Antimicrobial effects of Propolis, calcium Hydroxide, triple antibiotic paste, and modified triple antibiotic paste on tubular dentin inoculated with a dual-species biofilm: an ex vivo study

Abstract

Objectives

This study aimed to compare the antimicrobial effects of Propolis, Calcium Hydroxide (CH), Triple Antibiotic Paste (TAP), and modified TAP (mTAP) as intracanal medicaments on tubular dentin inoculated with a dual-species biofilm.

Materials and methods

In this ex vivo study, dentin cylinders were obtained from the root canals of 56 single-rooted extracted teeth, which were instrumented and inoculated with Enterococcus faecalis (E. faecalis) and Actinomyces naeslundii (A. naeslundii) suspensions to allow biofilm formation. After that, the cylinders were randomly assigned to five groups for a 7-day exposure to Propolis, TAP, mTAP (penicillin G), and Calcium Hydroxide (CH), and a no-medicament negative control group. Colonies were counted in dentin samples obtained from 200 μm to 400 μm depths and analyzed using ANOVA, Shapiro-Wilk, Levene, Tukey, robust Welch, Games-Howell, Fisher’s exact, Wilcoxon, and paired t tests (α = 0.05).

Results

All medicaments significantly decreased the colony counts (P < 0.05). CH, TAP, and mTAP showed similar optimal efficacy at both depths, whereas Propolis caused a significant reduction in bacterial count only at 200 μm (P = 0.047). Effect sizes were very high for both microorganisms. Dentin depth had a significant effect on the bacterial count of both microorganisms, and the load of both microorganisms was significantly lower at 400 μm than 200 μm depth (P < 0.001).

Conclusion

TAP and mTAP demonstrated high efficacy, and CH showed acceptable efficacy for elimination of E. faecalis and A. naeslundii dual-species biofilm. Propolis showed lower efficacy, highlighting the need for further modifications to enhance its penetration depth.

Introduction

Regenerative Endodontic Procedures (REPs) are increasingly used for managing immature teeth with pulp necrosis, aiming to restore the biological functions of the pulp–dentin complex and promote continued root development [1–3]. Successful clinical outcomes heavily depend on achieving an adequate level of root canal disinfection prior to the regenerative process, as residual microorganisms have been directly associated with compromised tissue regeneration and impaired apical development [4–6].(Figs. 1, 2, 3, and 4).

Fig. 1.

SEM micrograph indicating smear layer removal; open dentinal tubules can be observed at different magnifications (×2000, ×5000, and ×12000)

Fig. 2.

SEM micrograph of bacterial biofilm formed within the dentinal tubules after 1 week

Fig. 3.

SEM micrograph of bacterial biofilm formed at the opening of dentinal tubules after 3 weeks at different magnifications (×2000, ×5000, and ×12000)

Fig. 4.

SEM micrograph of biofilm growth in dentinal tubules after 3 weeks (both A. naeslundii and E. faecalis) at different magnifications (×2000, ×5000, and ×12000)

A wide range of microorganisms has been detected in infected root canal systems [7], among which Enterococcus faecalis (E. faecalis) and Actinomyces naeslundii (A. naeslundii) are frequently identified in persistent and secondary infections [8]. E. faecalis is particularly resistant due to its ability to survive in nutrient-deprived conditions, tolerate extreme pH levels, and deeply penetrate dentinal tubules [9–11].

A. naeslundii, a facultative Gram-positive rod, has strong adhesion capacity facilitated by fimbriae and has recently been reported as one of the most prevalent microorganisms in immature necrotic teeth [7, 12]. Both species are capable of forming biofilms and establishing persistent intratubular infections, contributing to treatment failure in REPs [7, 8, 13].

