Next Article in Journal
Comprehensive Bioinformatics Analysis Reveals the Potential Role of the hsa_circ_0001081/miR-26b-5p Axis in Regulating COL15A1 and TRIB3 within Hypoxia-Induced miRNA/mRNA Networks in Glioblastoma Cells
Previous Article in Journal
Common Variants in the TYR Gene with Unclear Pathogenicity as the Cause of Oculocutaneous Albinism in a Cohort of Russian Patients
Previous Article in Special Issue
In Silico Investigation of the Interlaminar and Mechanical Fracture of Arteries with Atheromatic Plaque during Angioplasty Treatment
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Biomedicines
Volume 12
Issue 10
10.3390/biomedicines12102235
biomedicines-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Influence of Polymerization Protocol on Adhesion and Proliferation of Streptococcus mutans on Three Dental Composite Resins

by
Francesco De Angelis
1,*,†,
Camillo D’Arcangelo
1,†,
Silvia Di Lodovico
2,
Edoardo Sorrentino
1,
Matteo Buonvivere
1,‡ and
Simonetta D’Ercole
1,‡
1
Department of Medical, Oral and Biotechnological Sciences, “G. d’Annunzio” University of Chieti–Pescara, Via dei Vestini 31, 66100 Chieti, Italy
2
Department of Pharmacy, “G. d’Annunzio” University of Chieti–Pescara, Via dei Vestini 31, 66100 Chieti, Italy
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
‡
These authors also contributed equally to this work.
Biomedicines 2024, 12(10), 2235; https://doi.org/10.3390/biomedicines12102235
Submission received: 5 August 2024 / Revised: 23 September 2024 / Accepted: 27 September 2024 / Published: 1 October 2024
(This article belongs to the Collection Feature Papers in Biomedical Materials)
Browse Figures
Versions Notes

Abstract

:
Background/Objectives: The aim of this in vitro study was to analyze and compare the Streptococcus mutans ability to adhere and form biofilm on the surface of light-cured VS heat-cured dental composite resins; Methods: Three composite resins with different chemical formulations were selected: GrandioSO (GR), Venus Diamond (VD) and Enamel Plus Hri Biofunction (BF). Disk-shaped specimens were manufactured by light-curing the composite resins (light-cured subgroups) and subjecting them to a further heat-curing cycle at 80° for 10 min (heat-cured subgroups). Specimens were analyzed for planktonic CFU count (CFU/mL), sessile CFU count (CFU/mL) and for biomass quantification (OD570nm); Results: The planktonic CFU count was higher in all the light-cured subgroups than in the heat-cured subgroups (light-cured: GR = 7.23 × 106, VD = 2.14 × 107, BF = 4.40 × 107; heat-cured: GR = 4.89 × 106, VD = 4.95 × 106, BF = 2.80 × 107), with a statistically significant increase for BF and VD. Focusing on the sessile CFUs, both GR (light-cured = 7.49 × 106; heat-cured = 3.97 × 106) and VD (light-cured = 2.93 × 107; heat-cured = 6.07 × 106) showed a significantly increased number of colonies in the light-cured subgroups. The OD570nm values recorded for the light-cured BF subgroup (0.4280) were significantly increased compared to the heat-cured BF subgroup (0.1931); Conclusions: A more complete polymerization protocol seems to lead to a potential reduction in the risk of secondary caries.

1. Introduction

Dental decay is a disease with multifactorial etiology. It is caused by cariogenic bacteria in the presence of fermentable carbohydrates, but is also influenced by external factors and individual susceptibility. The Gram-positive Streptococcus mutans is one of the main cariogenic bacteria. It survives at low pH and its pathogenesis is related to the production of organic acids following carbohydrate metabolism [1,2]. Streptococcus mutans acid release seems to act even on dental composites, thus predisposing pre-existing restorations to the risk of secondary caries [3,4]. The longevity of the restoration can also be affected by the shrinkage of the composite during polymerization, which may promote the occurrence of tooth decay [5]. Depending on the more or less virulent microbial strain, the esterase activity of Streptococcus mutans can deteriorate [6] and modify the surface morphology of composite resins [7]. Also, it may inhibit the activity of glycolysis-related enzymes [8]. Streptococcus mutans is able to form biofilm on solid surfaces [9,10,11,12] following glucosyltransferases (GTFs) production to catalyze extracellular polysaccharides synthesis (EPS) [13,14]. Its ability to form biofilm is greater than many other Streptococcus spp. that populate the oral cavity environment [15,16]. Some specific adhesins of Streptococcus mutans, known as dual antigen I/II, promote biofilm formation, mediating bacterial attachment to the tooth’s salivary pellicle [17,18,19] and interacting with other bacteria and host proteins [17,18,19,20]. Biofilm roughens and degrades the surfaces of composite resins [7], promoting bacterial colonization at the tooth–composite interface [21] and resulting in secondary caries [22] and pulpal disease [23]. Surface roughness (Ra) and surface free energy can affect bacterial adhesion [24]. A Ra greater than 0.2 µm is associated with greater bacterial colonization [25]. Lifshitz–Van der Waals forces, acid-base and electrostatic interactions generally affect bacterial adhesion to surfaces [26]. According to the DLVO (Derjaguin–Landau–Verweij e Overbeek) theory, these forces can be combined [27].
A polymerization reaction is defined as the degree of conversion of monomers into polymers. Direct resin composite restorations are light-cured chairside for 20–40 s, while indirect restorations typically undergo additional extra-oral heat-curing cycles before an effective intra-oral adhesive cementation [28,29,30]. Light-curing reactions never guarantee total monomeric conversion [31]; in fact, up to 45% of unreacted monomers may remain unpolymerized [32]. A heat-curing procedure promotes greater, even if not complete, monomer conversion into polymer chains [33,34], enhancing properties such as microhardness, flexural strength, fracture toughness, wear resistance, tensile strength and color stability [35,36,37,38].
Heat-curing seems also to reduce monomer leachability into saliva [39], which appears as relevant since previous research suggested that the release of ethylene glycol-dimethyl acrylate (EGDMA) and triethylene glycol-dimethacrylate (Teg-GDMA) from composite resins might supposedly enhance the growth of some Streptococcus spp. [40]. It has been observed that the light-curing time may affect bacteria surface colonization on resin composites [41]. For Kraigsley and coll. [42], the degree of conversion of cross-linked dimethacrylate polymers alters the biofilm metabolic activity. Furthermore, according to D’Ercole and coll. [43], in vitro bacterial adhesion and proliferation can be affected by the composite composition. Despite the well-known improvements in mechanical properties achieved following heat-curing protocols, studies concerning their effects on bacterial adhesion and proliferation are still lacking. Thus, the aim of this preliminary in vitro study was to analyze and compare the ability of Streptococcus mutans to adhere, proliferate and form biofilm on the surface of three commercially and nanohybrid available light- and heat-cured dental composite resins (GrandioSO: GR; Venus Diamond: VD; Enamel Plus Hri Biofunction: BF), each having different chemical formulations. These particular materials were selected because they had been already tested in presence of another common commensal cariogenic agent [44]. The null hypothesis was that no difference in terms of antibacterial and antibiofilm properties among chemically different and differently cured resin composites could be detected.

2. Materials and Methods

A summary of the experimental groups and subgroups, together with a list of the materials included in the present experimental design, is given in Table 1.

2.1. Realization of Composite Disks

Disk-shaped specimens were manufactured by placing uncured composite resin in polyvinylsiloxane molds (2 × 4 mm). The filled molds were positioned between two glass slides held in place with a paper clip to extrude the surplus material. As summarized in Table 1, for light-cured subgroups, a light-emitting diode curing unit (Celalux 3, VOCO, Cuxhaven, Germany) with an 8 mm tip diameter and an output power of 1300 mW/cm2 was used to cure each disk for 20 s from the top surface and 20 s from the bottom surface [43]. Each composite disk had a total surface area of 50.27 mm2. All disks were washed in an ultrasonic bath with distilled water for 10 min.
For heat-cured subgroups, disk-shaped specimens were made following the same protocol described for light-cured subgroups. Moreover, following the ultrasonic bath, samples received additional heat-polymerization in a composite heat-curing unit at 80 °C for 10 min (LaborLux, Micerium, Avegno, Genova, Italy).
The sample size for the present research was established, taking into account previous similar studies [43,44]. On this basis, a total of 339 light-cured and 339 heat-cured composite disks were manufactured for each different resin composite under investigation and used for the subsequent experimental steps.

