|Year : 2021 | Volume
| Issue : 1 | Page : 39-44
Effect of degree of conversion of different resin composite monomers (methacrylate and silorane) on two caries-associated bacteria: “An in Vitro study”
Marwa Elsheikh1, AS Elkady2, W Abdel Fatah1, Ahmed Musrati2
1 Department of Conservative Dentistry, Faculty of Dentistry, University of Alexandria, Alexandria, Egypt
2 Department of Pediatric Dentistry, Faculty of Dentistry, University of Benghazi, Benghazi, Libya
|Date of Submission||23-Nov-2019|
|Date of Decision||29-Jun-2020|
|Date of Acceptance||11-Jan-2021|
|Date of Web Publication||14-May-2021|
Department of Pediatric Dentistry, Faculty of Dentistry, University of Benghazi, Benghazi
Source of Support: None, Conflict of Interest: None
Background and Objectives: The biological response of oral bacteria to dental restorative polymer composites is mediated by the release of unpolymerized residual monomers. The aims of the present study were first, evaluating the effect of composite elutes from methacrylate composite resins and silorane-based composites on two cariogenic bacterial pathogens: Streptococcus mutans and Lactobacillus acidophilus and second, determination of degree of conversion (DC) of standardized discs made from the low-shrinkage silorane-based composite (FiltekTM P90) resin and methacrylate-based composite resin (FiltekTM Z350XT nanocomposite, FiltekTM Z350 Flow) using Fourier transform infrared spectroscopy (FTIR). Materials and Methods: Thirty-six composite specimens were prepared for the agar diffusion test and dissolved in a solvent (dimethyl sulfoxide) to attain a suspension, which was used to assess the effect of the residual monomers from methacrylate composite resins and silorane-based composites on the growth of S. mutans and L. acidophilus. To avoid more dilutions of the specimens, the colonies of bacteria were counted by the naked eye. Thirty composite specimens of 2 mm × 6 mm were polymerized for DC test, and the DC was measured using FTIR, also for unpolymerized composite resin. Results: The growth of S. mutans bacteria was inhibited when cultured with Filtek Z350 Flow composite resin, while the growth of L. acidophilus bacteria was stimulated by the Filtek Silorane p90 and Filtek Z350 Flow composite resin. The DC test with the Filtek Silorane composite resin showed highest DC (58%), then the Filtek Z350XT (Nanocomposite) (42%) followed by the Filtek Z350 Flow (Flowable) showed lowest (35%). Conclusions: Our results demonstrate that composite filling materials have a versatile nature of effect on oral pathogenic bacteria, which could modulate their pathogenesis. Dentists may thus select the appropriate type of composite filling according to the caries susceptibility of patients.
Keywords: Bacterial growth, biocompatibility, composite monomer
|How to cite this article:|
Elsheikh M, Elkady A S, Fatah W A, Musrati A. Effect of degree of conversion of different resin composite monomers (methacrylate and silorane) on two caries-associated bacteria: “An in Vitro study”. Dent Med Res 2021;9:39-44
|How to cite this URL:|
Elsheikh M, Elkady A S, Fatah W A, Musrati A. Effect of degree of conversion of different resin composite monomers (methacrylate and silorane) on two caries-associated bacteria: “An in Vitro study”. Dent Med Res [serial online] 2021 [cited 2021 Jun 17];9:39-44. Available from: https://www.dmrjournal.org/text.asp?2021/9/1/39/315964
| Introduction|| |
The biocompatibility of a dental resin composite is determined by several factors. To determine these factors, one must especially consider the amounts and types of compounds that were eluted from the set material.
Various studies have shown that residual monomers, for example, Bis-GMA, TEGDMA, UDMA, and other components, such as initiators, activators, or inhibitors, are released from polymerized composites depending on the monomer–polymer conversion rate.
As with any ecological niche present in nature, the microbial community of dental plaque is prone to physiological and compositional shifts as a result of environmental stresses and selective pressures. Streptococci and Lactobacilli were specially identified in a plaque located at the margins of composite fillings.
The low degree of conversion (DC) was an indication reported for the incomplete polymerization of resin composites, the finding that unpolymerized monomers can be extracted and used to accelerate the growth of cariogenic bacteria. However, it is still debatable whether polymerized resin composites accelerate Streptococcus mutans growth in vitro.
The DC of light-cure composite resins is the degree by which carbon double bond C = C is converted into a single carbon bond C–C and the extent to which the monomer is converted to polymer during the polymerization reaction.
Silorane-based composite restorative (Filtek P90 Silorane) exhibits good biocompatibility characteristics with low mutagenic potential; it also has a significantly lower susceptibility to adhere to Streptococci than conventional methacrylate-based composite resins. This might be due to its increased hydrophobicity because of their siloxane backbone.