Intracanal medicaments such as Calcium Hydroxide (CH) and antibiotic-based pastes are widely used to assist in microbial elimination. Triple Antibiotic Paste (TAP) is the most common medicament in REPs; however, concerns about dentin discoloration, cytotoxicity at high concentrations, and the need for alternatives with improved biocompatibility have encouraged investigation of modified TAP (mTAP: consisting of penicillin G, metronidazole, and ciprofloxacin) formulations and herbal medicaments such as Propolis [6, 14–23]. Propolis, a biocompatible natural resin with documented antimicrobial and anti-inflammatory properties, has recently gained attention as a potential intracanal medicament in regenerative endodontics [9, 24, 25]. Despite extensive research on these materials, most previous studies have focused primarily on single-species biofilms—mainly E. faecalis—which do not fully represent the polymicrobial nature of clinical infections. Dual-species biofilms offer a model that better simulates the microbial interactions and resistance profiles present in vivo. The combination of E. faecalis and A. naeslundii is particularly relevant due to their strong synergistic ability to form persistent biofilms in immature necrotic teeth [7].

Therefore, the present study aimed to compare the antimicrobial effects of Propolis, CH, TAP, and modified TAP (mTAP) on dentinal tubules inoculated with an E. faecalis–A. naeslundii dual-species biofilm. The null hypothesis was that no significant difference would exist among the tested medicaments in reducing the bacterial load at different dentinal depths.

Materials and methods

This ex vivo study was conducted on dentin cylinders prepared from 56 extracted single-rooted teeth with single canals. All extractions were performed with the patient’s informed consent, at the Department of Oral and Maxillofacial Surgery, Shahid Beheshti Dental School. The study protocol was approved by the university ethics committee (IR.SBMU.DRC.REC.1403.064), and the research was conducted in accordance with the Declaration of Helsinki.

Inclusion and exclusion criteria

Inclusion criteria were intact single-rooted teeth without prior endodontic treatment, caries, cracks, resorption, or calcification [16]. Exclusion criteria included teeth with curved, ribbon-shaped, or short roots.

Sample size

Based on one-way ANOVA (α = 0.05, β = 0.20) using PASS 21 and effect estimates from Edara Lillygrace et al. [24], the required sample size was 10 specimens per group. An additional six specimens were allocated for SEM evaluation.

Specimen preparation

A modified model of the method described by Haapasalo and Orstavik [26] was used in this study. Crowns were removed at the cementoenamel junction, and 6-mm root segments from the middle third were obtained. Cementum was removed and canals were enlarged to the diameter of a #3 Gates–Glidden drill (Mani, Takanezawa, Japan). The smear layer was eliminated using 17% EDTA followed by 5.25% sodium hypochlorite (Chamin, Tehran, Iran). Final dentin cylinders were sterilized and stored in sterile saline (Darupakhsh, Tehran, Iran) until use [16].

Biofilm formation

Sterile dentin specimens were inoculated with standardized suspensions of E. faecalis (ATCC 29212; 10⁸ CFU/mL) and A. naeslundii (PTCC 1201; 10⁶ CFU/mL), adjusted to 0.5 McFarland and verified spectrophotometrically [11, 12]. To facilitate intratubular penetration, specimens were centrifuged using a multi-stage protocol [7]. They were then incubated in BHI broth (Merck, Germany) with 1% sucrose at 37 °C for 21 days, with medium refreshed every 48 h [7]. All specimens were inoculated using the same standardized bacterial suspensions to ensure equal initial microbial loads. The concentrations were verified both visually (0.5 McFarland) and spectrophotometrically at 600 nm wavelength to confirm consistency among samples. To ensure reproducibility of biofilm formation, colony counts from randomly selected specimens were verified to show comparable bacterial loads, and SEM observations at one and three weeks confirmed consistent biofilm development within dentinal tubules across specimens.

SEM assessment

Six specimens were randomly selected for SEM analysis (FEIESEM QUANTA 200; EDAX SILICON DRIFT 2017) to verify smear layer removal and confirm biofilm formation at 1 and 3 weeks. Specimens were longitudinally split, fixed, dehydrated through graded ethanol, sputter-coated with gold, and examined under SEM.