2.2. Saliva Collection

According to a previously described protocol, human saliva samples were taken from healthy volunteers with an age > 18 years. The Ethics Committee of University “G. d’Annunzio”, Chieti–Pescara, Italy (approval code SALI, N. 19 of the 10 September 2020), approved the collection and the use of saliva [45].
Saliva was pooled, mixed, centrifuged (16.000× g for 1 h at 4 °C) and filtered from microorganisms (filters diameters: 0.8 μm, 0.45 μm, and 0.2 μm). To consider saliva samples sterile, no detected bacterial growth in both aerobic and anaerobic atmospheres after incubation for 48 h at 37 °C is required [45]. Sterile saliva was collected and kept frozen in sterile tubes until the study was carried out.

2.3. Bacterial Strain

The clinical bacterial strain Streptococcus mutans CH02 was isolated from the oral cavity of patients at the dental clinic of the University “G. d’Annunzio”, Chieti–Pescara [43]. The frozen (−80 °C) strain was recovered in Brain Heart Infusion broth (BHI, Oxoid, Milan, Italy) overnight at 37 °C under an anaerobic condition. The broth culture was diluted 1:10 in BHI broth containing 1% (w/v) sucrose and refreshed for 2 h at 37 °C in a shaking thermostatic water bath (120 rpm). Bacterial suspension was prepared using a spectrophotometer (Eppendorf) to obtain an optical density of OD600 = 0.125 corresponding to 9 × 106 CFU/mL.
All composite disk specimens were placed in 96-well polystyrene microtiter plates and the top and bottom surfaces were sterilized through the action of ultraviolet UV lights for 30 min.
To form the protein pellicle layer on the surface, sterile specimens were inoculated for 2 h in saliva at 37 °C in a shaking incubator. Biofilms were grown on each composite disk by inoculating 200 µL broth culture of Streptococcus mutans CH02 and incubating at 37 °C for 24 h under anaerobic conditions, and then for another 24 h in an aerobic atmosphere. As a negative control, salivary film-coated disks were incubated in bacteria-free culture media to confirm the sterility of the disks and the adsorption of the biomass dye.
After incubation, microbial growth was quantitatively analyzed for the following aspects:
(I)
Planktonic CFU count of the bacterial cells (CFU/mL);
(II)
Sessile CFU count of the cultivable cells on composite disks (CFU/mL);
(III)
Biomass evaluation of the biofilm produced on composite disks using Hucker’s crystal violet staining method (OD570nm).
Each quantitative test was performed in triplicate for three independent experiments.
Moreover, representative specimens from each subgroup were qualitatively analyzed to assess the biofilm morphology via Scanning Electron Microscope (SEM) evaluation.

2.4. Planktonic CFU Count

For the planktonic CFU count, the planktonic bacterial phase removed from the wells containing the resin disks was vortexed, diluted in PBS and spread on Tryptic Soy Agar (TSA) plates. Then, it was incubated in anaerobic conditions for 48 h at 37 °C. The CFU/mL count was performed. For the detection, 9 disks for the light-cured and 9 disks for the heat-cured subgroups of each composite resin material in triplicate were analyzed (9 light-cured GR and 9 heat-cured GR disks, 9 light-cured VD and 9 heat-cured VD disks, 9 light-cured BF and 9 heat-cured BF disks) for a total of 81 light-cured and 81 heat-cured disks.

2.5. Sessile CFU Count

The disks were placed in a sterile test tube containing 1 mL PBS. Then, each test tube was placed in a 4 kHz ultrasonic water bath (Euronda, Vicenza, Italy) for 3 min, followed by vortex mixing for 2 min to detach the Streptococcus mutans CH02 adhering to the surface of each disk. The live/dead analysis was carried out to evaluate the non-negative effect of the sonication and the vortex. The samples were diluted with PBS and spread on TSA plates. Finally, they were incubated for 48 h under anaerobic conditions at 37 °C, followed by the counting of CFU/mL. For this detection, 9 disks for the light-cured and 9 disks for the heat-cured subgroups of each composite resin material in triplicate were analyzed (9 light-cured GR and 9 heat-cured GR disks, 9 light-cured VD and 9 heat-cured VD disks, 9 light-cured BF and 9 heat-cured BF disks) for a total of 81 light-cured and 81 heat-cured disks.

2.6. Biomass Quantification by Optical Density (OD570nm)

Crystal-violet (CV) staining [44,46,47,48] was used to evaluate relative biofilm biomass formed by Streptococcus mutans CH02 on composite surfaces after 48 h for biofilm formation. The disks were washed three times with PBS, left to air dry, stained with crystal-violet 0.1% (Sigma–Aldrich, Milan, Italy) for 1 min and washed with PBS. Then, the CV was eluted with ethanol and the biofilm formation was quantified by measuring absorbance at 570 nm with a microplate reader (SAFAS, Munich, Germany). For this detection, 18 disks for each light-cured and 18 disks for each heat-cured subgroup were analyzed in triplicate, including negative controls (9 light-cured GR and 9 light-cured GR negative controls, 9 heat-cured GR and 9 heat-cured GR negative controls, 9 light-cured VD and 9 light-cured VD negative controls, 9 heat-cured VD and 9 heat-cured VD negative controls, 9 light-cured BF and 9 light-cured BF negative controls, 9 heat-cured BF and 9 heat-cured BF negative controls), for a total of 162 light-cured and 162 heat-cured disks.

2.7. Scanning Electron Microscope (SEM) Analysis

After 48 h of in vitro biofilm formation, representative specimens from each subgroup were fixed for 1 h in 2.5% glutaraldehyde, dehydrated in six ethanol washes (10%, 25%, 50%, 75% and 90% for 20 min and 100% for 1 h), and dried overnight in a bacteriological incubator at 37 °C. Then, they were observed using a SEM (EVO 50 XVP LaB6, Carl Zeiss SMT Ltd., Oberkochen, Germany) at 15 kV, under 500×, 1000× and 2000× magnifications after gold-sputter coating (Emitech K550, Emitech Ltd., Montigny Le Bretonneu, France). A total of 5 composite disks for each light-cured subgroup and 5 disks for each heat-cured subgroup were addressed via SEM analysis.

2.8. Statistical Analysis

Means and standard deviations for data coming from the quantitative experiments (planktonic CFU count, sessile CFU count, biomass quantification by OD570nm) were calculated in each subgroup. Statistical analysis was performed using SPSS for Windows version 21 (IBM SPSS Inc., Armonk, NY, USA), by means of Two-Way Analysis of Variance (ANOVA) and Tukey tests for post hoc intergroup comparisons. p-values less than 0.05 were considered significant.

3. Results

The detailed results (means and standard deviations) of the quantitative tests performed on the three composites that were analyzed and polymerized according to the two curing protocols under investigation are summarized in Table 2.
The planktonic CFU counts recorded for BF (light-cured = 4.40 × 107 CFU/mL; heat-cured = 2.80 × 107 CFU/mL) were significantly higher than those obtained for GR (light-cured = 7.23 × 106 CFU/mL; heat-cured = 4.89 × 106 CFU/mL) and VD (light-cured = 2.14 × 107 CFU/mL; heat-cured: 4.95 × 106 CFU/mL), concerning both curing protocols. No statistically significant differences were recorded between the heat-cured GR subgroup and the heat-cured VD subgroup (p < 0.05).
The sessile CFU counts recorded on both GR (light-cured = 7.49 × 106 CFU/mL; heat-cured = 3.97 × 106 CFU/mL) and VD (light-cured = 2.93 × 107 CFU/mL; heat-cured = 6.07 × 106 CFU/mL) were significantly higher in the light-cured subgroups compared to the heat-cured subgroups. No significant differences were recorded between the light-cured (6.71 × 106 CFU/mL) and heat-cured (6.38 × 106 CFU/mL) BF subgroups (p < 0.05). Specifically, the light-cured VD subgroup had the highest CFU count for the sessile phase cells ever.
The biomass quantification by optical density recorded in the light-cured BF subgroup (0.4280 OD570nm) was significantly higher than for the same heat-cured material (0.1931 OD570nm) (p < 0.05). No significant differences were recorded between the light-cured (0.1325 OD570nm) and the heat-cured (0.1464 OD570nm) GR subgroups and between the light-cured (0.1457 OD570nm) and the heat-cured (0.1731 OD570nm) VD subgroups.
SEM microphotographs, taken at 1000× and 3000× magnifications, showing the Streptococcus mutans CH02 cells adherent on the surface of the three light and heat-cured composite resins, are given in Figure 1, Figure 2 and Figure 3. An increased number of sessile colonies were observed in the light-cured subgroups.

