative nature, the result of direct contact test is not disturbed by the size of the inoculum and relatively insensitive to the size of inoculum that is brought in contact with the tested material. It eases standardized measurements of a large number of specimens and their respective control, simultaneously on the same microtiter plate and can monitor the microorganism's growth.[12]

Results of this study against S. mutans, Group A showed a mean bacterial inhibition value of 0.193 (P < 0.01) which is statistically significant at 24 h interval. At 48 h of the interval, there was no statistical difference found between Group A, Group B, and Group C. At 72 h of the interval, Group A is with a mean bacterial inhibition value of 0.142 (P < 0.01) and Group C with a mean of bacterial inhibition 0.151 (P < 0.01) which is statistically significant.

Results of this study against E. faecalis, Group B showed a mean of bacterial inhibition values of 0.15, 0.14, and 0.14 (P < 0.01) which is statistically significant at 24, 48, and 72 h of intervals.

Results of this study correlate with Taha et al.[16] who studied the antibacterial effect of three dentin bonding agents at different time intervals (24, 48, and 72 h) against S. mutans and E. faecalis and arrived at the conclusion that G Bond was able to delay bacterial growth during restorative treatment of dental caries.

Studies done by Giammanco et al. and Imazato et al. compared the antibacterial effect of dentin/enamel adhesive systems containing MDPB and not containing MDPB and found that the antibacterial effect does not seem to be entirely linked to the presence of the immobilized antimicrobial MDPB, since the resin not incorporating MDPB also exhibit comparable bacteriostatic activity. They concluded that both materials tested delay bacterial growth equally.[17],[18]

The difference noted in the antibacterial activity of the adhesives against the bacteria tested could be related either to their acidity or their chemical composition. The pH value of G-Bond and Clearfil SE Bond is 2, and this may explain why the self-etching adhesives showed inhibitory effects against the test bacteria used in the study. A study done by Harper and Loesche on the pH values of dentin adhesives, state that pH of 2.3 for Lactobacillus casei and 3.0 for S. mutans caused 100% killing over a 3 h period.[19] In this present study, Group A and Group B displayed statistically higher antibacterial effect. The pH of these adhesives was 2.0 or lower which might be effective enough to kill, or at least inactivate, the bacteria.

On the other hand, the pH values of the bonding systems increase after contact with dentinal substrate by buffering action. The solution is diluted by dentinal fluid as it works its way into the deeper area of the lesion and subsequently reduces its antibacterial effect. Therefore, for successful restoration, it is beneficial to provide a bonding system with intrinsic antimicrobial properties.

It seems preferable to develop new antibacterial dentin bonding systems that form a perfect seal between the tooth and the resin interface, thus preventing exposure of the pulp-dentin complex to bacteria and their toxins. Therefore, it is reasonable to suggest that restoration longevity might be improved using restorative or liner materials with antibacterial properties.[20]

The growth inhibitory effects of dentin adhesives observed in this study would be similar in vivo.[21] Moreover, the duration of this effect is not known, nor can it be hypothesized from this assay system. The long-term effect might be useful in case of future microleakage and bacterial infiltration.

Therefore, long-term clinical studies are unavailable to determine whether antibacterial effects of these bonding systems observed in vitro suffice to increase the longevity of dental restorations.


  Conclusion Top


On the basis of this in vitro study, it may be concluded that G Bond and Clearfil SE Bond reveal antibacterial effect against S. mutans and E. faecalis. Furthermore, G Bond and Clearfil SE Bond are able to inhibit the bacteria growth more effectively than the fluoride containing adhesive Prime and Bond NT.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Duzyol M, Karamese M, Akgul N, Gul P, Celik N, Duzyol E, et al. Comparison of the antibacterial efficacy of several dentin bonding agents: Two different in vitro studies. Int J Dent Sci Res 2016;4:1-4.  Back to cited text no. 1
    
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Lukomska-Szymańska M, Zarzycka B, Zurawska-Olszewska J, Olbert-Sieroszewsk V, Sokolowski K, Krzeminski Z, et al. Antibacterial activity of eight different dentine bonding systems. Int Organ Sci Res J Dent Med Sci 2014;13:48-54.  Back to cited text no. 2
    
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Imazato S, Kuramoto A, Takahashi Y, Ebisu S, Peters MC.In vitro antibacterial effects of the dentin primer of clearfil protect bond. Dent Mater 2006;22:527-32.  Back to cited text no. 3
    
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Chen C, Weir MD, Cheng L, Lin NJ, Lin-Gibson S, Chow LC, et al. Antibacterial activity and ion release of bonding agent containing amorphous calcium phosphate nanoparticles. Dent Mater 2014;30:891-901.  Back to cited text no. 4
    
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Zhang K, Wang S, Zhou X, Xu HH, Weir MD, Ge Y, et al. Effect of antibacterial dental adhesive on multispecies biofilms formation. J Dent Res 2015;94:622-9.  Back to cited text no. 5
    
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Browne RM, Tobias RS. Microbial microleakage and pulpal inflammation: A review. Endod Dent Traumatol 1986;2:177-83.  Back to cited text no. 11
    
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Sampath PB, Hegde MN, Hegde P. Assessment of antibacterial properties of newer dentin bonding agents: An in vitro study. Contemp Clin Dent 2011;2:165-9.  Back to cited text no. 12
[PUBMED]  Medknow Journal  
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Yazici AR, Baseren M, Dayangaç B. The effect of current-generation bonding systems on microleakage of resin composite restorations. Quintessence Int 2002;33:763-9.  Back to cited text no. 13
    
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André CB, Gomes BP, Duque TM, Stipp RN, Chan DC, Ambrosano GM, et al. Dentine bond strength and antimicrobial activity evaluation of adhesive systems. J Dent 2015;43:466-75.  Back to cited text no. 14
    
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Hamouda IM, Al-Khodary AM, El Shami FM. Degree of conversion and antimicrobial activity of etch-and-rinse versus self-etching adhesives. J Adhes Dent 2010;12:33-8.  Back to cited text no. 15
    
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Giammanco GM, Cumbo EM, Luciani A, Gallina G, Mammina C, Pizzo G.In vitro evaluation of the antibacterial activity of cured dentin/enamel adhesive incorporating the antimicrobial agent MDPB. New Microbiol 2009;32:385-90.  Back to cited text no. 17
    
18.
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20.
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21.
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8]


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