Preparation of medicaments

TAP and mTAP (penicillin G replacing minocycline) (Merck, Germany) were prepared as 0.1% (equal to 1 mg/mL) gels using a 6% methylcellulose carrier. Methylcellulose was selected as a neutral, biocompatible gel carrier at a 6% concentration, which has been shown in previous studies to lack intrinsic antimicrobial effects at this level [16]. CH paste was prepared by mixing 1 g of CH powder (Merck, Germany) with 1 mL of saline, resulting in a final concentration of a 1 g/mL (1000 mg/mL) with a creamy consistency [27–29]. Propolis (Gyah Kala, Tehran, Iran) paste was prepared from spray-dried Iranian Propolis powder mixed with saline to a final concentration of approximately 0.67 mg/mL with a creamy consistency [24]. The ethanol used in the extraction process was completely removed during spray drying, resulting in a dry powder with no residual solvent content. A no-medicament group served as the negative control.

Application of medicaments

After biofilm maturation (3–weeks), canals were dried and sealed externally with nail varnish. Specimens were randomly assigned to five groups (n = 10) and filled with the assigned medicament using a Lentulo spiral (Mani, Takanezawa, Japan). Both canal orifices were sealed with paraffin, and specimens were incubated at 37 °C for 7 days [30].

Intracanal sampling

Specimens were rinsed with sterile saline, and dentin chips were collected from depths of 200 μm and 400 μm using #4 and #5 Gates–Glidden drills. Samples were weighed, vortexed in 1 mL of BHI broth (Merck, Germany), serially diluted, and cultured on selective media: bile esculin agar (Merck, Germany) for E. faecalis and CFAT agar (Merck, Germany) for A. naeslundii. After 24 h of incubation at 37 °C, the colony-forming units (CFUs) were visually counted [16]. The final number of colonies in each sample was divided by the weight of the dentin chips corresponding to the same sample, and the results were reported as CFUs/mg. Colony counts were expressed as log₁₀ CFU/mg.

The operator who applied the medicaments was blinded to the study objectives and type of medicaments. The operator who cultured the samples and counted the colonies was also unaware of the group allocation of the specimens.

Statistical analysis

Statistical analysis was performed using SPSS (IBM Corp., USA). The Shapiro–Wilk test was used to assess data normality, and Levene’s test confirmed homogeneity of variances. A two-way repeated-measures ANOVA was applied to evaluate the effects of medicament type, dentin depth, and their interaction on colony counts (log₁₀ CFU/mg). Pairwise comparisons were conducted using Bonferroni and Tukey tests. For intra-group analyses, paired t-test or Wilcoxon test was used according to data distribution. To assess bacterial growth versus non-growth, and to determine the frequency of sterile samples, Fisher’s Exact Test was employed. Effect sizes (η²) and 95% confidence intervals were calculated to assess the magnitude and precision of observed differences. A significance level of P < 0.05 was adopted for all analyses.

Results

SEM validation

SEM imaging confirmed smear layer removal and the presence of a mature dual-species biofilm at 1 and 3 weeks. As these observations served only as methodological verification, no further ultrastructural description is provided [31].

Colony counts at 200 μm and 400 μm

Across both depths, TAP, mTAP, and CH produced significant reductions in bacterial load compared with the control group (P < 0.05). Propolis showed a modest reduction at 200 μm (E. faecalis: 8.11 ± 0.72 vs. 8.88 ± 0.27 log₁₀ CFU/mg; A. naeslundii: 8.09 ± 0.68 vs. 8.83 ± 0.43 log₁₀ CFU/mg, P < 0.05) but did not significantly differ from the control at 400 μm for either microorganism (E. faecalis: 7.82 ± 0.38 vs. 7.94 ± 0.28; A. naeslundii: 7.61 ± 0.44 vs. 7.55 ± 0.33, P > 0.05) (Tables 1 and 2). E. faecalis counts decreased significantly from 200 to 400 μm in all groups except Propolis (Tables 1 and 2). A. naeslundii exhibited a similar depth-dependent reduction across all groups. A combined summary of colony counts at both depths is shown in (Tables 1 and 2). Pairwise comparisons demonstrated that TAP and mTAP consistently had the greatest antimicrobial activity, followed closely by CH. Propolis exhibited the weakest effect.

Table 1.

Mean colony count (log 10 weight adjusted CFUs) of E. faecalis at 200 and 400 μm depths in the study groups (n = 10)

Depth	Group	Mean ± SD
200 μm	control	8.88 ± 0.27
	Propolis	8.11 ± 0.72
	m-TAP	7.56 ± 0.55
	TAP	7.52 ± 0.40
	CH	7.46 ± 0.37
400 μm	control	7.94 ± 0.28
	Propolis	7.82 ± 0.38
	m-TAP	7.08 ± 0.56
	TAP	6.81 ± 0.25
	CH	6.72 ± 0.31

Table 2.