4. Discussion

The null hypothesis tested in the present study had to be rejected. In the present study, significant differences in terms of bacterial growth and biofilm formation in the presence of chemically different and differently cured resin composites were recorded. Both the specific composite resins composition and the polymerization protocol proved to be relevant factors, able to significantly influence the behavior of Streptococcus mutans.
In the present study, the three composite resins tested (Table 1) were selected because they are well-known and widely used nanohybrid dental composites that significantly differ one another in terms of formulation, both concerning the resin matrix chemicals and the filler load, size and composition. Furthermore, previous research [44] has recently analyzed the effect of these three resin composites on the adhesion and proliferation of a common commensal cariogenic agent, Candida albicans [49,50,51,52], making it relevant to further investigate the behavior of the main cariogenic bacteria, Streptococcus mutans, in the presence of the same materials.
Specific characteristics of each different material, surface properties in particular, may influence bacterial adhesion [25]. A reduced organic matrix microhardness favors inorganic fillers exposure following material wear by attrition [53]. Nanometric-sized composite fillers lead to superior esthetic characteristics due to their reduced dimensions and better distribution [54,55]. Also, the amount, shape and size of the filler can affect the bacterial adhesion to the composites surface [53,54,56]. A larger filler dimension promotes greater roughness [55,57,58,59,60]. Smooth surfaces show a reduced bacterial adherence and accumulation than rough surfaces [59], retarding biofilm adherence and growth [61]. In the present study, the small size of the micrometric filler in GR (max 1 µm) and BF (max 3 µm) composites compared to the VD composite (max 20 µm) could hypothetically justify the increased sessile proliferation of Streptococcus mutans CH02 on the latter material. In order to limit surface differences, in the present protocol, all composite disks were light-cured within polyvinylsiloxane molds and between two glass slides, which guaranteed standardized and predictably smooth surfaces, with no need of any final polishing steps. Consequently, it was possible to exclude the potential confounding effect of any difference in surface roughness among hand-polished materials, focusing the attention just on the two variables under investigations (i.e., curing protocol and composite formulation). Moreover, the use of molds and glass slides allowed to prevent the composite material from coming into contact with oxygen, avoiding the formation of any oxygen inhibition layer (OIL) on the composite surface. OIL may negatively affect the polymerization reaction, reducing the monomeric degree conversion [62,63,64,65,66]. Additionally, the tested composite samples were coated with salivary film, which promotes oral bacteria binding to salivary receptors [67].
Low volumetric shrinkage, good reactivity and mechanical properties are among the main features that justify the use of bisphenol A-glycidyl methacrylate (Bis-GMA) as the most common monomer for dental composite formulation [68]. However, in resin-based dental materials, Bis-GMA is generally mixed with other monomers, such as TEG-DMA and EGDMA, in order to optimize its low viscosity [69,70,71,72,73,74]. In recent years, concerns have been raised as a result of the observed estrogenic action of bisphenol A (BPA) [75] following the salivary hydrolysis of Bis-GMA-based resins [76]. Thus, Bis-GMA free dental resin composites have been introduced, replacing Bis-GMA with different monomers that do not release BPA, such as urethane-dimethacrylate (UDMA). Bis-GMA replacement still needs further investigation [77]. Based on Kim et al., Bis-GMA seems to inhibit the planktonic growth and the viability of Streptococcus mutans [78]. Accordingly, Lin et al. [79] recorded a reduced Streptococcus mutans activity and biofilm formation in the presence of Bis-GMA. For these reasons, in the present study, a (Bis-GMA)-based (GR) and two (Bis-GMA)-free dental resin composites (VD and BF) were selected. Hansel et al. [40], on the other hand, observed that the release of other co-monomers (such as TEGDMA and EGDMA) could be somehow able to promote the growth of some Streptococcus spp. This second mechanism, combined with the well-known reduction in monomers released from heat-cured composites [80], might hypothetically justify the reduced number of sessile Streptococcus mutans colonies herein observed on heat-cured materials. Regarding the planktonic CFU count, data recorded on the (Bis-GMA)-free BF were significantly higher than those obtained on the (Bis-GMA)-based GR, both in light-cured and heat-cured subgroups, supporting the potential inhibitory role of Bis-GMA. However, significantly reduced planktonic colonies were observed also with the (Bis-GMA)-free VD, suggesting that such a planktonic Streptococcus mutans decrease could also be related to the action of other monomers, or a combination of them [43]. Focusing on the Streptococcus mutans sessile colonies, the light-cured VD (Bis-GMA-free) resulted in a significant increase in the CFU/mL count compared to the light-cured BF (Bis-GMA-free as well) and to the light-cured GR (Bis-GMA-based).
Various heat-curing protocols have been described in the literature, whose times ranged between 10 and 6 h, with different maximum temperature values [81,82,83,84]. A 10 min heat-curing treatment has been shown to be adequate to increase composite properties [85]. Also, in the oral cavity, temperatures can change [86]. Dental materials that resist oral cavity variations up to a temperature of 77.4 °C (i.e., drinking hot fluids) daily can preserve their physical and chemical properties [86]. High temperatures can be responsible of an irreversible deformation and changes in the materials properties [86], justifying the chosen temperature (80 °C) used in our experimental study. Heat-curing protocols promote greater monomer-to-polymer conversion [33,34], limiting monomer leaching [87,88,89]. Brambilla et al. [41] already showed how reduced curing times could be responsible for the increased in vitro colonization of composite surfaces by Streptococcus mutans. The authors suggested that this phenomenon could be related to the presence of unpolymerized monomers on the material surface, but the exact biomechanical mechanisms were not fully clarified [90,91]. The results of the present preliminary in vitro study confirmed a general trend towards the reduction for both sessile and planktonic CFUs of heat-cured resin composites.
Regarding the biomass quantification by optical density (OD570nm), data recorded in the light-cured BF subgroup were significantly higher than for the same material subjected to a heat-curing protocol. Bacterial colonization is the main agent for caries onset [41]. Streptococcus mutans is one of the main promoters of biofilm formation by controlling the matrix development formed by insoluble exopolysaccharides as α1,3-glucans [92,93]. Insoluble exopolysaccharides prevent saliva neutralization [13]. Secondary agents in caries lesions process, such as Candida albicans [51], may also promote exopolysaccharide increase and accumulation, enhancing the formation of microcolonies by Streptococcus mutans [50,94]. A reduced biofilm biomass is considered less resistant to the effect of a salivary buffer [43]. The long-term outcome of composite restorations is negatively affected by the presence of thick biofilms [95]. Acids and bacteria enzymatic degradation can jeopardize the function and the esthetic longevity of a restoration [96,97,98]. Further composite degradation is related to the Streptococcus mutans esterase gene that can catalyze the uncured monomers, causing degradation and marginal bacterial colonization [99], as well as an alteration of surface topography [7].
As already mentioned, Streptococcus mutans is not the only microorganism involved in oral biofilm formation and decay development. In recent studies, the high prevalence of Candida albicans was recovered in bacterial biofilms together with Streptococcus mutans [49,50,100,101]. The presence of Candida albicans induces the expression of Streptococcus mutans glucosyltransferases and genes to withstand environmental stress [50]. Indeed, a greater biofilm complexity makes microorganisms more tolerant to environmental stresses [44]. Further studies should better investigate Candida albicans adhesion and proliferation on differently cured composite resins, mainly focusing on the potential interactions between Streptococcus mutans and Candida albicans. Among the limitations of this study, the use of a mono-species and static bacterial model could limit the generalizability of the obtained findings. Further studies should be carried out to confirm the adhesion properties with multispecies clinical biofilms.
The clinical relevance of the present results should be carefully interpreted, also considering other paramount properties related to the peculiar composition of any different resin-based materials. Whenever a material comes into contact with live tissues and cells they can interact, generating a biological response, and many components of composite resins have shown potential cytotoxic activity [102]. For instance, the residues of free methacrylate monomers following polymerization may trigger the production of prostaglandin E2, cyclooxygenase 2 (COX2) and an increase in interleukin-1B (IL-1B), IL-6 and nitric oxide (NO) [103,104,105]. Residual monomers may be eluted after composite placement [106,107] following both chemical and physical degradation over time [108].
In the present research, composites were treated using already known and well referenced methodologies. Nevertheless, the improvement of the study is related to the observed effect of the different curing treatments on the interaction between the composite disks and Streptococcus mutans.
The results obtained in this preliminary in vitro study highlight further potential benefits from an effective resin composite heat-curing cycle. Besides the already known advantages in terms of mechanical properties such as microhardness, flexural strength, wear resistance, tensile strength, fracture toughness and color stability [35,36,37,38], the present results highlighted the following:
  • The intrinsic ability of Streptococcus mutans to proliferate seems impaired in the presence of composites subjected to additional heat-curing protocols, with a general trend towards a reduction in both sessile and planktonic CFUs;
  • Where there is an inability to reduce sessile CFUs, heat-curing still seems to inhibit the production of biofilm glycoproteins.
From a clinical perspective, a reduced adhesion, proliferation and/or matrix production by Streptococcus mutans, achieved as a result of additional heat-curing polymerization protocols, could significantly decrease its pathogenicity, thereby reducing the risk of secondary caries. A significant role seems also to be played by the filler particle size, with small-sized fillers leading to reduced Streptococcus mutans proliferation.

