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Table of Contents   
ORIGINAL RESEARCH  
Year : 2021  |  Volume : 24  |  Issue : 2  |  Page : 214-218
Effect of dental acid etchant-mediated photodynamic therapy on bacterial reduction and microshear bond strength of composite to dentin – An in vitro study


1 Department of Conservative Dentistry and Endodontics, I.T.S.-C.D.S.R, Ghaziabad, Uttar Pradesh, India
2 Department of Oral Pathology, I.T.S.-C.D.S.R, Ghaziabad, Uttar Pradesh, India

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Date of Submission22-Dec-2020
Date of Decision03-Feb-2021
Date of Acceptance14-Jul-2021
Date of Web Publication09-Oct-2021
 

   Abstract 


Objective: The objective of this study was to assess the effect of dental acid etchant (DAE)-mediated photodynamic therapy on bacterial reduction and microshear bond strength of composite to dentin.
Materials and Methods: Eighty permanent third molars after sample preparation were exposed to a cariogenic challenge with Streptococcus mutans. After incubation, specimens were randomly divided into four groups (n = 20): Group I – DAE, Group II – low-level laser (LLL), Group III – diode laser + methylene blue (MB + L), and Group IV – diode laser + DAE (DAE + L). Half of the specimens from each group were selected for bacterial reduction assessment and the other half for microshear bond strength. All the samples for assessment of bacterial reduction (before and after intervention) were seeded onto the surface of mitis-salivarius-bacitracin medium. After incubation, the viable bacterial count was determined in colony-forming unit/mL. For microshear bond strength assessment, samples were subjected to various treatment modalities and then bonding and debonding procedure was performed for blocks of composite and values were recorded.
Results: Significant reductions in S. mutans were observed in all the groups – Group I (DAE) 68.50%, Group II (LLL) 55.90%, Group III (MB + L) 88.60%, and Group IV (DAE + L) 87% with comparable bacterial reduction between Group III (MB + L) and Group IV (DAE + L). Furthermore, a significant difference in bond strength values was seen in Group III (MB + L) 10.99 MPa and Group IV (DAE + L) 17.99 MPa whereas an insignificant difference was found between Group I (DAE) 20.74 MPa, Group II (LLL) 18.27 MPa, and Group IV (DAE + L).
Conclusion: DAE caused a comparable reduction in bacterial count to MB-assisted PDT and also there was no adverse effect on bond strength values. PDT can be performed while acid etchant containing MB dye is being applied in the cavity, thus reducing operative time and enhancing cavity disinfection.

Keywords: Cavity disinfection; dental acid etchant; microshear bond strength; photodynamic therapy

How to cite this article:
Gupta J, Taneja S, Jain A. Effect of dental acid etchant-mediated photodynamic therapy on bacterial reduction and microshear bond strength of composite to dentin – An in vitro study. J Conserv Dent 2021;24:214-8

How to cite this URL:
Gupta J, Taneja S, Jain A. Effect of dental acid etchant-mediated photodynamic therapy on bacterial reduction and microshear bond strength of composite to dentin – An in vitro study. J Conserv Dent [serial online] 2021 [cited 2021 Nov 30];24:214-8. Available from: https://www.jcd.org.in/text.asp?2021/24/2/214/327859



   Introduction Top


Minimally invasive dentistry has emphasized the importance of maximum tissue preservation in the management of deep dentinal carious lesions. Mechanical instrumentation of infected carious lesions provides gross removal of cavity pathogens, but in order to achieve a higher level of bacterial eradication for the last pathogens found in the diseased tissue (responsible for recurrence and postoperative sensitivity), a variety of antimicrobial agents are used.

A lot of antibacterial treatment modalities such as sodium hypochlorite, chlorhexidine, hydrogen peroxide, povidone-iodine, as well as the use of lasers, halogens, and oral ozone machines have all been investigated for their antimicrobial treatment effectiveness over a long period of time. Literature shows that a large number of bacteria remain viable after the use of these modalities, and moreover, the effective concentration of these disinfectants may pose a risk for toxicity followed by adverse tissue reactions.[1],[2] Therefore, in order to find alternative therapeutic approaches to substitute these chemicals, antimicrobial photodynamic therapy (aPDT) has emerged as an effective and promising alternative antibacterial treatment modality.