Mean colony count (log 10 weight adjusted CFUs) of A. naeslundii at 200 μm and 400 μm depths in the study groups

Depth	Group	Mean ±SD
200 μm	control	8.83 ± 0.43
	Propolis	8.09 ± 0.68
	m-TAP	7.63 ± 0.41
	TAP	7.50 ± 0.28
	CH	7.35 ± 0.51
400 μm	control	7.55 ± 0.33
	Propolis	7.61 ± 0.44
	m-TAP	6.85 ± 0.35
	TAP	6.64 ± 0.47
	CH	6.49 ± 0.46

Within-group comparison of bacterial count at different depths

E. faecalis: In the control group, the colony count at 400 μm was significantly lower than at 200 μm (P = 0.000). No significant difference was observed in the Propolis group (P = 0.202). Significant reductions at 400 μm compared to 200 μm were observed in the CH (P = 0.04), TAP (P = 0.03), and mTAP (P = 0.021) groups.

A. naeslundii: Colony counts at 400 μm were significantly lower than at 200 μm in all groups: control (P = 0.000), Propolis (P = 0.047), CH (P = 0.009), TAP (P = 0.001), and mTAP (P = 0.002).

Comparison of microorganism-free specimens

No significant difference was found between groups in terms of complete elimination of bacterial growth (P > 0.05), indicating the persistence of biofilm even after medicament application (Table 3).

Table 3.

Growth status of E. faecalis at 400 μm and A. naeslundii at 200 and 400 μm depths in different groups

		no growth	growth	Total
A. naeslundii at 200 μm	control	1	9	10
	Propolis	0	10	10
	m-TAP	1	9	10
	TAP	1	9	10
	CH	1	9	10
	Total	4	46	50
A. naeslundii at 400 μm	control	1	9	10
	Propolis	0	10	10
	m-TAP	4	6	10
	TAP	2	8	10
	CH	2	8	10
	Total	9	41	50
E. faecalis at 400 μm	control	0	10	10
	Propolis	0	10	10
	m-TAP	1	9	10
	TAP	1	9	10
	CH	1	9	10
	Total	3	47	50

Effect size (partial Eta squared)

Partial eta-squared values indicated large effect sizes for the impact of medicament type on bacterial counts: 0.57–0.60 for A. naeslundii and 0.57–0.68 for E. faecalis across both depths.

Discussion

This study evaluated the antibacterial effectiveness of Propolis, CH, TAP, and mTAP against a dual-species biofilm of E. faecalis and A. naeslundii within dentinal tubules. Consistent with previous research, antibiotic-based medicaments (TAP and mTAP) demonstrated the greatest antimicrobial efficacy at both depths, whereas Propolis showed the weakest performance, particularly at 400 μm. These results confirm that mature biofilms present substantial resistance and require medicaments with deeper penetrability and sustained antibacterial activity [16, 32].

The strong performance of TAP and mTAP aligns with previous studies demonstrating their broad-spectrum activity, deep dentin penetration, and ability to disrupt mature biofilms. mTAP, in particular, has been proposed as a promising alternative to conventional TAP due to reduced discoloration potential and the inclusion of penicillin G, which shows strong activity against Gram-positive endodontic pathogens. In the present study, TAP and mTAP showed comparable results, supporting earlier reports indicating that replacement of minocycline with penicillin G does not compromise antibacterial efficacy in multi-species models [1, 7, 12, 16, 22, 32–34].

Interestingly, our study demonstrated the antibiofilm effect of Calcium Hydroxide (CH) at both 200 and 400 μm depths, which contrasts with some previous reports, particularly regarding E. faecalis. This discrepancy may be explained by several biological and physical factors: the particle size and vehicle of CH may enhance intratubular penetration, thereby improving its efficacy [16, 21, 35–45]. Additionally, in dual-species biofilm conditions, bacterial proton pump activity may be partially impaired and the protective biofilm matrix weakened, which can relatively increase CH effectiveness, making it comparable to TAP and mTAP (21). These findings suggest that proper placement and sufficient contact time allow CH to be more effective under dual-species conditions than in single-species models.

