Antibacterial effects of bioactive restorative dental materials on Streptococcus mutans: An in vitro study using the direct contact test

Abstract

This in vitro study aimed to evaluate the antibacterial activity of various restorative dental materials against Streptococcus mutans, a major cariogenic pathogen. The materials tested included a resin composite (Estelite Sigma Quick), conventional glass ionomer cement (Fuji IX), resin-modified glass ionomer cement (Fuji II LC), a bioactive resin-based material (Activa BioACTIVE Restorative), and a calcium silicate-based material (Biodentine). Antibacterial activity was assessed using the direct contact test (DCT). Each material was tested against S. mutans at 3, 6, 16, and 24-h intervals. Colony-forming units (CFU) were quantified following serial dilution and culture on BHI agar. Statistical comparisons were conducted using the Kruskal–Wallis test. All materials except Estelite demonstrated significant antibacterial effects. Biodentine exhibited the greatest inhibition (P ≤ 0.001), followed by Fuji IX (P ≤ 0.001), and Fuji II LC (P ≤ 0.01). Activa BioACTIVE showed significant bacterial reduction at 16 and 24 h (P ≤ 0.05). Estelite showed no significant antibacterial effect (P > 0.05). Biodentine displayed sustained and pronounced antibacterial effects, suggesting its suitability for patients at high risk of caries. Fuji IX and Fuji II LC also exhibited antibacterial properties, though to a lesser extent. The findings support the use of bioactive restorative materials in managing bacterial presence and enhancing restoration longevity. The superior antibacterial performance of Biodentine highlights its potential role in preventing secondary caries, particularly in high-risk populations. Clinicians are encouraged to consider bioactive materials as part of comprehensive caries management strategies.

Introduction

Dental caries is a biofilm-related disease which is identified by the demineralization of enamel, dentin and microbial activity within the oral cavity (Ribeiro and Paster 2023). Streptococcus mutans is a normal flora in the oral cavity which can be cariogenic when the oral condition is favorable for its growth. A previous study reported that the prevalence of S. mutans in visible occlusal plaque ranged from 96 to 100%.(Thitisakyothin et al.2025). It is recognized as a key cariogenic microorganism due to its acidogenicity, aciduricity, and ability to synthesize extracellular polysaccharides, facilitating biofilm formation and lesion progression (Yadav and Prakash 2017; Gao et al. 2024). Acid production from bacterial metabolism lowers the pH, leading to enamel and dentin demineralization, cavitation, and progressive structural damage (Nedeljkovic et al. 2020). Successful caries management depends not only on achieving sealed restorations but also on the remineralization of affected lesions (Melo et al. 2013). Effective caries management requires both remineralization of demineralized hard tissues and elimination of residual cariogenic bacteria at the tooth–restoration interface (Frencken et al. 2012; Chen et al. 2013). Remineralization restores the mineral content and mechanical integrity of dental tissues, while antibacterial activity reduces the bacterial load and disrupts biofilm formation, thereby preventing secondary caries (Chen et al. 2013). Despite advances in restorative dentistry, no currently available restorative material provides both sustained remineralization and durable antibacterial activity (Stewart and Finer 2019). Resin composite is routinely used for direct restorative materials due to their optical and mechanical properties (Baroudi and Rodrigues 2023). However, the lack of inherent antibacterial properties and the degradation of the bonding interface can lead to the failure of resin composite restorations (Bourgi et al. 2024). In contrast to resin composites, glass ionomer cement is recognized for its superior capacity to support lesion healing. It is regarded as a bioactive material that releases fluoride and adheres to tooth structure via ion exchange (Sidhu and Nicholson 2016). Nevertheless, the reduction of these properties over time has driven the search for superior materials (Dutra et al. 2024). Calcium silicate cements were generally used in endodontic treatment because of their antibacterial effects and capacity to induce remineralization. However, the downside of a long setting time has impeded its application in restorative dentistry (Jang et al. 2023). Advances in calcium silicate materials with reduced setting times present a promising remedy to facilitate their application in restorative dentistry (Eskandari et al. 2022).