aPDT is a technique that relies on the use of a low-energy light source and a dye, also termed photosensitizing (PS) agent, for the production of reactive oxygen species (ROS) which is highly electrophilic and can directly oxidize double bonds in biological molecules and macromolecules, thus playing an important role in cytotoxicity by causing cell damage and death.[3],[4] Among the photosensitizers used in aPDT, erythrosine and methylene blue (MB) have shown promising results in the inactivation of cariogenic bacteria.[5],[6],[7],[8],[9]

MB belongs to the phenothiazine group with absorption in the red light range of 600–660 nm and has demonstrated a significant bactericidal action against cariogenic bacteria.[7],[10] The dental acid etchants (DAEs) used to etch enamel and dentin prior to application of adhesive systems consist of a 35%–37% solution of phosphoric acid with added MB dye which imparts color to it. The presence of this MB dye in the etchant may act as an effective alternative photosensitizer.

Furthermore, there are few reports available in literature regarding the effect of caries detection dye on the bond strength of sound and carious affected dentin.[11],[12] The photosensitizer dye used in photodynamic therapy may affect the bonding of composite to dentin as its residual structural molecules may interfere during the adhesion process.

Hence, the aim of the present study was to assess the effect of photodynamic therapy on Streptococcus mutans in dentinal carious lesions using DAE as a PS agent and its influence on microshear bond strength of composite to dentin – an in vitro study.

The null hypothesis for the study was that there is no effect of photodynamic therapy on dentinal carious lesions using DAE as a PS agent on bacterial count and microshear bond strength when compared with photodynamic therapy using MB, low-level laser (LLL), and etchant alone.


   Materials and Methods Top


Sample collection

After receiving internal ethical committee clearance, this study was conducted in the Department of Conservative Dentistry and Endodontics in collaboration with the Department of Oral Surgery and Centre For Advanced Research. A total of eighty samples of extracted permanent third molars with no visible cracks or fractures were collected for the study.

Sample preparation

The occlusal third from coronal portion of all the samples were removed in order to expose the dentin surface using a double-sided diamond disk in a low-speed handpiece ( API,Delhi,India) under continuous flow of water. The dentin surfaces were polished with wet silicon carbide sandpaper sheets, P600 grit. 4 mm × 4 mm labels were placed onto the dentin surface of all the specimens to mark the working area and the rest of the sample's surfaces were sealed using nail polish. The label was then removed to enable the generation of the carious lesion.

Formation of Streptococcus mutans biofilm

All the samples were then exposed to a cariogenic challenge in brain–heart infusion (BHI) broth (HiMedia Laboratories, Mumbai, India) supplemented with 1% glucose, 1% sucrose, 0.5% yeast extract, and S. mutans type strain ATCC 25175 (HiMedia Laboratories, Mumbai, India), standardized to 0.5 McFarland turbidity. Samples were incubated at 37°C under strictly anaerobic conditions within an anaerobic system (N2 85%, H2 5%, CO2 10%) using anaerobic gas jar and gas pack (HiMedia Laboratories, Mumbai, India) and stored in a bacteriological incubator for 16 days. BHI broth was replaced every 24 h during the incubation period.

Experimental groups

After 16 days, samples were taken out from the incubator and were divided into four groups:

  • Group I (n = 20) – DAE
  • Group II (n = 20) – LLL group
  • Group III (n = 20) – Diode laser + MB (MB + L)
  • Group IV (n = 20) – Diode laser + DAE (DAE + L).


Half of the specimens from each group were evaluated for bacterial count and the other half were assessed for microshear bond strength. Preoperative samples for bacterial count were taken from the 40 specimens (10 from each group) and were collected from the marked surface on the tooth using spoon excavator (API, Delhi, India).

Treatment modalities

  • Group I (DAE) – DAE (DeTrey Conditioner 36, Dentsply Sirona) was applied onto the carious dentin for 15 s without LLL irradiation. The acid solution was then removed by rinsing with distilled water
  • Group II (LLL) – The carious dentin on the sample's surface was irradiated with low-level laser (SiroLaser Blue, Dentsply Sirona, Germany) for 15 s; LLL was administered at a spot energy of 4 J/cm2, wavelength = 660 nm, and power = 5 W
  • Group III (MB + L) – In this group, MB (HiMedia Laboratories, Mumbai, India) was used as the photosensitizer agent, which was prepared with distilled water to obtain a final concentration of 0.01% (100 mg/L, 268 uM). After that, solution was applied onto the sample's surface and was irradiated with LLL in a similar manner as in Group II for 15 s and afterward the dye solution was rinsed off with distilled water
  • Group IV (DAE + L) – In this group, DAE which will act as a PS agent was applied onto the carious dentin and was simultaneously exposed by LLL (similarly as in Group II) for 15 s. Afterward, etchant was rinsed with distilled water.