Propolis demonstrated notable antibacterial activity at 200 μm but showed markedly reduced efficacy at 400 μm. This is consistent with its physicochemical limitations—high viscosity, low solubility, and limited diffusion capacity—which restrict its ability to penetrate deep into dentinal tubules [32–34, 39]. Furthermore, the antimicrobial potency of Propolis is highly dependent on geographic origin and chemical profile, especially flavonoid concentration. These variations may explain why some studies report results comparable to TAP, whereas others—like the present study—show weaker performance, particularly against mature biofilms [32, 40].

The depth-dependent decrease in bacterial counts observed across most groups is consistent with prior work showing lower microbial colonization and viability in deeper dentin regions due to reduced nutrient availability and limited tubule diameter. However, complete bacterial elimination was not achieved by any medicament, reinforcing the known resistance of organized biofilm structures and emphasizing that chemical medicaments alone may not fully eradicate intratubular infection [46–48].

Taken together, the findings support the superior efficacy of antibiotic-based medicaments, highlight the clinically acceptable and possibly underestimated potential of CH, and indicate that while Propolis is biocompatible and biologically attractive, its antimicrobial penetration remains insufficient without formulation enhancement. These outcomes are clinically relevant for regenerative endodontic procedures, where both antimicrobial efficacy and cell compatibility are critical for predictable outcomes [49].

This study had several limitations. First, despite using human dentin, ex vivo conditions cannot fully replicate the dynamic oral environment. Second, the antimicrobial properties of Propolis vary by geographic origin and chemical composition, which could influence its performance. Third, only a dual-species biofilm model and a single 7-day time point were evaluated. Therefore, future studies should investigate multi-species biofilms, different medicament concentrations and formulations, and multiple time intervals. In addition, advanced analytical methods (e.g., CLSM, PCR and HPLC) as well as well-designed clinical trials are required to further validate and generalize these findings.

From a clinical perspective, these findings indicate that Propolis and mTAP may serve as biocompatible alternatives to CH and conventional TAP, particularly when prolonged intracanal dressing is desired. The seven-day application period used in this study reflects a clinically relevant balance between antimicrobial effectiveness and cytocompatibility reported in previous literature.

Conclusion

According to the obtained results, although all the tested medicaments led to a significant reduction in bacterial counts, their effectiveness varied depending on dentin depth and biofilm resistance. Within the limitations of this ex vivo study, TAP and mTAP demonstrated greater antibacterial activity at different dentin depths and against resistant biofilms compared with the other tested medicaments. CH, due to its biological properties and antimicrobial potential, may be considered an alternative option when non-antibiotic intracanal medicaments are preferred. Although Propolis exhibits notable biological properties, its antimicrobial effectiveness against complex biofilms remains inconclusive and requires further investigation.

Abbreviations

TAP

Triple Antibiotic Paste

mTAP

Modified Triple Antibiotic Paste

CH

Calcium Hydroxide

CFU

Colony-Forming Unit

SD

Standard Deviation

REP

Regenerative Endodontic Procedures

SEM

Scanning Electron Microscopy

BHI

Brain Heart Infusion

Authors’ contributions

Dr. Zohreh Ahangari: designed the study and contributed to the language editing of the manuscript.Dr. Nazanin Zargar: contributed to the study design, data analysis, interpretation of results, and critical revision of the manuscript.Dr. Maryam Pourhajibagher: performed the experiments, assisted with microbiological procedures, and contributed to the manuscript’s language editing.Dr. Rezvan Shahhosseini: conducted the experiments, wrote the initial draft of the manuscript, and was responsible for sample preparation.All authors read and approved the final version of the manuscript.

Funding

Not applicable.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All extractions were performed with the patient’s informed consent, at the Department of Oral and Maxillofacial Surgery, Shahid Beheshti Dental School. The study protocol was approved by the university ethics committee (IR.SBMU.DRC.REC.1403.064), and the research was conducted in accordance with the Declaration of Helsinki.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.