Antibacterial activity of materials is commonly assessed in research by the agar diffusion test (Esteki et al. 2021). However, the test does not accurately assess the materials as in clinical application, which may lead to misinterpretation of the results. The direct contact method is considered suitable for testing materials with low solubility such as calcium silicate cements (Janini et al. 2021).

The absence of a single material with both remineralization activity and long-lasting antibacterial effect reflects the need for further evaluation of bioactive restorative materials. This study aimed to assess the antibacterial efficacy of various restorative dental materials including resin composite, conventional and resin-modified glass ionomer cement, a bioactive resin-based material, and a calcium silicate-based material against Streptococcus mutans. This study utilized the direct contact method to approximate the clinical performance of the materials. The null hypothesis stated that there would be no significant difference in antibacterial activity among the tested materials at 3, 6, 16, and 24 h.

Methods

Antibacterial testing

The antibacterial activity of each material was assessed using the direct contact test (DCT). A total of six groups were included in the experiment: one control group and five experimental groups representing each restorative material, with five specimens per group (n = 5). All groups were tested against Streptococcus mutans at intervals of 3, 6, 16, and 24 h. Colony-forming unit (CFU) counts were determined following serial dilution and plating on brain heart infusion (BHI) agar. The study included six groups including five experimental groups and one control group. The control group was the bacterial suspension only.

Preparation of material discs

The restorative materials used in the study are listed in Table 1. Each material was prepared according to the manufacturer's instructions with n = 12 discs per group (3 discs for each time point). A sterilized Teflon mold (6 × 2 mm³) was used to fabricate the discs. During polymerization, the discs were covered with celluloid strips and glass slides to eliminate oxygen inhibition. Excess edges were trimmed using a No. 15 blade.

Table 1.

List of tested restorative dental materials and their manufacturers

Material Name	Manufacturer	Instructions for Use
Fuji IX, Gold Label Capsule	GC Corp., Tokyo, Japan	• Shake capsule • Activate capsule • Mix for 10 s • Apply directly to cavity • Protect with varnish or coat
Estelite Sigma Quick	Tokuyama Dental Corp., Japan	• Etch tooth surface • Apply adhesive • Place in ≤ 2 mm increments • Light cure each layer for 10 s
Fuji II LC, Gold Label Capsule	GC Corp., Tokyo, Japan	• Shake capsule • Activate capsule • Mix for 10 s • Apply to cavity • Light cure for 20 s • Protect surface
Activa BioACTIVE Restorative	PULPDENT Corp., MA, USA	• Etch tooth surface • Apply bonding agent • Apply from syringe • Light cure for 20 s • Self-cures as backup • Polish after full set (4–6 min)
Biodentine	SEPTODONT, Saint-Maur-des-Fossés, France	• Mix for 30 s in capsule mixer • Apply with spatula or gun • Wait approximately 12 min for full setting

Bacterial inoculum preparation

S. mutans (ATCC 25175) was provided by the Department of Stomatology, Faculty of Dentistry, Srinakharinwirot University. The bacteria were cultured on BHI agar using the streak plate technique and incubated at 37 °C for 48–72 h. A single colony was transferred to 5 mL of BHI broth and incubated at 37 °C for 24 h to prepare a pure inoculum. The bacterial suspension was adjusted to match the 0.5 McFarland standard (~ 1.5 × 10⁸ CFU/mL), verified using a spectrophotometer (600 nm, OD 0.08–0.10). The inoculum was used within 15 min of preparation (Fig. 1).

Fig. 1.

Preparation of Streptococcus mutans inoculum adjusted to 0.5 McFarland standard

Direct contact test procedure

The experiment was performed at the Department of Stomatology, Faculty of Dentistry, Srinakharinwirot University. Each test material disc was placed horizontally at the bottom of a 48-well microplate. A 10 µL aliquot of bacterial suspension (~ 1.5 × 10⁶ CFU/mL) was pipetted onto the surface of each disc and allowed to react with materials over 1 h. This step ensured direct contact between the bacterial cells and the test material surface. Subsequently, 400 µL of BHI broth was added to each well, and the plates were incubated at 3, 6, 16, and 24 h.