Postoperative samples for bacterial reduction were collected from specimens previously selected from each group.

Assessment of reduction in bacterial count/viable cell count

All the collected samples were immediately placed in BHI transport medium and homogenized for 3 min in a tube shaker. Immediately after homogenization, samples were diluted two folds into test tubes, and 25-μL aliquots from each dilution were seeded with a micropipette onto the surface of mitis-salivarius-bacitracin medium. All plates were incubated in anaerobic jars at 37°C for 48 h. After incubation, the viable bacterial count was determined in colony-forming unit (CFU)/mL.

Sample preparation for microshear bond strength

For microshear bond strength measurement, the other half of the samples (apart from bacterial reduction ones) were mounted in acrylic resin blocks and then were treated in a similar way as the abovementioned modalities. After this, DAE (DeTrey Conditioner 36, Dentsply Sirona) was applied on all the samples of Group II and Group III (samples of Group I and Group IV were already etched during aPDT) for 15 s. After rinsing of etchant, bonding agent (One Coat Bond SL, Coltene Whaledent Private Ltd., Mumbai, India) was applied and was light cured for 20 s followed by placement of composite (Filtek Z250 XT, 3M ESPE, USA) over the marked areas using mold (4 mm × 4 mm × 2 mm) and light curing for 20 s. Test samples were stored in distilled water (24 h, 37°C) and later subjected to thermocycling (5°C–55°C, 1,500 cycles).

Testing of microshear bond strength

Debonding procedure was performed on Instron universal testing machine at a crosshead speed of 0.5 mm/min until the composite block gets separated from the sample's surface.[11]

Statistical analysis

The data were compiled using Microsoft Excel Spreadsheet and subjected to statistical analysis using Statistical Package for Social Sciences version 20 (IBM Corp., Armonk, N.Y., USA). Shapiro–Wilk test was done to determine the normality of the data. Data were found to be normally distributed, thus parametric tests were done. One-way ANOVA was done to determine whether there was any statistically significant difference in the mean CFU count and shear bond strength between the four groups. This was followed by Tukey's post hoc test for multiple pairwise comparisons. P <0.05 was considered statistically significant.


   Results Top


Significant reduction in bacterial count were observed after treatment in all the four groups. The highest percentage reductions in S. mutans count were observed in Group III (MB + L) and Group IV (DAE + L) and there was an insignificant difference when both of these groups were compared stating that almost comparable reduction in S. mutans count was observed when PDT was done with MB and when it was done with DAE. Conversely, when microshear bond strength was compared within all the groups, a significant difference in bond strength was observed between Group III (MB + L) and Group IV (DAE + L). In Group I, Group II, and Group IV, there was an insignificant difference in bond strength values.


   Discussion Top


Photodynamic therapy is a promising therapy for deep dentinal caries because it can rapidly reduce the bacterial load from the cavities, thus reducing the excessive removal of affected dentin and favoring dental tissue repair. DAE is very commonly used in the world of adhesive dentistry to create micromechanical tags for bonding. The blue color of the etchant is provided by MB dye for precise application during placement, and this MB dye can act as a potent photosensitizer during PDT. The results of the present study rejected the null hypothesis as there was a significant difference in S. mutans colony count and microshear bond strength between all the four groups.

In our study, significant reductions in S. mutans colony counts were observed when aPDT was done with the help of DAE (Group IV) containing MB dye as a photosensitizer [Table 1] and [Figure 1]d, [Figure 2]d. Similarly, when aPDT was done with MB dye, significant %age reductions in S. mutans count were observed and there was an insignificant difference in Group III (MB + L) and Group IV (DAE + L) [Table 1] and [Figure 1]c, [Figure 2]c. aPDT is a two-step process in which neither the light source nor the photosensitizer exerts an effective antimicrobial effect when used alone, but their combination triggers a sequence of biological events that culminate in the microorganism death.[13] ROS are generated upon photosensitizer irradiation with light of appropriate wavelength in the presence of oxygen. The free radicals produced, in turn, promote a bactericidal action by damaging of the cellular plasma membrane and/or damaging of the cell DNA.[14],[15] These results are also in accordance with the findings of the study of Pinheiro et al.[16] and Guglielmi et al.[17] which also claimed a significant bacterial reduction in colony count after PDT.
Figure 1: Streptococcus mutans count before treatment. (a) Group I- DAE (b) Group II- LLL (c) Group III- MB+L (d) Group IV- DAE+L