Flowable composite restorative materials are made flowable by the addition of lower molecular weight resin diluents which may exhibit increased mass release and therefore increased cytotoxicity. The fact that the flowable composite has less filler and more monomers than the conventional composite could also affect the components leached from it and subsequently its biocompatibility. Different substances such as residual monomers, additives, and degradation products may irritate the oral soft tissues, which stimulate the growth of bacteria and promote allergic reactions.
TEGDMA can potentially interfere with intracellular functions by easily penetrating membranes and cross-reacting with intracellular molecules such as glutathione and possibly depleting the ability of cells to partake in detoxification reactions.
In this in vitro study, we tested the hypothesis that the capability of residual resin monomers leached from three different composite resins (Z350 XT, Silorane, and Z350 flow) inhibits, in a differential pattern, the growth of cariogenic bacteria S. mutans and Lactobacillus acidophilus.
| Materials and Methods|| |
In this study, three composite restoration materials were used: FiltekTM Silorane p90 (Microhybrid composite), FiltekTM Z350 XT (Nanofilled composite), and FiltekTM Z350 flow (Flowable composite) [Table 1]. Pure potassium bromide (KBR) and chloroform were used for DC measurement. For microbiology test S. mutans (ATCC 25175), L. acidophilus (ATCC 4356), 99% Dimethyl sulfoxide (DMSO) C2H6OS, brain heart infusion (BHI) broth and agar and anaerobic gas packs are used.
Degree of conversion test
Thirty composite specimens for DC test were prepared and divided as follows: 10 specimens FiltekTM Silorane p90, 10 specimens FiltekTM Z350XT, and 10 specimens FiltekTM Z350 flow.
Composite specimens were 2 mm in height and 6 mm in diameter, formed in prefabricated special-split Teflon mold that had two halves. The composite materials were condensed incrementally in the Teflon mold and covered with celluloid strip, then cured for 40 s for each increment by MiniLED light-curing unit (600 mW/cm2) intensity in such a way that the curing tip was placed in direct contact with mold and perpendicular to it.
The DC of composite resin was measured using Fourier transform infrared spectroscopy (FTIR).
Four milligram of the composite resin powder, crushed manually in agate mortar, were measured by a digital microbalance and mixed with 200 mg of pure KBr in an agate mortar to obtain a total of 204 mg which was suitable for achieving good spectra. KBr is transparent in the mid-IR region (from 400 to 4000 cm−1 from the spectrum).
The mixture was then pressed under a hydrolytic vacuum pump at 80 kN for 2 min to form a disc of 12 mm diameter of the KBR containing the composite material to be tested. The composite discs were put in holder attachment and inserted in the FTIR spectrometer, and then, they were exposed to 120 scans at a resolution of 4 cm−1.
The uncured specimens were prepared by taking 4 mg composite resin from each composite resin syringe using a digital balance, then diluted in 2 ml of chloroform, and poured in a liquid cell, then inserted inside the FTIR where they were exposed to 120 scans at a resolution of 4 cm−1. The solution spectrum was recorded.
Degree of conversion determination
The DC for methacrylate-based composite resin (Filtek Z350XT, Filtek Z350 flow) was determined by calculating the absorbance peak intensities of aliphatic C = C at 1638 cm−1 and carbonyl C = O at 1720 cm−1, before and after polymerization.
The carbonyl C = O band intensities were used as an internal standard because it was remained constant and not affected by the polymerization process.
On the other hand, the aliphatic C = C peak intensities were reduced after polymerization due to the breakdown of these bonds during the polymerization process.
The DC% was calculated according to the following equation:
C = C aliphatic was at an absorption peak at 1638 cm−1 of the cured specimen. C = O carbonyl was at an absorption peak at 1720 cm−1 of the cured specimen.
C = C aliphatic was at an absorption peak at 1638 cm−1 of the uncured specimen. C = O carbonyl was at an absorption peak at 1720 cm−1 of the uncured specimen.
The DC for the silorane-based composite resin (Filtek Silorane P90) was determined by calculating the absorbance peak intensities of oxirane C-O-C at 882 cm−1 and aromatic C-H at 1275 cm−1, before and after polymerization, the aromatic C-H peak intensities used as an internal standard, because it was remained constant as an aromatic ring and not affected by the polymerization process, also the oxirane C-O-C peak intensities were reduced after polymerization due to the opening of the oxirane ring during the polymerization process.
The DC percent was calculated according to the following equation:
C-O-C oxirane was at an absorption peak at 882 cm−1 of the cured specimen. Aromatic C-H was at an absorption peak at 1257 cm−1 of the cured specimen.
C-O-C oxirane was at an absorption peak at 882 cm−1 of the uncured specimen. Aromatic C-H was at an absorption peak at 1257 cm−1 of the uncured specimens.
Thirty-six composite specimens for microbiology test were prepared 12 specimens for each type of composite.