After incubation, the medium was resuspended 20 times using a micropipette. A 50 µL aliquot was transferred onto an agar plate, spread using sterile glass beads, and incubated at 37 °C for 48 h. CFUs were counted and converted to CFU/mL using the formula:

$$CFU/mL=\left(n,u,m,b,e,r,,o,f,,c,o,l,o,n,i,e,s,\times ,d,i,l,u,t,i,o,n,,f,a,c,t,o,r\right)/volumeofcultureplated\left(m,L\right)$$

A material was considered to have antibacterial properties if it achieved a ≥ 99% bacterial reduction (2-log₁₀ reduction) compared to the control, as defined by JIS Z 2801 (Fig. 2).

Fig. 2.

Schematic of direct contact test (DCT) procedure for antibacterial evaluation

Statistical analysis

The experiment was done in triplicate. Data were analyzed using SPSS version 25.0. Non-parametric tests were selected due to the non-normal distribution of CFU data, as confirmed by the Shapiro–Wilk test. The Kruskal–Wallis test was done to determine overall differences among groups at each time interval. When a significant difference was found, Dunn’s post hoc pairwise comparisons were performed to identify which specific groups differed. Exact p-values were reported for each comparison, with p < 0.05 considered statistically significant.

Results

Direct contact test

Based on the Kruskal–Wallis test followed by Dunn’s post hoc comparisons, the direct contact test (DCT) showed that, except for Estelite, all restorative materials significantly reduced S. mutans growth compared to the control. The Kruskal–Wallis test indicated significant overall differences in CFU counts among materials at every time interval (p < 0.001). Dunn’s post hoc test was then performed to evaluate pairwise differences. Results from the direct contact test (DCT) is shown in Fig. 3. The results revealed that, apart from Estelite, all materials produced significant reductions in S. mutans growth when in contact with the tested materials. Notably, relative to the control group, Biodentine exhibited the highest antibacterial activity (P ≤ 0.001), followed by Fuji IX (P ≤ 0.001), Fuji II LC (P ≤ 0.01), and Activa BioACTIVE (P ≤ 0.05) though exhibit the significance after 16-h period. Moreover, Biodentine completely inhibited bacterial growth at all time points, with no detectable CFU counts observed over the 24-h incubation period, as shown in Fig. 4, 5, 6, 7. Biodentine and Fuji IX consistently exhibited the strongest antibacterial effects against S. mutans across all time points.

Fig. 3.

The comparison of CFU/ml differences among each material by each hour by Kruskal–Wallis test and symbol (*) showed a significant level after Dunn’s post-hoc comparison with (*) = P ≤ 0.05, (**) = P ≤ 0.01, and (***) = P ≤ 0.001

Fig. 4.

Representative images of bacterial colony formation on agar plates at 3 h: (a) Biodentine, (b) Fuji IX, (c) Fuji II LC, (d) Activa BioACTIVE, (e) Estelite and (f) control

Fig. 5.

Representative images of bacterial colony formation on agar plates at 6 h: (a) Biodentine, (b) Fuji IX, (c) Fuji II LC, (d) Activa BioACTIVE, (e) Estelite and (f) control

Fig. 6.

Representative images of bacterial colony formation on agar plates at 16 h: (a) Biodentine, (b) Fuji IX, (c) Fuji II LC, (d) Activa BioACTIVE, (e) Estelite and (f) control

Fig. 7.

Representative images of bacterial colony formation on agar plates at 24 h: (a) Biodentine, (b) Fuji IX, (c) Fuji II LC, (d) Activa BioACTIVE (e) Estelite and (f) control

Discussion

Biodentine showed a strong antibacterial effect against S. mutans for 24 h (p < 0.001), matching earlier studies that reported its long-lasting antimicrobial activity (Rajasekharan et al. 2014; Farrugia et al. 2018). Rajasekharan et al. (2014) described this effect to the material’s high initial alkalinity and prolonged calcium hydroxide release, creating an environment unfavorable for bacterial survival. Similarly, Farrugia et al. (2018) reported that the surface characteristics of Biodentine contributed to its antimicrobial performance. Both studies mainly evaluated calcium silicate cements for endodontic use. which include pulp capping or root-end filling. In these procedures, the material is placed close to vital pulp or periapical tissues, which are mostly soft tissues. In contrast, the present study evaluated Biodentine as a restorative material, directly exposed to cariogenic bacteria in a simulated restorative environment. As S. mutans grows optimally at a pH of 4–9, pH values above this range can retard bacterial growth or cause bacterial death (Nakajo et al. 2006). Therefore, the high pH of Biodentine, exceeding 11, is likely responsible for its strong bactericidal action against S. mutans (Aminoshariae et al. 2022). This property may explain why Biodentine demonstrated superior and prolonged antibacterial activity compared with the other restorative materials tested in the current study. This difference in application context highlights the novelty of our findings and suggests that Biodentine’s antibacterial efficacy may be useful not only in endodontic procedures but also in restorative treatments. Such benefits are especially important where bacterial control in deep carious lesions is critical.