Author Contributions

Conceptualization, F.D.A., C.D. and S.D.; methodology, F.D.A., C.D., S.D.L., E.S., M.B. and S.D.; validation, F.D.A., C.D. and S.D.; formal analysis, F.D.A., C.D., S.D.L., E.S., M.B. and S.D.; investigation, F.D.A., C.D., S.D.L., E.S., M.B. and S.D.; resources, C.D. and S.D.; data curation, F.D.A., S.D.L., E.S. and M.B.; writing—original draft preparation, F.D.A., E.S., S.D.L. and M.B.; writing—review and editing, F.D.A., C.D., S.D.L. and S.D.; visualization, F.D.A., S.D.L., E.S. and M.B.; supervision, F.D.A., C.D. and S.D.; project administration, F.D.A., C.D. and S.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of University “G. d’Annunzio”, Chieti–Pescara, Italy (approval code SALI, N. 19 of the 10 September 2020).

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Beighton, D. The complex oral microflora of high-risk individuals and groups and its role in the caries process. Community Dent. Oral Epidemiol. 2005, 33, 248–255. [Google Scholar] [CrossRef] [PubMed]
  2. Zhu, W.; Liu, S.; Zhuang, P.; Liu, J.; Wang, Y.; Lin, H. Characterization of acid-tolerance-associated small RNAs in clinical isolates of Streptococcus mutans: Potential biomarkers for caries prevention. Mol. Med. Rep. 2017, 16, 9242–9250. [Google Scholar] [CrossRef]
  3. Hahnel, S.; Henrich, A.; Rosentritt, M.; Handel, G.; Burgers, R. Influence of artificial ageing on surface properties and Streptococcus mutans adhesion to dental composite materials. J. Mater. Sci. Mater. Med. 2010, 21, 823–833. [Google Scholar] [CrossRef] [PubMed]
  4. Hannig, M. Transmission electron microscopy of early plaque formation on dental materials in vivo. Eur. J. Oral Sci. 1999, 107, 55–64. [Google Scholar] [CrossRef] [PubMed]
  5. Schneider, L.F.; Cavalcante, L.M.; Silikas, N. Shrinkage Stresses Generated during Resin-Composite Applications: A Review. J. Dent. Biomech. 2010, 2010, 131630. [Google Scholar] [CrossRef] [PubMed]
  6. Bourbia, M.; Ma, D.; Cvitkovitch, D.G.; Santerre, J.P.; Finer, Y. Cariogenic bacteria degrade dental resin composites and adhesives. J. Dent. Res. 2013, 92, 989–994. [Google Scholar] [CrossRef]
  7. Beyth, N.; Bahir, R.; Matalon, S.; Domb, A.J.; Weiss, E.I. Streptococcus mutans biofilm changes surface-topography of resin composites. Dent. Mater. Off. Publ. Acad. Dent. Mater. 2008, 24, 732–736. [Google Scholar] [CrossRef]
  8. Hasan, S.; Danishuddin, M.; Adil, M.; Singh, K.; Verma, P.K.; Khan, A.U. Efficacy of E. officinalis on the cariogenic properties of Streptococcus mutans: A novel and alternative approach to suppress quorum-sensing mechanism. PLoS ONE 2012, 7, e40319. [Google Scholar] [CrossRef]
  9. Huang, L.; Xu, Q.A.; Liu, C.; Fan, M.W.; Li, Y.H. Anti-caries DNA vaccine-induced secretory immunoglobulin A antibodies inhibit formation of Streptococcus mutans biofilms in vitro. Acta Pharmacol. Sin. 2013, 34, 239–246. [Google Scholar] [CrossRef]
  10. Nomura, H.; Isshiki, Y.; Sakuda, K.; Sakuma, K.; Kondo, S. Effects of oakmoss and its components on biofilm formation of Legionella pneumophila. Biol. Pharm. Bull. 2013, 36, 833–837. [Google Scholar] [CrossRef]
  11. Wojtyczka, R.D.; Kępa, M.; Idzik, D.; Kubina, R.; Kabała-Dzik, A.; Dziedzic, A.; Wąsik, T.J. In Vitro Antimicrobial Activity of Ethanolic Extract of Polish Propolis against Biofilm Forming Staphylococcus epidermidis Strains. Evid. Based Complement. Altern. Med. Ecam 2013, 2013, 590703. [Google Scholar] [CrossRef] [PubMed]
  12. Zhao, K.; Tseng, B.S.; Beckerman, B.; Jin, F.; Gibiansky, M.L.; Harrison, J.J.; Luijten, E.; Parsek, M.R.; Wong, G.C.L. Psl trails guide exploration and microcolony formation in Pseudomonas aeruginosa biofilms. Nature 2013, 497, 388–391. [Google Scholar] [CrossRef]
  13. Xiao, J.; Klein, M.I.; Falsetta, M.L.; Lu, B.; Delahunty, C.M.; Yates, J.R., 3rd; Heydorn, A.; Koo, H. The exopolysaccharide matrix modulates the interaction between 3D architecture and virulence of a mixed-species oral biofilm. PLoS Pathog. 2012, 8, e1002623. [Google Scholar] [CrossRef] [PubMed]
  14. Yang, Y.; Mao, M.; Lei, L.; Li, M.; Yin, J.; Ma, X.; Tao, X.; Yang, Y.; Hu, T. Regulation of water-soluble glucan synthesis by the Streptococcus mutans dexA gene effects biofilm aggregation and cariogenic pathogenicity. Mol. Oral Microbiol. 2019, 34, 51–63. [Google Scholar] [CrossRef] [PubMed]
  15. Jaykus, L.-A.; Wang, H.H.; Schlesinger, L.S. Food-Borne Microbes: Shaping the Host Ecosystem; ASM Press: Washington, DC, USA, 2009. [Google Scholar]
  16. Tamura, S.; Yonezawa, H.; Motegi, M.; Nakao, R.; Yoneda, S.; Watanabe, H.; Yamazaki, T.; Senpuku, H. Inhibiting effects of Streptococcus salivarius on competence-stimulating peptide-dependent biofilm formation by Streptococcus mutans. Oral Microbiol. Immunol. 2009, 24, 152–161. [Google Scholar] [CrossRef] [PubMed]
  17. Brady, L.J.; Maddocks, S.E.; Larson, M.R.; Forsgren, N.; Persson, K.; Deivanayagam, C.C.; Jenkinson, H.F. The changing faces of Streptococcus antigen I/II polypeptide family adhesins. Mol. Microbiol. 2010, 77, 276–286. [Google Scholar] [CrossRef]
  18. Jakubovics, N.S.; Strömberg, N.; van Dolleweerd, C.J.; Kelly, C.G.; Jenkinson, H.F. Differential binding specificities of oral streptococcal antigen I/II family adhesins for human or bacterial ligands. Mol. Microbiol. 2005, 55, 1591–1605. [Google Scholar] [CrossRef]
  19. Larson, M.R.; Rajashankar, K.R.; Crowley, P.J.; Kelly, C.; Mitchell, T.J.; Brady, L.J.; Deivanayagam, C. Crystal structure of the C-terminal region of Streptococcus mutans antigen I/II and characterization of salivary agglutinin adherence domains. J. Biol. Chem. 2011, 286, 21657–21666. [Google Scholar] [CrossRef]
  20. Sullan, R.M.; Li, J.K.; Crowley, P.J.; Brady, L.J.; Dufrêne, Y.F. Binding forces of Streptococcus mutans P1 adhesin. ACS Nano 2015, 9, 1448–1460. [Google Scholar] [CrossRef]
  21. Carvalho, R.M.; Pereira, J.C.; Yoshiyama, M.; Pashley, D.H. A review of polymerization contraction: The influence of stress development versus stress relief. Oper. Dent. 1996, 21, 17–24. [Google Scholar]
  22. Collins, C.J.; Bryant, R.W.; Hodge, K.L. A clinical evaluation of posterior composite resin restorations: 8-year findings. J. Dent. 1998, 26, 311–317. [Google Scholar] [CrossRef]
  23. Pashley, D.H. Clinical considerations of microleakage. J. Endod. 1990, 16, 70–77. [Google Scholar] [CrossRef] [PubMed]
  24. Song, F.; Koo, H.; Ren, D. Effects of Material Properties on Bacterial Adhesion and Biofilm Formation. J. Dent. Res. 2015, 94, 1027–1034. [Google Scholar] [CrossRef] [PubMed]