An agar diffusion test was used to assess the effect of the residual monomers from methacrylate composite resins and silorane-based composites on the growth of cariogenic bacterial pathogens S. mutans and L. acidophilus.
The mean count of S. mutans control group was 15.0 ± 2.83, Filtek Silorane was 10.0 ± 1.87, Filtek Z350XT was 10.20 ± 2.49, and Filtek Z350 flow composite was 6.0 ± 1.58. The difference between Filtek Z350 flow composite and the control groups was statistically significant (P = 0.002), while the differences between the Filtek Silorane composite and both the control group and Filtek Z350XT composite were not statistically significant (P = 0.087 and P = 0.104; respectively) [Table 3] and [Figure 2].
|Figure 1: Comparison between different composite groups according to the degree of conversion %|
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|Figure 2: Comparison between the effect of composite on Streptococcus mutans|
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|Table 2: Comparison between the degree of conversion of Filtek Silorane, Filtek Z350XT, and Filtek Z350 flow composite|
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|Table 3: Comparison between different studied composite groups (Filtek Silorane, Filtek Z350XT, and Filtek Z350 flow) effect on Streptococcus mutans|
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The media was prepared by suspending 37 g BHI powder in deionized water soaked for 10 min then dissolved with gentle heat before dispensing into test tubes followed by autoclaving at 121°C for 15 min. The BHI agar was prepared by adding 0.2% agar to the BHI or suspending 52 g BHI agar/liter to broth followed by autoclaving at 121°C for 15 min.
Blood agar plates were prepared by suspending 40 g in 1 l of purified water, mixed well and dissolved by heating with frequent agitation, boiled for 1 min until complete dissolution, sterilized by autoclaving for 15 min at 121°C, cooled to 45°C–50°C, then added 5% defibrinated blood and homogenized gently. The blood agar was evenly distributed over the surface of 15 cm in diameter sterile Petri dishes to a thickness of 5 mm.
Preparation of bacteria
Two lyophilized cariogenic bacteria stocks S. mutans (ATCC 25175) and L. acidophilus (ATCC 4356) were cultured according to the standard instructions given by the suppliers and cultured on blood and BHI agar Petri dishes by incubation in an anaerobic jar (at 37°C in the incubator for 72 h).
Composite specimens were prepared in the Teflon mold in tablet shape with 2 mm × 6 mm dimensions in a strict sterile condition, and then the specimen was crushed with a sterile orthodontic plier.
For preparation of composite suspension (CS), after 1 h of curing of a composite specimen, 0.3 g composite powder had been dissolved in 1.5 ml of 99% concentration of DMSO (C2H6OS) composite solvent used for monomer elusion in a sterile test tube and kept for 24 h to allow residual monomer elusion.
Agar diffusion test
One liter of BHI media was prepared and distributed in sterile test tubes, 9.8 ml in each tube.
S. mutans and L. acidophilus were taken from the BHI agar plates and each suspended by a calibrated sterile loop in 5 sterile test tubes containing the previously prepared BHI media to give turbidity equal to 0.5 McFarland.
Two hundred microliter of the tested CS was added by automatic pipette to 9.8 ml BHI media containing the S. mutans in five sterile tubes to get 2% of DMSO in the media.
For each type of bacteria, two control tubes were prepared: one contained bacteria and DMSO by 2% concentration in BHI media and the other control contained the bacteria and BHI media. The tubes were then incubated anaerobically at 37°C for 48 h. This was done for each type of composite against each type of bacteria separately.
Tenfold serial dilutions were done in sterile Eppendorf for each tube by taking 100 µl from each tube and adding to 900 µl of BHI to give a dilution of 10−1, this was repeated to reach the concentration of 10−6.
Each petri dish was divided into six sections indicating the concentrations, then 10 µl of each diluted Eppendorf taken by automatic pipet and spread in its marked section on the petri dish, followed by incubation of the Petri dishes anaerobically at 37°C for 72 h.
Colony counting was performed on blood and BHI agar plates by vision for both S. mutans and L. acidophilus. The number of colonies was counted in each sector and multiplied by dilution factor to get the final count.
| Results|| |
Degree of conversion
The mean DC % of Filtek Silorane was 58.30 ± 3.33, Filtek Z350XT: 42.90 ± 2.67, and Filtek Z350 flow: 35.82 ± 4.75 [Table 2] and [Figure 1]. The differences between the three composite groups were statistically significant (P < 0.001*).
The mean count of S. mutans control group was 15.0 ± 2.83, Filtek Silorane was 10.0 ± 1.87, Filtek Z350XT was 10.20 ± 2.49, and Filtek Z350 flow composite was 6.0 ± 1.58. The difference between Filtek Z350 flow composite and the control groups was statistically significant (P = 0.002), while the differences between the Filtek Silorane composite and both the control group and Filtek Z350XT composite were not statistically significant (P = 0.087 and P = 0.104; respectively).