Among the tested materials, Fuji IX exhibited strong antibacterial activity, comparable to Biodentine at most time points. Fuji IX was included as a representative of traditional GICs, which are known to possess antibacterial properties (Farrugia and Camilleri 2015). Statistical analysis showed a significant difference between Fuji IX and the control group, as well as other tested materials (3 h: p < 0.001; 6 h: p < 0.001; 16 h: p = 0.004; 24 h: p < 0.001). A study by Chen et al. (2020) used the direct contact test to evaluate the antimicrobial effects of Fuji IX modified with reduced graphene–silver nanoparticles (R-GNs/Ag), with conventional Fuji IX serving as the negative control against S. mutans. The results indicated an increase in viable bacterial counts at 12 h and 24 h in the non-metallized Fuji IX group. In the present study, however, a similar growth pattern was observed at 3 h and 6 h, followed by a decrease in viable colonies at 16 h and 24 h. A literature review by Hafshejani et al. (2017) also supported that the immediate antibacterial effect of GICs was mainly attributable to their fluoride release. The fluoride release showed an initial peak within the first 24–48 h, followed by a steady state at a reduced level. According to Cabral et al. (2015), fluoride release from GIC was related to the composition rather than the type of cement. They also reported that the highest fluoride release occured within the first 24 h following application. Then the fluoride release gradually decreased over time. While this initial release is beneficial for antibacterial effects and remineralization, the subsequent reduction in fluoride release may compromise the long-term efficacy of GICs. This result indicated that, despite their short-term efficacy, the long-term antibacterial activity of GICs may be less sustainable. The finding in the current study also suggested that a longer observation period may be necessary to evaluate the stability of the antibacterial effect of tested materials.

Fuji II LC, a resin-modified glass ionomer cement (RMGIC), showed significantly lower bacterial counts compared with the control and the Estelite resin composite at all tested intervals (p-values ranging from 0.002 to 0.055). However, its antibacterial capability was lower than that of Fuji IX. Francois et al. (2020) reported an in-depth analysis of fluoride-releasing restorative materials, describing their modes of action, fluoride-releasing potential, and clinical applications. They reported that, although Fuji II LC demonstrated antimicrobial activity, its fluoride release was less effective than that of Fuji IX. This difference was likely due to the resin-based constituents of Fuji II LC, which may reduce fluoride ion release.

ACTIVA BioACTIVE, a bioactive glass-containing restorative material, significantly reduced bacterial counts compared with the control at 16 h (p = 0.043) and 24 h (p = 0.049). Its antibacterial effectiveness was comparable to both Fuji II LC and Estelite during the tested time intervals. This finding conformed with previous studies using different methods. Conti et al. (2023) reported that the antibacterial activity of ACTIVA was comparable to Ketac Silver when using agar diffusion method. The study evaluated materials within 48 h. ACTIVA’s antibacterial effect may derive from ion release. As bioactive glass can raise pH, disrupt bacterial membranes, and inhibit S. mutans biofilm formation (Drago et al. 2018). However, its resin content may limit this effect. This finding was consistent with previous report of no inhibitory activity in resin-based cements (Lila-Krasniqi et al. 2022).