  25. Bollen, C.M.; Lambrechts, P.; Quirynen, M. Comparison of surface roughness of oral hard materials to the threshold surface roughness for bacterial plaque retention: A review of the literature. Dent. Mater. Off. Publ. Acad. Dent. Mater. 1997, 13, 258–269. [Google Scholar]
  26. Busscher, H.J.; Rinastiti, M.; Siswomihardjo, W.; van der Mei, H.C. Biofilm formation on dental restorative and implant materials. J. Dent. Res. 2010, 89, 657–665. [Google Scholar] [CrossRef] [PubMed]
  27. Van Oss, C.J. Hydrophobicity of biosurfaces—Origin, quantitative determination and interaction energies. Colloids Surf. B Biointerfaces 1995, 5, 91–110. [Google Scholar] [CrossRef]
  28. D’Arcangelo, C.; De Angelis, F.; Vadini, M.; Carluccio, F.; Vitalone, L.M.; D’Amario, M. Influence of curing time, overlay material and thickness on three light-curing composites used for luting indirect composite restorations. J. Adhes. Dent. 2012, 14, 377–384. [Google Scholar]
  29. De Angelis, F.; D’Arcangelo, C.; Buonvivere, M.; Rondoni, G.D.; Vadini, M. Shear bond strength of glass ionomer and resin-based cements to different types of zirconia. J. Esthet. Restor. Dent. Off. Publ. Am. Acad. Esthet. Dent. 2020, 32, 806–814. [Google Scholar] [CrossRef]
  30. Vadini, M.; D’Amario, M.; De Angelis, F.; Falco, A.; D’Arcangelo, C. No-Prep Rehabilitation of Fractured Maxillary Incisors with Partial Veneers. J. Esthet. Restor. Dent. Off. Publ. Am. Acad. Esthet. Dent. 2016, 28, 351–358. [Google Scholar] [CrossRef]
  31. Peutzfeldt, A. Resin composites in dentistry: The monomer systems. Eur. J. Oral Sci. 1997, 105, 97–116. [Google Scholar] [CrossRef]
  32. Amirouche-Korichi, A.; Mouzali, M.; Watts, D.C. Effects of monomer ratios and highly radiopaque fillers on degree of conversion and shrinkage-strain of dental resin composites. Dent. Mater. Off. Publ. Acad. Dent. Mater. 2009, 25, 1411–1418. [Google Scholar] [CrossRef] [PubMed]
  33. Al-Zain, A.O.; Marghalani, H.Y. Influence of Light-curing Distances on Microflexural Strength of Two Resin-based Composites. Oper. Dent. 2020, 45, 297–305. [Google Scholar] [CrossRef] [PubMed]
  34. Al-Zain, A.O.; Platt, J.A. Effect of light-curing distance and curing time on composite microflexural strength. Dent. Mater. J. 2021, 40, 202–208. [Google Scholar] [CrossRef] [PubMed]
  35. Cruz, F.L.; Carvalho, R.F.; Batista, C.H.T.; Siqueira-Júnior, H.M.; Queiroz, J.R.C.; Leite, F.P. Efecto del tratamiento térmico y de fibras de polietileno en la resistencia a la flexión de resinas compuestas. Acta Odontológica Venez. 2014, 52, 3–4. [Google Scholar]
  36. Grazioli, G.; Francia, A.; Cuevas-Suárez, C.E.; Zanchi, C.H.; Moraes, R.R. Simple and Low-Cost Thermal Treatments on Direct Resin Composites for Indirect Use. Braz. Dent. J. 2019, 30, 279–284. [Google Scholar] [CrossRef] [PubMed]
  37. Lapesqueur, M.; Surriaga, P.; Masache, M.E.; Vásquez, B.; Peña, M.; Gómes, O.M.M.; Domínguez, J.A. Efectos sobre microdureza y grado de conversión de dos tipos de resinas sometidas a tratamientos de pospolimerización. Rec. Nac. Odontol. 2015, 11, 49–56. [Google Scholar] [CrossRef]
  38. Zamalloa-Quintana, M.; López-Gurreonero, C.; Santander-Rengifo, F.M.; Ladera-Castañeda, M.; Castro-Pérez Vargas, A.; Cornejo-Pinto, A.; Cervantes-Ganoza, L.; Cayo-Rojas, C. Effect of Additional Dry Heat Curing on Microflexural Strength in Three Types of Resin Composite: An In Vitro Study. Crystals 2022, 12, 1045. [Google Scholar] [CrossRef]
  39. Bağis, Y.H.; Rueggeberg, F.A. Mass loss in urethane/TEGDMA- and Bis-GMA/TEGDMA-based resin composites during post-cure heating. Dent. Mater. Off. Publ. Acad. Dent. Mater. 1997, 13, 377–380. [Google Scholar] [CrossRef]
  40. Hansel, C.; Leyhausen, G.; Mai, U.E.; Geurtsen, W. Effects of various resin composite (co)monomers and extracts on two caries-associated micro-organisms in vitro. J. Dent. Res. 1998, 77, 60–67. [Google Scholar] [CrossRef]
  41. Brambilla, E.; Gagliani, M.; Ionescu, A.; Fadini, L.; García-Godoy, F. The influence of light-curing time on the bacterial colonization of resin composite surfaces. Dent. Mater. Off. Publ. Acad. Dent. Mater. 2009, 25, 1067–1072. [Google Scholar] [CrossRef]
  42. Kraigsley, A.M.; Tang, K.; Lippa, K.A.; Howarter, J.A.; Lin-Gibson, S.; Lin, N.J. Effect of polymer degree of conversion on Streptococcus mutans biofilms. Macromol. Biosci. 2012, 12, 1706–1713. [Google Scholar] [CrossRef] [PubMed]
  43. D’Ercole, S.; De Angelis, F.; Biferi, V.; Noviello, C.; Tripodi, D.; Di Lodovico, S.; Cellini, L.; D’Arcangelo, C. Antibacterial and Antibiofilm Properties of Three Resin-Based Dental Composites against Streptococcus mutans. Materials 2022, 15, 1891. [Google Scholar] [CrossRef] [PubMed]
  44. De Angelis, F.; D’Ercole, S.; Di Giulio, M.; Vadini, M.; Biferi, V.; Buonvivere, M.; Vanini, L.; Cellini, L.; Di Lodovico, S.; D’Arcangelo, C. In Vitro Evaluation of Candida albicans Adhesion on Heat-Cured Resin-Based Dental Composites. Materials 2023, 16, 5818. [Google Scholar] [CrossRef] [PubMed]
  45. Petrini, M.; Giuliani, A.; Di Campli, E.; Di Lodovico, S.; Iezzi, G.; Piattelli, A.; D’Ercole, S. The Bacterial Anti-Adhesive Activity of Double-Etched Titanium (DAE) as a Dental Implant Surface. Int. J. Mol. Sci. 2020, 21, 8315. [Google Scholar] [CrossRef] [PubMed]
  46. Di Lodovico, S.; Gasparri, F.; Di Campli, E.; Di Fermo, P.; D’Ercole, S.; Cellini, L.; Di Giulio, M. Prebiotic Combinations Effects on the Colonization of Staphylococcal Skin Strains. Microorganisms 2020, 9, 37. [Google Scholar] [CrossRef]
  47. Ommen, P.; Zobek, N.; Meyer, R.L. Quantification of biofilm biomass by staining: Non-toxic safranin can replace the popular crystal violet. J. Microbiol. Methods 2017, 141, 87–89. [Google Scholar] [CrossRef]
  48. Xu, Z.; Liang, Y.; Lin, S.; Chen, D.; Li, B.; Li, L.; Deng, Y. Crystal Violet and XTT Assays on Staphylococcus aureus Biofilm Quantification. Curr. Microbiol. 2016, 73, 474–482. [Google Scholar] [CrossRef]
  49. Metwalli, K.H.; Khan, S.A.; Krom, B.P.; Jabra-Rizk, M.A. Streptococcus mutans, Candida albicans, and the human mouth: A sticky situation. PLoS Pathog. 2013, 9, e1003616. [Google Scholar] [CrossRef]
  50. Falsetta, M.L.; Klein, M.I.; Colonne, P.M.; Scott-Anne, K.; Gregoire, S.; Pai, C.H.; Gonzalez-Begne, M.; Watson, G.; Krysan, D.J.; Bowen, W.H.; et al. Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo. Infect. Immun. 2014, 82, 1968–1981. [Google Scholar] [CrossRef]
  51. Pereira, D.; Seneviratne, C.J.; Koga-Ito, C.Y.; Samaranayake, L.P. Is the oral fungal pathogen Candida albicans a cariogen? Oral Dis. 2018, 24, 518–526. [Google Scholar] [CrossRef]