For L. acidophilus, the composite groups appeared to exert stimulatory effects on the bacteria [Table 4] and [Figure 3].
|Figure 3: Comparison between effects of composite groups on Lactobacillus acidophilu|
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|Table 4: Comparison between different studied composite groups (Filtek Silorane, Filtek Z350XT, and Filtek Z350 flow) effect on L. acidophilus bacteria|
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The mean count of L. acidophilus control group was 0.82 ± 0.96, Filtek Silorane was 5.0 ± 1.58, Filtek Z350XT was 3.80 ± 0.84, and Filtek Z350 flow composite was 4.80 ± 0.84. Both differences between each of Filtek Z350 flow composite and the control groups, Filtek Silorane composite and the control groups were statistically significant (P = 0.009, P = 0.0006; respectively), while the difference between the Filtek Z350XT composite and control was not statistically significant (P = 0.104).
| Discussion|| |
A major concern exists about composite toxicity and consequently their effect on human health. Different substances such as residual monomers, additives, and degradation products may irritate the oral soft tissues, stimulate the growth of bacteria, reach the pulpal tissues, and promote allergic reactions.
This study was conducted to compare the DC of Filtek Silorane P90, Filtek Z350XT (nanocomposite), and Filtek Z350 flow composite and to compare the effect of their residual monomers on the growth of two main cariogenic bacterial pathogens, i.e., S. mutans and L. acidophilus.
Degree of polymerization is among the factors that critically affect the clinical performance of resin composites to determine the DC %. FTIR has been widely used as a reliable method as it detects the C = C stretching vibrations directly before and after curing of materials.
Different studies, indicated the important role of the type of the solvent, and that irreversible processes, such as leaching of components, may occur (e.g., in the presence of DMSO). A close match in the solubility parameter resulted in maximum softening of the resin.,
The DC% of Filtek Silorane ranged from 55% to 65%, which was comparable to the range found in another study under the same conditions.
In agreement with the present study, Durner et al. postulated that Filtek Z350XT composite with DC % in the range of 46.5 after 40 s of polymerization has a high significant inverse correlation between the DC% and the amount of eluted camphorquinone (CQ), EGDMA, and TEGDMA. This implies that the amount of CQ in the elution medium significantly decreased; thereby, a higher DC was expressed by Filtek Z350XT composite resin.
The increase in DC% of Filtek P90 composite resin than Filtek Z350XT composite resin and Filtek Z350 flow was due to limitations on the mobility of the reactive species imposed by the rapid formation of a cross-linked polymeric network in methacrylate-based composites in comparison to increased chain mobility in silorane-based dental composite.
The release of substances from polymerized dental composites is due to the low DC of methacrylate-based monomers provided that the monomers remain fixed in the polymer network and it was unlikely that the monomer can be eluted undestroyed.
The initial aim of the present study was to evaluate either growth stimulation or inhibition of S. mutans and L. acidophilus) by various composite types in vitro. The bacteria were inoculated in liquid cultures.
The present study results showed statistically significant inhibition of S. mutans bacterial strains growth with Filtek Z350 flow composite in comparison with statistical insignificant inhibition Filtek Silorane and Filtek Z350XT composite which, on the other side, means more cytotoxic effect on the pulp.
Kraigsley et al. evaluated results from biofilms cultured on polymer disks with varying DC values; they demonstrated that the metabolic activity of S. mutans was decreased as DC decreased. Leachable components from low DC polymers had a negative effect on the metabolic activity of S. mutans similar to that seen in biofilms cultured directly on low DC polymers.
In our study, all the three composite groups resulted in a marked increase in the bacterial count of L. acidophilus bacteria compared to the control suspensions with the different composite tested, there was no significant statistical difference between the Filtek Z350XT composite and the control, but there was a significant statistical difference between Filtek Z350 flow composite and the control.
It is obvious that there was a direct correlation between the resin (DC %) and the number of eluted monomers and thus the cytotoxic effect of different resin monomers. This was probably because the lower DC can cause either growth inhibition or stimulation of two caries-associated bacteria (S. mutans and L. acidophilus) since S. mutans was considered as the main initiating bacteria in the caries process, while L. acidophilus helps in the progression of caries.
| Conclusions|| |
Within the limitations of the current study, the following were concluded:
- According to DC test, the Filtek Silorane composite resin showed the highest DC (58%) compared with Filtek Z350XT (nanocomposite) (42%) and Filtek Z350 Flow (Flowable) which showed the lowest (35%)
- The growth of S. mutans bacteria was inhibited when cultured with Filtek Z350 Flow composite resin
- L. acidophilus bacteria growth was stimulated by the Filtek Silorane and Filtek Z350 Flow composite resin.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4]