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Figure 2: Streptococcus mutans count after treatment. (a) Group I- DAE (b) Group II-LLL (c) Group III-MB+L (d) Group IV-DAE+L

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Table 1: Comparative table showing percentage reduction in bacterial count and microshear bond strength (mean±standard deviation) MPa between the four experimental groups

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S. mutans colony count reduction was also seen in Group I (DAE) and Group II (LLL) but significantly less when compared to Group III (MB + L) and Group IV (DAE + L) [Table 1] and [Figure 1]a, [Figure 1]b, [Figure 2]a, [Figure 2]b. The use of DAE alone (Group I) resulted in bacterial reduction, and the reason can be attributed to low pH of phosphoric acid. Acid etching using 32%–37% phosphoric acid (pH 0.1–0.4) not only simultaneously etches enamel and dentin, but also the low pH kills many residual bacteria. Settembrini et al.[18] determined the antimicrobial activity of eight commercially available etchant materials and indicated that all of the etchants demonstrated antimicrobial activity against the bacteria tested. Similarly, Ozel et al.[19] concluded that the use of cavity disinfectants would not be necessary when a phosphoric acid etchant alone is used. The cause of bacterial reduction in Group II, i.e., when only LLL was used, can be explained by the disinfecting property imparted by laser light, but when it is used along with photosensitizer as in Group III and Group IV, its antibacterial efficacy increases many folds. Another possible explanation for microbial reduction in this group is the presence of endogenous dyes produced by bacterial metabolism in the carious dentin which gets activated by diode laser imparting antibacterial effect.[20],[21]

Now when the second parameter, i.e., microshear bond strength of composite to dentin, was assessed after aPDT in all the groups, a significant difference in bond strength values was obtained between Group III (MB + L) and Group IV (DAE + L). There was an insignificant difference in bond strength values between Group I (DAE), Group II (LLL), and Group IV (DAE + L).

In relation to the influence of dyes on the adhesion of composite, the decrease in the bond strength may be due to dye solution remaining in sound and affected dentin as mentioned in other studies.[22] Dye remaining trapped in dentin may adversely affect the wetting of dentin by materials, thereby decreasing micromechanical retention of these adhesive materials. Resin materials are sensitive to previous dentin contamination.[23] During our study, it was observed that even after being rinsed and acid etched, MB dye was not completely removed, as evidenced by some samples remaining lightly colored which might have influenced the results. Contrary to this, a study by Housseiney et al.[12] and Legler et al.[24] showed that the use of caries detector dyes before acid etching did not interfere with the etching patterns as evidenced by scanning electron microscope or composite bonding to either enamel or dentin and attributed this result to either lack of substantial effect of the dyes or to acid etching which dissolves the superficial layers of enamel and dentin which might have allowed removal of the smear layer and the debris left on the surface.

In the present study, the application time of DAE during PDT was 15 s which is the standard time used for etching of dentin prior to the application of adhesive systems. Thus, it can be concluded that DAE-assisted PDT caused a comparable reduction in bacterial count as compared to MB-assisted PDT and also there was no adverse effect on bond strength values. Hence, dentists can perform both procedures simultaneously; that is, PDT can be performed while acid etchant containing MB dye is being applied in the cavity, thus reducing operative time and enhancing cavity disinfection. The tabletop design of our study was in accordance with the studies done previously,[11],[16],[22] so it was possible to calculate bacterial reduction and microshear bond strength values while keeping all the other variables constant. The final objective of our tabletop design was to gather data in prediction of the eventual clinical outcome. However, the limitations were that we had not simulated the oral conditions such as role of saliva and intrapulpal pressure; therefore, further clinical studies/trials are needed to corroborate the results clinically.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Dr. Jaya Gupta
Department of Conservative Dentistry and Endodontics, I.T.S.-C.D.S.R. Muradnagar, Ghaziabad - 201 206, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JCD.JCD_620_20

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