  52. Xiao, J.; Huang, X.; Alkhers, N.; Alzamil, H.; Alzoubi, S.; Wu, T.T.; Castillo, D.A.; Campbell, F.; Davis, J.; Herzog, K.; et al. Candida albicans and Early Childhood Caries: A Systematic Review and Meta-Analysis. Caries Res. 2018, 52, 102–112. [Google Scholar] [CrossRef] [PubMed]
  53. Pereira, C.A.; Eskelson, E.; Cavalli, V.; Liporoni, P.C.; Jorge, A.O.; do Rego, M.A. Streptococcus mutans biofilm adhesion on composite resin surfaces after different finishing and polishing techniques. Oper. Dent. 2011, 36, 311–317. [Google Scholar] [CrossRef] [PubMed]
  54. Uçtaşli, M.B.; Arisu, H.D.; Omürlü, H.; Eligüzeloğlu, E.; Ozcan, S.; Ergun, G. The effect of different finishing and polishing systems on the surface roughness of different composite restorative materials. J. Contemp. Dent. Pract. 2007, 8, 89–96. [Google Scholar] [PubMed]
  55. Attar, N. The effect of finishing and polishing procedures on the surface roughness of composite resin materials. J. Contemp. Dent. Pract. 2007, 8, 27–35. [Google Scholar] [CrossRef]
  56. Motevasselian, F.; Zibafar, E.; Yassini, E.; Mirzaei, M.; Pourmirhoseni, N. Adherence of Streptococcus Mutans to Microhybrid and Nanohybrid Resin Composites and Dental Amalgam: An In Vitro Study. J. Dent. 2017, 14, 337–343. [Google Scholar]
  57. Barbosa, S.H.; Zanata, R.L.; Navarro, M.F.; Nunes, O.B. Effect of different finishing and polishing techniques on the surface roughness of microfilled, hybrid and packable composite resins. Braz. Dent. J. 2005, 16, 39–44. [Google Scholar] [CrossRef]
  58. Jung, M.; Eichelberger, K.; Klimek, J. Surface geometry of four nanofiller and one hybrid composite after one-step and multiple-step polishing. Oper. Dent. 2007, 32, 347–355. [Google Scholar] [CrossRef]
  59. Ikeda, M.; Matin, K.; Nikaido, T.; Foxton, R.M.; Tagami, J. Effect of surface characteristics on adherence of S. mutans biofilms to indirect resin composites. Dent. Mater. J. 2007, 26, 915–923. [Google Scholar] [CrossRef]
  60. Koh, R.; Neiva, G.; Dennison, J.; Yaman, P. Finishing systems on the final surface roughness of composites. J. Contemp. Dent. Pract. 2008, 9, 138–145. [Google Scholar] [CrossRef]
  61. Ono, M.; Nikaido, T.; Ikeda, M.; Imai, S.; Hanada, N.; Tagami, J.; Matin, K. Surface properties of resin composite materials relative to biofilm formation. Dent. Mater. J. 2007, 26, 613–622. [Google Scholar] [CrossRef]
  62. Suh, B.I. Oxygen-inhibited layer in adhesion dentistry. J. Esthet. Restor. Dent. Off. Publ. Am. Acad. Esthet. Dent. 2004, 16, 316–323. [Google Scholar] [CrossRef] [PubMed]
  63. Faria-e-Silva, A.L.; Lima, A.F.; Moraes, R.R.; Piva, E.; Martins, L.R. Degree of conversion of etch-and-rinse and self-etch adhesives light-cured using QTH or LED. Oper. Dent. 2010, 35, 649–654. [Google Scholar] [CrossRef] [PubMed]
  64. Arrais, C.A.; Pontes, F.M.; Santos, L.P.; Leite, E.R.; Giannini, M. Degree of conversion of adhesive systems light-cured by LED and halogen light. Braz. Dent. J. 2007, 18, 54–59. [Google Scholar] [CrossRef] [PubMed]
  65. Navarra, C.O.; Cadenaro, M.; Codan, B.; Mazzoni, A.; Sergo, V.; De Stefano Dorigo, E.; Breschi, L. Degree of conversion and interfacial nanoleakage expression of three one-step self-etch adhesives. Eur. J. Oral Sci. 2009, 117, 463–469. [Google Scholar] [CrossRef] [PubMed]
  66. Cadenaro, M.; Antoniolli, F.; Sauro, S.; Tay, F.R.; Di Lenarda, R.; Prati, C.; Biasotto, M.; Contardo, L.; Breschi, L. Degree of conversion and permeability of dental adhesives. Eur. J. Oral Sci. 2005, 113, 525–530. [Google Scholar] [CrossRef]
  67. Ahn, S.J.; Ahn, S.J.; Wen, Z.T.; Brady, L.J.; Burne, R.A. Characteristics of biofilm formation by Streptococcus mutans in the presence of saliva. Infect. Immun. 2008, 76, 4259–4268. [Google Scholar] [CrossRef]
  68. Gajewski, V.E.; Pfeifer, C.S.; Fróes-Salgado, N.R.; Boaro, L.C.; Braga, R.R. Monomers used in resin composites: Degree of conversion, mechanical properties and water sorption/solubility. Braz. Dent. J. 2012, 23, 508–514. [Google Scholar] [CrossRef]
  69. Asmussen, E. NMR-analysis of monomers in restorative resins. Acta Odontol. Scand. 1975, 33, 129–134. [Google Scholar] [CrossRef]
  70. Ruyter, I.E.; Sjoevik, I.J. Composition of dental resin and composite materials. Acta Odontol. Scand. 1981, 39, 133–146. [Google Scholar] [CrossRef]
  71. Vankerckhoven, H.; Lambrechts, P.; van Beylen, M.; Vanherle, G. Characterization of composite resins by NMR and TEM. J. Dent. Res. 1981, 60, 1957–1965. [Google Scholar] [CrossRef]
  72. Ruyter, I.E.; Oysaed, H. Composites for use in posterior teeth: Composition and conversion. J. Biomed. Mater. Res. 1987, 21, 11–23. [Google Scholar] [CrossRef] [PubMed]
  73. Gonçalves, F.; Azevedo, C.L.; Ferracane, J.L.; Braga, R.R. BisGMA/TEGDMA ratio and filler content effects on shrinkage stress. Dent. Mater. Off. Publ. Acad. Dent. Mater. 2011, 27, 520–526. [Google Scholar] [CrossRef] [PubMed]
  74. Lee, J.H.; Um, C.M.; Lee, I.B. Rheological properties of resin composites according to variations in monomer and filler composition. Dent. Mater. Off. Publ. Acad. Dent. Mater. 2006, 22, 515–526. [Google Scholar] [CrossRef] [PubMed]
  75. Söderholm, K.J.; Mariotti, A. BIS-GMA—Based resins in dentistry: Are they safe? J. Am. Dent. Assoc. 1999, 130, 201–209. [Google Scholar] [CrossRef] [PubMed]
  76. Kingman, A.; Hyman, J.; Masten, S.A.; Jayaram, B.; Smith, C.; Eichmiller, F.; Arnold, M.C.; Wong, P.A.; Schaeffer, J.M.; Solanki, S.; et al. Bisphenol A and other compounds in human saliva and urine associated with the placement of composite restorations. J. Am. Dent. Assoc. 2012, 143, 1292–1302. [Google Scholar] [CrossRef]
  77. Hampe, T.; Liersch, J.; Wiechens, B.; Wassmann, T.; Schubert, A.; Alhussein, M.; Bürgers, R.; Krohn, S. A Pilot Study on Monomer and Bisphenol A (BPA) Release from UDMA-Based and Conventional Indirect Veneering Composites. Polymers 2022, 14, 4580. [Google Scholar] [CrossRef]
  78. Kim, K.; An, J.S.; Lim, B.S.; Ahn, S.J. Effect of Bisphenol A Glycol Methacrylate on Virulent Properties of Streptococcus mutans UA159. Caries Res. 2019, 53, 84–95. [Google Scholar] [CrossRef]
  79. Lin, N.J.; Keeler, C.; Kraigsley, A.M.; Ye, J.; Lin-Gibson, S. Effect of dental monomers and initiators on Streptococcus mutans oral biofilms. Dent. Mater. Off. Publ. Acad. Dent. Mater. 2018, 34, 776–785. [Google Scholar] [CrossRef]
  80. De Angelis, F.; Sarteur, N.; Buonvivere, M.; Vadini, M.; Šteffl, M.; D’Arcangelo, C. Meta-analytical analysis on components released from resin-based dental materials. Clin. Oral Investig. 2022, 26, 6015–6041. [Google Scholar] [CrossRef]