The resin composite Estelite demonstrated negligible antibacterial activity, with growth patterns comparable to the controls. Its inability to inhibit S. mutans activity is consistent with previous findings. Calderón et al. (2019) highlighted that surface roughness, stemming from factors such as filler size and polishing technique, facilitated bacterial adhesion and biofilm formation in resin composites. However, the material itself has no antibacterial activity according to previous reports. A recent review reported that the composition of resin composite does not exhibit antibacterial activity. The concentration of monomers released from composites is too low to induce antibacterial activity, and the inorganic fillers are silica with no antimicrobial properties (Algarni 2024). Similarly, Chen et al. (2018) emphasized that standard resin composites lack intrinsic antibacterial effects, and any observed antimicrobial activity results from incorporated agents rather than the base material itself.

Streptococcus mutans was selected for this study because it plays a well-known role in starting and advancing dental caries, especially secondary caries. It produces glucosyltransferase and synthesizes extracellular polysaccharides. It also adheres strongly to tooth surfaces and co-aggregates with other bacteria. These abilities contribute greatly to the formation of cariogenic biofilm (Gao et al. 2024). Using a single bacterial strain ensures a genetically uniform culture, simplifying interpretation of experimental outcomes. This approach is widely adopted in in vitro studies because it allows precise control and clarity in assessing the effects of test materials (Khere et al. 2019; Conti et al. 2023).

The direct contact test (DCT) was used to evaluate antibacterial properties of the restorative materials. This method closely simulates bacterial exposure to material surfaces in clinical conditions. The procedure was a modified version described by Tavassoli Hojati et al. (2013), using material disc specimens prepared by the methodology of Miki et al. (2016). Antibacterial activity was assessed at 3, 6, 16, and 24 h to monitor bacterial growth patterns and the duration of antibacterial effects. Although the DCT was used in this study, it has inherent limitations. It is not suitable for long-term observation because bacterial viability may decline due to insufficient nutrient or test condition changing which may not relevant to the material’s antibacterial properties. Additionally, DCT results are difficult to compare across studies, as the method has been modified between laboratories and lacks of long-term stability (Cunliffe et al. 2021).

This study has several limitations including a short incubation period, the use of a single bacterial strain, and an in vitro design. Newer microbial analysis methods, such as biofilm models using various bacterial strains, could provide more detailed information about antibacterial effects. In clinical practice, the choice of restorative materials should consider long-term fluoride release, bioactivity, antibacterial capacity, and mechanical performance. Biodentine showed strong antibacterial properties in this study. However, its use as a permanent restorative material should also consider its mechanical strength, wear resistance, and aesthetic limitations before clinical recommendation. Currently, this material is most suitable for pulp protection in deep carious lesions or root caries in elderly patients, where antibacterial and remineralizing activities are prioritized over the mechanical strength of restorations.

Conclusion

Based on the findings of this study, clinicians are encouraged to incorporate bioactive restorative materials such as Biodentine in restorative treatments, particularly for patients at high risk of developing secondary caries. Biodentine, a calcium silicate-based material, demonstrated the most consistent and potent antibacterial effect against Streptococcus mutans, maintaining its efficacy throughout a 24-h period.

The direct contact test employed in this investigation provided a model for antibacterial activity study by simulating material-bacteria interaction under in vitro conditions. The results indicated the critical role of material selection in influencing bacterial growth and managing microbial colonization at the restoration interface. It would also be necessary to consider more bacterial species and longer clinical observation to confirm the long-term antimicrobial efficacy of these materials in various clinical settings.

Clinical relevance

The use of restorative materials with superior antibacterial properties, such as Biodentine, may significantly reduce the risk of secondary caries and enhance the long-term success of restorations, particularly in patients with high caries activity. Clinicians should consider the antibacterial potential of restorative materials as a key factor in treatment planning, especially for patients with high caries risk.

Author contributions

SB and SC Conceptsualization and methodology. OK and CS investigation. SB,SC, and OK data curation and visualization, OK and SC writing original draft. SC,SB and OK review and editing. All the authors have read and approved the manuscript.

Funding

This research was funded by the Faculty of Dentistry, Srinakharinwirot University (grant number 351/2567). The funder had no role in the design, data collection, data analysis, or reporting of the study.

Data availability

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding authors.

Declarations

Ethics

The study has been granted an exemption from requiring ethics approval by Srinakharinwirot University. (This research is considered as non-human research according to the university protocal).

Conflict of interest

The authors have no conflicts of interest to declare.