  81. Lucena-Martín, C.; González-López, S.; Navajas-Rodríguez de Mondelo, J.M. The effect of various surface treatments and bonding agents on the repaired strength of heat-treated composites. J. Prosthet. Dent. 2001, 86, 481–488. [Google Scholar] [CrossRef]
  82. Uzay, C.; Boztepe, M.H.; Bayramoğlu, M.; Geren, N. Effect of post-curing heat treatment on mechanical properties of fiber reinforced polymer (FRP) composites. Mater. Test. 2017, 59, 366–372. [Google Scholar] [CrossRef]
  83. Takeshige, F.; Kinomoto, Y.; Torii, M. Additional heat-curing of light-cured composite resin for inlay restoration. J. Osaka Univ. Dent. Sch. 1995, 35, 59–66. [Google Scholar] [PubMed]
  84. Malta, D.A.; Magne, P.; Monteiro-Junior, S. Bond strength and monomer conversion of indirect composite resin restorations, Part 1: Light vs heat polymerization. J. Adhes. Dent. 2014, 16, 517–522. [Google Scholar] [PubMed]
  85. Ferracane, J.L.; Condon, J.R. Post-cure heat treatments for composites: Properties and fractography. Dent. Mater. Off. Publ. Acad. Dent. Mater. 1992, 8, 290–295. [Google Scholar] [CrossRef] [PubMed]
  86. Marcinkowska, A.; Gauza-Wlodarczyk, M.; Kubisz, L.; Hedzelek, W. The electrical properties and glass transition of some dental materials after temperature exposure. J. Mater. Sci. Mater. Med. 2017, 28, 186. [Google Scholar] [CrossRef]
  87. Caughman, W.F.; Caughman, G.B.; Shiflett, R.A.; Rueggeberg, F.; Schuster, G.S. Correlation of cytotoxicity, filler loading and curing time of dental composites. Biomaterials 1991, 12, 737–740. [Google Scholar] [CrossRef]
  88. Rueggeberg, F.A.; Hashinger, D.T.; Fairhurst, C.W. Calibration of FTIR conversion analysis of contemporary dental resin composites. Dent. Mater. Off. Publ. Acad. Dent. Mater. 1990, 6, 241–249. [Google Scholar] [CrossRef]
  89. Shono, Y.; Ogawa, T.; Terashita, M.; Carvalho, R.M.; Pashley, E.L.; Pashley, D.H. Regional measurement of resin-dentin bonding as an array. J. Dent. Res. 1999, 78, 699–705. [Google Scholar] [CrossRef]
  90. Khalichi, P.; Cvitkovitch, D.G.; Santerre, J.P. Effect of composite resin biodegradation products on oral streptococcal growth. Biomaterials 2004, 25, 5467–5472. [Google Scholar] [CrossRef]
  91. Geurtsen, W. Biocompatibility of resin-modified filling materials. Crit. Rev. Oral Biol. Med. Off. Publ. Am. Assoc. Oral Biol. 2000, 11, 333–355. [Google Scholar] [CrossRef]
  92. Hajishengallis, E.; Parsaei, Y.; Klein, M.I.; Koo, H. Advances in the microbial etiology and pathogenesis of early childhood caries. Mol. Oral Microbiol. 2017, 32, 24–34. [Google Scholar] [CrossRef] [PubMed]
  93. Bowen, W.H.; Koo, H. Biology of Streptococcus mutans-derived glucosyltransferases: Role in extracellular matrix formation of cariogenic biofilms. Caries Res. 2011, 45, 69–86. [Google Scholar] [CrossRef] [PubMed]
  94. Hwang, G.; Liu, Y.; Kim, D.; Li, Y.; Krysan, D.J.; Koo, H. Candida albicans mannans mediate Streptococcus mutans exoenzyme GtfB binding to modulate cross-kingdom biofilm development in vivo. PLoS Pathog. 2017, 13, e1006407. [Google Scholar] [CrossRef] [PubMed]
  95. Kusuma Yulianto, H.D.; Rinastiti, M.; Cune, M.S.; de Haan-Visser, W.; Atema-Smit, J.; Busscher, H.J.; van der Mei, H.C. Biofilm composition and composite degradation during intra-oral wear. Dent. Mater. Off. Publ. Acad. Dent. Mater. 2019, 35, 740–750. [Google Scholar] [CrossRef]
  96. Delaviz, Y.; Finer, Y.; Santerre, J.P. Biodegradation of resin composites and adhesives by oral bacteria and saliva: A rationale for new material designs that consider the clinical environment and treatment challenges. Dent. Mater. Off. Publ. Acad. Dent. Mater. 2014, 30, 16–32. [Google Scholar] [CrossRef]
  97. Demarco, F.F.; Collares, K.; Coelho-de-Souza, F.H.; Correa, M.B.; Cenci, M.S.; Moraes, R.R.; Opdam, N.J. Anterior composite restorations: A systematic review on long-term survival and reasons for failure. Dent. Mater. Off. Publ. Acad. Dent. Mater. 2015, 31, 1214–1224. [Google Scholar] [CrossRef]
  98. Opdam, N.J.; van de Sande, F.H.; Bronkhorst, E.; Cenci, M.S.; Bottenberg, P.; Pallesen, U.; Gaengler, P.; Lindberg, A.; Huysmans, M.C.; van Dijken, J.W. Longevity of posterior composite restorations: A systematic review and meta-analysis. J. Dent. Res. 2014, 93, 943–949. [Google Scholar] [CrossRef]
  99. Huang, B.; Sadeghinejad, L.; Adebayo, O.I.A.; Ma, D.; Xiao, Y.; Siqueira, W.L.; Cvitkovitch, D.G.; Finer, Y. Gene expression and protein synthesis of esterase from Streptococcus mutans are affected by biodegradation by-product from methacrylate resin composites and adhesives. Acta Biomater. 2018, 81, 158–168. [Google Scholar] [CrossRef]
  100. Jenkinson, H.F.; Lala, H.C.; Shepherd, M.G. Coaggregation of Streptococcus sanguis and other streptococci with Candida albicans. Infect. Immun. 1990, 58, 1429–1436. [Google Scholar] [CrossRef]
  101. Kolenbrander, P.E.; Andersen, R.N.; Blehert, D.S.; Egland, P.G.; Foster, J.S.; Palmer, R.J., Jr. Communication among oral bacteria. Microbiol. Mol. Biol. Rev. MMBR 2002, 66, 486–505. [Google Scholar] [CrossRef]
  102. De Angelis, F.; Mandatori, D.; Schiavone, V.; Melito, F.P.; Valentinuzzi, S.; Vadini, M.; Di Tomo, P.; Vanini, L.; Pelusi, L.; Pipino, C.; et al. Cytotoxic and Genotoxic Effects of Composite Resins on Cultured Human Gingival Fibroblasts. Materials 2021, 14, 5225. [Google Scholar] [CrossRef] [PubMed]
  103. Kuan, Y.H.; Huang, F.M.; Lee, S.S.; Li, Y.C.; Chang, Y.C. Bisgma stimulates prostaglandin E2 production in macrophages via cyclooxygenase-2, cytosolic phospholipase A2, and mitogen-activated protein kinases family. PLoS ONE 2013, 8, e82942. [Google Scholar] [CrossRef] [PubMed]
  104. Kuan, Y.H.; Huang, F.M.; Li, Y.C.; Chang, Y.C. Proinflammatory activation of macrophages by bisphenol A-glycidyl-methacrylate involved NFκB activation via PI3K/Akt pathway. Food Chem. Toxicol. Int. J. Publ. Br. Ind. Biol. Res. Assoc. 2012, 50, 4003–4009. [Google Scholar] [CrossRef] [PubMed]
  105. Huang, F.M.; Chang, Y.C.; Lee, S.S.; Yeh, C.H.; Lee, K.G.; Huang, Y.C.; Chen, C.J.; Chen, W.Y.; Pan, P.H.; Kuan, Y.H. BisGMA-induced cytotoxicity and genotoxicity in macrophages are attenuated by wogonin via reduction of intrinsic caspase pathway activation. Environ. Toxicol. 2016, 31, 176–184. [Google Scholar] [CrossRef] [PubMed]
  106. Emmler, J.; Seiss, M.; Kreppel, H.; Reichl, F.X.; Hickel, R.; Kehe, K. Cytotoxicity of the dental composite component TEGDMA and selected metabolic by-products in human pulmonary cells. Dent. Mater. Off. Publ. Acad. Dent. Mater. 2008, 24, 1670–1675. [Google Scholar] [CrossRef]
  107. Chang, H.H.; Chang, M.C.; Wang, H.H.; Huang, G.F.; Lee, Y.L.; Wang, Y.L.; Chan, C.P.; Yeung, S.Y.; Tseng, S.K.; Jeng, J.H. Urethane dimethacrylate induces cytotoxicity and regulates cyclooxygenase-2, hemeoxygenase and carboxylesterase expression in human dental pulp cells. Acta Biomater. 2014, 10, 722–731. [Google Scholar] [CrossRef]
  108. Santerre, J.P.; Shajii, L.; Leung, B.W. Relation of dental composite formulations to their degradation and the release of hydrolyzed polymeric-resin-derived products. Crit. Rev. Oral Biol. Med. Off. Publ. Am. Assoc. Oral Biol. 2001, 12, 136–151. [Google Scholar] [CrossRef]
Biomedicines 12 02235 g001
Figure 1. Representative SEM microphotographs of Streptococcus mutans colonies formed on disk-shaped specimens from light-cured GR subgroup, at 1000× (A) and 3000× (B) magnifications, and from heat-cured GR subgroup, at 1000× (C) and 3000× (D) magnifications. Arrows in 3000× SEM images indicate examples of Streptococcus mutans colonies.
Figure 1. Representative SEM microphotographs of Streptococcus mutans colonies formed on disk-shaped specimens from light-cured GR subgroup, at 1000× (A) and 3000× (B) magnifications, and from heat-cured GR subgroup, at 1000× (C) and 3000× (D) magnifications. Arrows in 3000× SEM images indicate examples of Streptococcus mutans colonies.
Biomedicines 12 02235 g001
Biomedicines 12 02235 g002
Figure 2. Representative SEM microphotographs of Streptococcus mutans colonies formed on disk-shaped specimens from light-cured VD subgroup, at 1000× (A) and 3000× (B) magnifications, and from heat-cured VD subgroup, at 1000× (C) and 3000× (D) magnifications. Arrows in 3000× SEM images indicate examples of Streptococcus mutans colonies.
Figure 2. Representative SEM microphotographs of Streptococcus mutans colonies formed on disk-shaped specimens from light-cured VD subgroup, at 1000× (A) and 3000× (B) magnifications, and from heat-cured VD subgroup, at 1000× (C) and 3000× (D) magnifications. Arrows in 3000× SEM images indicate examples of Streptococcus mutans colonies.
Biomedicines 12 02235 g002
Biomedicines 12 02235 g003
Figure 3. Representative SEM microphotographs of Streptococcus mutans colonies formed on disk-shaped specimens from light-cured BF subgroup, at 1000× (A) and 3000× (B) magnifications, and from heat-cured BF subgroup, at 1000× (C) and 3000× (D) magnifications. Arrows in 3000× SEM images indicate examples of Streptococcus mutans colonies.
Figure 3. Representative SEM microphotographs of Streptococcus mutans colonies formed on disk-shaped specimens from light-cured BF subgroup, at 1000× (A) and 3000× (B) magnifications, and from heat-cured BF subgroup, at 1000× (C) and 3000× (D) magnifications. Arrows in 3000× SEM images indicate examples of Streptococcus mutans colonies.
Biomedicines 12 02235 g003
Table 1. Summary of experimental groups and materials employed in the study.
Table 1. Summary of experimental groups and materials employed in the study.
Experimental GroupCuring ProtocolMaterialManufacturerLot NumberComposition
GRLight-curing GrandioSO
Shade A2
(Nanohybrid)		Voco GmbH, Cuxhaven, Germany221377289% fillers (1 μm glass filler); 20–40 nm (silicon dioxide filler); pigments (iron oxide, titanium dioxide).
Bis-GMA, Bis-EMA, TEGDMA
Heat-curing
VDLight-curingVenus Diamond
Shade A2
(Nanohybrid)		Kulzer GmbH, Hanau, GermanyK01020164% fillers (5 nm–20 μm), barium aluminum fluoride glass (Ø 0.7 max. <2 μm), discrete nanoparticles.
TCD-DI-HEA, UDMA.
Heat-curing
BFLight-curingEnamel Plus
HRi Biofuntion
Shade BF2
(Nanohybrid)		Micerium SpA, Avegno, Italy202300099074% fillers (0.005 µm-0.05 µm silicon dioxide), (0.2–3.0 µm glassy filler). UDMA, Tricyclodecane dimethanol dimethacrylate.
Heat-curing
For all light-cured resin composites, a curing of 20 s on both specimen sides with a light-emitting diode curing unit (Celalux 3, VOCO, Cuxhaven, Germany)—output 1300 mW/cm2—was realized. For all heat-cured resin composites, after the light-curing process, a subsequent and further polymerization of 10 min in a heat-curing unit (LaborLux, Micerium, Avegno, Genova, Italy) at 80 °C was realized.
Table 2. Means (and standard deviations) for SM planktonic CFU count (CFU/mL), sessile CFU count (CFU/mL) and biomass quantification by optical density (OD570nm).
Table 2. Means (and standard deviations) for SM planktonic CFU count (CFU/mL), sessile CFU count (CFU/mL) and biomass quantification by optical density (OD570nm).
-Planktonic CFU Count (CFU/mL)
Material
Curing protocolGRVDBF
Light-cured 7.23 × 106 c1
(8.05 × 105)		2.14 × 107 b1
(4.03 × 106)		4.40 ×1 07 a1
(6.13 × 106)
Heat-cured 4.89 × 106 b1
(7.65 × 105)		4.95 × 106 b2
(9.19 × 105)		2.80 × 107 a2
(4.33 × 106)
-Sessile CFU Count (CFU/mL)
Material
Curing protocolGRVDBF
Light-cured 7.49 × 106 b1
(7.88 × 105)		2.93 × 107 a1
(6.31 × 106)		6.71 × 106 b1
(8.55 × 105)
Heat-cured 3.97 × 106 a2
(8.70 × 105)		6.07 × 106 a2
(8.19 × 105)		6.38 × 106 a1
(4.90 × 105)
-Biomass Quantification by Optical Density (OD570nm)
Material
Curing protocolGRVDBF
Light-cured 0.1325 b1
(0.0207)		0.1457 b1
(0.0376)		0.4280 a1
(0.0907)
Heat-cured 0.1464 a1
(0.0287)		0.1731 a1
(0.0241)		0.1931 a2
(0.0490)
Different superscript letters indicate a statistically significant difference between the levels of “Material” factor. The superscript letter “a” indicates highest values. The superscript letter “b” indicates higher values than “c”. The same numbers in subscript indicate no significant differences among the levels of “Curing protocol” factor.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

De Angelis, F.; D’Arcangelo, C.; Di Lodovico, S.; Sorrentino, E.; Buonvivere, M.; D’Ercole, S. Influence of Polymerization Protocol on Adhesion and Proliferation of Streptococcus mutans on Three Dental Composite Resins. Biomedicines 2024, 12, 2235. https://doi.org/10.3390/biomedicines12102235

AMA Style

De Angelis F, D’Arcangelo C, Di Lodovico S, Sorrentino E, Buonvivere M, D’Ercole S. Influence of Polymerization Protocol on Adhesion and Proliferation of Streptococcus mutans on Three Dental Composite Resins. Biomedicines. 2024; 12(10):2235. https://doi.org/10.3390/biomedicines12102235

Chicago/Turabian Style

De Angelis, Francesco, Camillo D’Arcangelo, Silvia Di Lodovico, Edoardo Sorrentino, Matteo Buonvivere, and Simonetta D’Ercole. 2024. "Influence of Polymerization Protocol on Adhesion and Proliferation of Streptococcus mutans on Three Dental Composite Resins" Biomedicines 12, no. 10: 2235. https://doi.org/10.3390/biomedicines12102235

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop