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

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 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 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An ex vivo study Zohreh Ahangari, Nazanin Zargar, Maryam Pourhajibagher, Rezvan Shahhosseini This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7502815/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Feb, 2026 Read the published version in BMC Oral Health → Version 1 posted 12 You are reading this latest preprint version 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 that were instrumented and inoculated with Enterococcus faecalis ( E. faecalis ) and Actinomyces naeslundii ( A. naeslundii ) susentions in order to biofilms formation. After that, the cylinders were randomly assigned to 5 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 and 400 µm depths and analyzed by ANOVA, Shapiro-Wilk, Levene, Tukey, robust Welch, Games-Howell, Fisher’s exact, Wilcoxon, and paired t tests (alpha=0.05). Results: All medicaments significantly decreased the colony count (P<0.05). CH, TAP, and mTAP had equally optimal efficacy at both depths; while, propolis caused a significant reduction in bacterial count only at 200 µm (P=0.047). The 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 showed high efficacy, and CH should acceptable efficacy for elimination of E. faecalis and A. naeslundii double-species biofilm. Propolis showed lower efficacy, highlighting the need for further modifications to enhance its penetration depth. Actinomyces naeslundii Antibiotic Biofilms Calcium Hydroxide Enterococcus faecalis Propolis Regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Regenerative endodontic procedures (REPs) are a novel therapeutic approach for treatment of immature developing teeth that have been traumatized by infection, trauma, or caries, and have developed irreversible pulpitis or pulp necrosis [ 1 – 3 ]. The main goal of REPs is to strengthen the root structure and facilitate its development in width and length. In REPs, presence of residual bacteria is significantly associated with impaired root development and incomplete closure of the apex [ 4 ]. Therefore, effective root canal disinfection plays a key role in supporting the proliferation and regeneration of pulp tissue [ 5 ]. Root canal disinfection, which involves the use of irrigating solutions and intracanal medicaments, is one of the main steps in endodontic treatment, especially REPs [ 6 ]. Also, preservation of viable cells during the treatment process is of great importance considering the possible toxic effects of irrigating solutions and intracanal medications, as the survival of these cells is essential for the successful regeneration of pulpal tissue [ 6 ]. A wide diversity has been identified in the root canal system microbiota [ 7 ]. Enterococcus faecalis ( E. faecalis ) and Actinomyces naeslundii ( A. naeslundii ) are among the bacterial species that have been frequently isolated from chronic and persistent root canal infections [ 8 ]. E. faecalis is the most common resistant bacterial species associated with dental infections, which has been abundantly observed and isolated from the root canals of permanent and primary teeth with failed endodontic treatment [ 9 ]. The high resistance of this microorganism to irrigating solutions and intracanal medicaments, the ability to grow in a wide range of pH, tolerance in nutritionally-deprived conditions, the ability to penetrate into dentinal tubules, and strong adhesion to dentin collagen are among the main reasons for its survival in chronic apical periodontitis. In addition to superficial penetration into dentinal tubules to a depth of 1000 µm [ 10 ], E. faecalis uses the root cementum as a long-term refuge for reinfection. Thus, this microorganism is commonly used for research purposes in endodontics [ 11 ]. On the other hand, A naeslundii is a Gram-positive, facultative rod-shaped anaerobic that has been increasingly recognized for its ability to form biofilm and its association with endodontic treatment failures [ 12 ]. Adhesion of A naeslundii to dentin collagen is mediated by its unique thin, hair-like surface structures (fimbriae) [ 12 ]. Recently, this microorganism was identified as the most common microorganism in immature permanent teeth with pulp necrosis [ 7 , 12 ], suggesting a possible association with failure of REPs. Both E. faecalis and A. naeslundii ​​ have the ability to form biofilm and penetrate into dentinal tubules, which allows them to withstand the adverse conditions of the root canal environment [ 7 , 8 , 13 ]. In addition, they are easily cultured in vitro, which makes them suitable for in vitro studies [ 13 ]. Calcium hydroxide (CH) has been widely used as an intracanal medicament, and has been recommended for REPs due to its favorable antibacterial properties. However, some concerns exist regarding its effectiveness as a potent and durable antibacterial agent in some cases of REPs. Also, canal debridement and cleaning after its application are challenging [ 6 ]. With the development of antibiotics, various intracanal medicaments are used in modern dentistry to reduce the microbial load of root canals [ 14 ]. Triple antibiotic paste (TAP) is the most commonly used intracanal medicament in REPs, which is a combination of ciprofloxacin, metronidazole, and minocycline [ 5 ]. However, the use of TAP is associated with problems such as coronal discoloration, changes in dentin structure, and cytotoxicity at high concentrations [ 15 ]. Using an antibiotic with comprehensive antibacterial effects against root canal pathogens with minimal discoloration potential and minimal cytotoxic effects on various stem cells may lead to successful clinical outcomes [ 16 ]. High concentrations of TAP are toxic for stem cells from the apical papilla [ 17 ] and negatively affect their adhesion and proliferation [ 18 ]. Therefore, a recent study recommended low concentrations of TAP to reduce its toxicity [ 19 ]. According to recent findings, a concentration of 20 mg/mL TAP can be effective for eliminating a 3-week mature E. faecalis biofilm up to 400 µm deep from the internal canal wall, while the antibacterial effect of CH is limited to only 200 µm depth from the internal canal wall with less efficacy [ 16 ]. Tooth discoloration is a major drawback of TAP. Therefore, in some cases, the use of dual antibiotic paste, which consists of only ciprofloxacin and metronidazole, is suggested [ 20 ]. However, a systematic review reported significantly lower antimicrobial efficacy of dual antibiotic paste than TAP [ 21 ]. Therefore, some modifications were made in the TAP combination. In one of these modified combinations, which consists of penicillin G, metronidazole, and ciprofloxacin, it was observed that replacing minocycline with penicillin G probably results in a higher efficacy of the drug against E. faecalis biofilm formation [ 22 ]. In addition, this combination was also effective against a diverse group of root canal microbiota [ 15 ]. In vitro studies have shown that Actinomyces species are sensitive to moxifloxacin (a fluoroquinolone) and amoxicillin [ 12 , 22 ]. Currently, evidence-based dentistry has attracted attention to the use of herbal products in endodontics, which are mainly recommended due to their high biocompatibility and fewer side effects than chemical and synthetic antimicrobial agents [ 23 ]. Propolis is a natural resinous substance with high biocompatibility, which is extracted from plant resins collected by honeybees. Due to the presence of flavonoids, propolis has significant antimicrobial and anti-inflammatory properties [ 9 ]. The toxicity of propolis has been reported to be approximately 10 times lower than that of CH [ 24 ]. It has been shown that propolis is more effective against resistant microorganisms and is more biocompatible with periradicular tissues than the conventional root canal medicaments [ 25 ]. Despite extensive efforts in this field, complete elimination of microorganisms from the root canal system remains challenging. Therefore, it is imperative to find a more effective medicament to remove the residual microorganisms from dentinal tubules. Most previous studies on this topic used single-species biofilms (mainly E. faecalis) to evaluate antimicrobial activity, and studies on multi-species biofilms formed on human dentin are limited in number [ 7 ]. Since single-species biofilms do not accurately reflect the clinical conditions, the present study focused on a dual-species biofilm of microorganisms that have a higher prevalence in immature teeth. Therefore, this study aimed to compare the antimicrobial effects of propolis, CH, TAP, and modified TAP (mTAP) as intracanal medicaments on tubular dentin inoculated with a dual-species biofilm. The null hypothesis of the study was that no significant difference would be found in the efficacy of different medicaments for inhibition of the dual-species biofilm. Materials and Methods This ex vivo study was conducted on dentin blocks obtained from the root canals of 56 single-rooted, single-canal teeth extracted as part of prosthodontic treatment or due to poor periodontal prognosis. 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). Eligibility criteria: Single-rooted single-canal extracted teeth with a circular-shaped canal and no history of endodontic treatment were included after ensuring absence of root caries, cracks, fractures, intracanal calcifications, and internal/external root resorption by their inspection under a microscope [ 16 ]. Teeth with gradual curvature extended to the apical region, and those with ribbon-shaped or short roots were excluded. Sample size: The sample size was calculated to be 10 in each group using one-way ANOVA menu of PASS 21 (due to quantitative nature of the dependent variable and presence of 5 groups), assuming alpha = 0.05, beta = 0.2 (study power of 80%), and mean and standard deviation values obtained from a study by Edara Lillygrace et al [ 24 ]. Also, 6 specimens were used for scanning electron microscopy (SEM) assessment. Specimen preparation: A modified model of the method described by Haapasalo and Qrstavik [ 26 ] was used in this study. Eligible teeth (n = 56) were disinfected by immersion in 5.25% sodium hypochlorite (Chamin, Tehran, Iran) for 48 hours. They were then stored in 0.09% sterile saline (Darupakhsh, Tehran, Iran) at room temperature until use. All crowns were cut at the cementoenamel junction by a rotary diamond disc operating at 700 rpm (Dorsa, Tehran, Iran) under copious water irrigation perpendicular to the longitudinal tooth axis. Root end was also cut such that a 6-mm cylinder was obtained from the middle-third of the root. The root cementum was also removed by a diamond fissure bur (Mani, Takanezawa, Japan) and highspeed handpiece under water coolant. Gates Glidden drills #1, 2 and 3 were then used to dilate the canals. In all phases of specimen preparation, copious irrigation was performed with 5.25% sodium hypochlorite and saline. Finally, cylinders with 6 mm length, 4 mm external diameter and equal internal diameter corresponding to the diameter of a #3 Gates Glidden drill were obtained. To eliminate the smear layer, the specimens were placed in an ultrasonic bath (Vector 55; Jeltraft, Jelenko) containing 17% EDTA (pH = 7.8) for 5 minutes, followed by immersion in 5.25% sodium hypochlorite for 5 minutes, and a final 10-minute rinse with distilled water [ 16 ]. Biofilm formation: The dentin cylinders were autoclave-sterilized in separate test tubes containing saline. Next, they were incubated at 37°C under aerobic conditions for one week to ensure absence of microbial colonies and accuracy of sterilization. Subsequently, sterile specimens were randomly placed in microtubes containing 300 mL A. naeslundii (PTCC1201) suspension with a final concentration of 10 6 colony forming units/milliliter (CFUs/mL) [ 12 ] and 300 mL of E. faecalis (ATCC29212) with a final concentration of 10 8 CFUs/mL. The concentrations were adjusted initially by visual comparison to 0.5 McFarland standard and then by reading their optical density by a spectrophotometer (Epoch, Germany) at 600 nm wavelength [ 11 ]. Optical density values between 0.08–0.13 correspond to 0.5 McFarland standard. To enhance bacterial penetration, the specimens were centrifuged in four consecutive phases at 1400 x g, 2000 x g, 3600 x g, and 5600 x g each for 5 minutes. After each cycle, the bacterial suspension was refreshed [ 7 ]. Inoculated specimens were incubated in microtubes containing 1 mL of fresh sterile brain heart infusion (BHI) broth (Merck, Germany) with 1% sucrose at 37°C for 21 days to allow biofilm formation. BHI was refreshed once every 2 days to ensure bacterial viability [ 7 ]. After incubation, two specimens were randomly selected for SEM assessment of biofilm growth. SEM assessment: Six specimens were randomly selected and prepared for SEM assessment; of which, two were assessed to ensure elimination of smear layer and opening of dentinal tubules. Two other specimens were removed from the incubator after one week, and the remaining two after 3 weeks of incubation to ensure biofilm formation within the dentinal tubules by SEM assessment. Next, a diamond fissure bur was used to create to longitudinal grooves on the buccal and lingual surfaces of the specimens such that they did not reach the root canal lumen [ 27 ]. The specimens were then split in half at the site of grooves by using a sterile chisel and a hammer. To fix the biofilm, the specimens were immersed in ethanol for 10 minutes. To prepare samples for SEM, the specimens were immersed in 2.5% glutaraldehyde solution (Merck, Germany) at 4°C for 30 minutes. Next, to remove glutaraldehyde, the specimens were entirely immersed in 1.5% sodium cacodylate buffer (Merck, Germany). Subsequently, they were immersed in 25% ethanol for 5 minutes (Merck, Germany), 50% ethanol for 5 minutes, 70% ethanol for 5 minutes, and 90% ethanol also for 5 minutes. Finally, they were immersed in 100% ethanol 3 times, each time for 5 minutes for dehydration. Next, they were exposed to air to dry and were then gold sputter coated and underwent SEM assessment (FEIESEM QUANTA 200; EDAX SILICON DRIFT 2017) to ensure biofilm formation within the dentinal tubules. 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. Preparation of medicaments: To prepare TAP and mTAP in the form of gel with equal standard concentration, 6% methyl cellulose powder and distilled water were used to synthesize a neutral carrier. The medicament powder and methyl cellulose were weighed by a digital scale (Sartorius, Germany) with 10 − 4 g accuracy. To prepare 20 mL of TAP with 0.1% concentration (equal to 1 mg/mL), 6.66 mg of metronidazole (Merck, Germany), ciprofloxacin (Merck, Germany), and minocycline (GALENpharma GmbH, Wittland13, Germany) in 1:1:1 ratio (a total of 20 mg of antibiotic powder) along with 1.2 g of 6% methyl cellulose powder were mixed in a sterile container. Sterile distilled water was then added to reach the final volume to 20 mL. The obtained mixture was homogenized with a magnetic stirrer (IKA, Germany) to obtain a suspension with 1 mg/mL final concentration of TAP [ 16 ]. The same was performed to prepare mTAP with the difference that ciprofloxacin, metronidazole, and penicillin G (Merck, Germany) were used in the abovementioned amounts and ratios. CH paste was prepared in a creamy consistency by mixing 1 g of CH powder (Merck, Germany) with 1 mL of saline on a shaker (Heidolph, Germany) for 1 minute to obtain a homogenous paste [ 28 ]. The propolis used in this study was dried powdered extract (Gyah Kala, Tehran, Iran) that was identified and purified using high-pressure liquid chromatography. The extract was first extracted using the Maceration method at a 1:10 ratio with an ethanol-water solvent and then dried and powdered using a spray dryer (Labplant SD-06, UK). The resulting powder was brown in color with a bulk density ranging from 40 to 70 g/100ml. To prepare propolis paste, 20 mg of propolis powder was mixed with sterile saline in 1:1.5 (w/v) ratio and placed in a shaker for 1 minute to obtain a paste with a creamy consistency [ 24 ]. The fifth group was the negative control group, in which biofilm was developed on the specimens, but no medicament was applied in the canal. Antibiogram by the agar well diffusion method: To examine bacterial susceptibility to certain concentration of medicaments, an antibiogram test was performed. For this purpose, plates were prepared from Mueller Hinton agar (Merck, Germany) and after cooling, E. faecalis and A. naeslundii ​​were cultured on different plates by the spread culture method. Immediately after that, a well was created by removing a small agar cylinder from the middle of each plate to place the medicaments, and 20 µL of the medicaments at the desired concentration were poured into each well. The plates were incubated at 37°C for 24 hours. After incubation, the diameter of the growth inhibition zone of each drug was measured and recorded separately [ 29 ]. Based on the results of the antibiogram test of the study groups, TAP and mTAP with 0.1% concentration, propolis 90%, and CH 100% were selected for this study. Application of medicaments: The experiments had to be performed on mature bacterial biofilm, defined as a 3-week-incubated biofilm in which the bacteria develop resistance to disinfectants [ 30 ]. After a 21-day period, the specimens were transferred to aseptic conditions for injection of the prepared medicaments next to the flame. The specimens were removed from the tube, and each specimen was individually rinsed with 10 mL of sterile saline to remove residual culture medium. The canal of each dentin cylinder was then dried with paper points #60 and #70 (MetaBiomed, Tehran, Iran). After drying of each specimen separately with sterile gauze aseptically, their external surface was covered with two layers of nail varnish to create conditions similar to the clinical environment and to allow the irrigating solutions to affect only the inside of the canal. The specimens were then randomly divided into 5 groups (n = 10). Under aseptic conditions, a certain amount of the medicaments was delivered into the canal of dentin cylinders with a size 40 Lentulo (Mani, Takanezawa, Japan) using the ENDO IT device (NSK, NAKANISHI Inc, Japan) such that it completely filled the specimen lumen from the apex to the coronal opening. The opening and the end of dentin cylinders were sealed with paraffin. Next, the specimens were placed upright in separate microtubes, and incubated at 37°C under humid conditions for 7 days (similar to the clinical environment). Intracanal sampling: Dentin cylinders were removed from the incubator and, in order to take samples under aseptic conditions, the following steps were performed next to the flame: First, each specimen was removed from the microtube, paraffin was removed from both ends, and the intracanal medicament was rinsed with 10 mL of sterile saline under pressure. Next, each specimen was held with sterile forceps, and the canal wall was first trimmed with #4 Gates Glidden drill (200 µm depth from inside the canal) followed by #5 Gates Glidden drill (400 µm depth from inside the canal). The dentin powder obtained from each depth was poured separately into microtubes that had been previously weighed with an accuracy of 10 − 4 g and autoclave-sterilized. Then, the weight of the microtube plus the dentin chips was measured again with an accuracy of 10 − 4 g, and by calculating the difference between these two values, the weight of the dentin chips was obtained. Next, 1 mL of BHI broth (Merck, Germany) was added to each microtube, and the contents of each microtube were serially diluted 3 times (1:10); 10 µL of the diluted solution was inoculated onto bile esculin agar (Merck, Germany) for E. faecalis growth, and CFAT (Merck, Germany) for A. naeslundii growth and spread-cultured. After 24 hours of incubation at 37°C, the formed colony units 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. Statistical analysis: The Shapiro-Wilk test was applied to assess the normality of data distribution in the study groups. Since the colony count data distribution was normal, and homogeneity of the variances was confirmed by the Levene test, a two-way repeated measures ANOVA was used to analyze the main effects of antibiotic type, depth, and their interaction effect on colony count. Pairwise comparisons were performed by the Bonferroni and Tukey tests. The nonparametric Wilcoxon test and the parametric Paired t-test were used for intra-group comparisons. To assess bacterial growth versus non-growth, and to determine the frequency of sterile samples, Fisher’s Exact Test was employed. It should be noted that in order to adjust the effect of sample weight, the colony count was first divided by the weight of the samples, and the logarithm to base 10 of the adjusted variables was used as the comparison criterion. P < 0.05 was considered statistically significant. Results SEM results: Smear layer removal from the dentinal canal wall of the specimens was confirmed by SEM micrographs. The opening of dentinal tubules was clearly visible on the micrographs at three different magnifications (×2000, ×5000, and ×12000) (Fig. 1 ). Observation of open dentinal tubules at different magnifications not only confirmed the success of the preparation method, but also indicated that the tubules served as pathways for bacterial penetration and colonization. After one week of bacterial inoculation, formation of a dual-species biofilm consisting of A naeslundii (rod-shaped) and E. faecalis (cocci-shaped) was clearly observed within the dentinal tubules (Fig. 2 ). Three weeks after culture, the specimens underwent SEM to assess the growth and maturity of the biofilm. The images obtained showed extensive bacterial colonization as a dual-species biofilm at the opening of dentinal tubules at three different magnifications (×2000, ×5000 and ×12000) (Fig. 3 ). High-magnification SEM micrographs showed simultaneous presence of two bacterial species, A. naeslundii (rod-shaped) and E. faecalis (cocci-shaped), on the dentin surface and within the dentinal tubules (Fig. 3 ). After 3 weeks, the association of these two species as a mature dual-species biofilm could be observed on different areas of the dentin surface and also within the dentinal tubules (Fig. 4 ). Morphological differences between the two species, including differences in cell morphology and their arrangement and attachment within the biofilm structure, were clearly discernible and indicated simultaneous participation of both species in biofilm formation [ 31 ]. Colonies formed on specific culture medium after inoculation of the medicaments: Assessment of colony growth in the specific culture medium clearly showed the difference in the efficacy of the tested medicaments. In the control group, extensive and unrestricted growth of E. faecalis and A. naeslundii indicated absence of any inhibitory factor and eligibility of this group for comparison with other groups. In contrast, the images of the medicament groups indicated a significant difference in the degree of inhibition of colony growth. E. faecalis colony count at 200 µm depth: Table 1 presents the mean colony count (log 10 weight adjusted CFUs) of E. faecalis at 200 µm depth. The colony count data had a normal distribution in all groups (P > 0.05) except in the mTAP group (P = 0.01). Homogeneity of the variances was also confirmed by the Levene test (P = 0.237). Thus, one-way ANOVA was applied, which showed a significant difference in colony count among the 5 groups (P < 0.001). Pairwise comparisons (Table 2 ) by the Tukey’s HSD test showed that the control group had a significantly higher mean E. faecalis colony count than the other four groups at 200 µm depth. The propolis group had a higher colony count than the CH group (P = 0.041). Other comparisons were not statistically significant (P > 0.05). 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 Std. Deviation Minimum Maximum 200 µm control 88.8 27.0 52.8 36.9 propolis 11.8 72.0 80.6 37.9 m-TAP 57.7 55.0 27.6 18.8 TAP 52.7 40.0 65.6 06.8 CH 46.7 38.0 90.6 21.8 400 µm control 94.7 28.0 44.7 40.8 propolis 82.7 38.0 13.7 44.8 m-TAP 08.7 56.0 15.6 63.7 TAP 81.6 25.0 36.6 10.7 CH 72.6 31.0 19.6 17.7 Table 2 Pairwise comparison of the groups regarding the mean E. faecalis count at 200 and 400 µm depths Depth Compared groups Mean Difference (|I-J|) Std. Error Sig. 200 µm control propolis 77.0 21.0 009.0 m-TAP 31.1 22.0 00.0 TAP 35.1 22.0 00.0 CH 41.1 21.0 00.0 propolis m-TAP 54.0 21.0 11.0 TAP 58.0 21.0 075.0 CH 64.0 21.0 04.0 m-TAP TAP 04.0 21.0 00.1 CH 10.0 21.0 99.0 TAP CH 05.0 21.0 99.0 400 µm control propolis 12.0 15.0 91.0 m-TAP 86.0 21.0 01.0 TAP 13.1 12.0 00.0 CH 22.1 13.0 00.0 propolis m-TAP 73.0 22.0 03.0 TAP 00.1 14.0 00.0 CH 10.1 16.0 00.0 m-TAP TAP 26.0 20.0 70.0 CH 36.0 21.0 48.0 TAP CH 09.0 13.0 95.0 E. faecalis colony count at 400 µm depth: Table 1 presents the mean colony count (log 10 weight adjusted CFUs) of E. faecalis at 400 µm depth. Due to non-homogeneity of the variances as shown by the Levene test, the robust Welch test was applied to compare the 5 groups, which revealed a significant difference (P 0.05), and both the control and propolis groups had a significantly higher bacterial count than the other three groups (P 0.05). Comparison of E. faecalis count at 200 and 400 µm depths after the application of different medicaments : Two-way ANOVA showed that penetration depth significantly affected the bacterial count of E. faecalis (P < 0.001). The mean bacterial count at 400 µm depth was significantly lower than that at 200 µm depth. This reduction in bacterial count was observed in all groups (P < 0.001). On the other hand, a statistically significant difference was also observed among the study groups (P < 0.001). The control and propolis groups showed the highest bacterial count at both depths, and the difference between these two groups was not statistically significant (P = 0.082); however, both had a significantly higher bacterial count than the CH, TAP, and mTAP groups. The interaction between depth and group was not significant (P = 0.082), indicating that the pattern of bacterial count reduction from 200 to 400 µm depth was similar in all groups, and the intensity of the effect of depth was almost the same, regardless of the type of medicament. A. naeslundii colony count at 200 µm depth : Table 3 presents the mean colony count (log 10 weight adjusted CFUs) of A. naeslundii at 200 µm depth. The colony count data had a normal distribution in all groups (P > 0.05). Due to normal data distribution and homogeneity of the variances, one-way ANOVA was applied, which revealed a significant difference in colony count among the 5 groups (P < 0.001). Pairwise comparisons (Table 4 ) by the Tukey’s HSD test showed that the control group had a significantly higher mean count than the other four groups (P 0.05). Table 3 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 Std. Deviation Minimum Maximum 200 µm control 83.8 43.0 40.8 71.9 propolis 09.8 69.0 70.6 42.9 m-TAP 63.7 41.0 67.6 08.8 TAP 50.7 28.0 10.7 00.8 CH 35.7 51.0 26.6 05.8 400 µm control 55.7 33.0 05.7 16.8 propolis 61.7 44.0 71.6 35.8 m-TAP 85.6 35.0 22.6 18.7 TAP 64.6 47.0 95.5 63.7 CH 49.6 46.0 79.5 17.7 Table 4 Pairwise comparison of the groups regarding the mean A. naeslundii count at 200 and 400 µm depths Depth Compared groups Mean Difference (|I-J|) Std. Error Sig. 200 µm control propolis 73.0 22.0 01.0 m-TAP 20.1 23.0 00.0 TAP 34.1 23.0 00.0 CH 48.1 23.0 00.0 propolis m-TAP 46.0 22.0 26.0 TAP 60.0 22.0 07.0 CH 74.0 22.0 01.0 m-TAP TAP 14.0 23.0 97.0 CH 28.0 23.0 74.0 TAP CH 14.0 23.0 97.0 400 µm control propolis 06.0 19.0 99.0 m-TAP 70.0 22.0 02.0 TAP 91.0 20.0 001.0 CH 05.1 20.0 00.0 propolis m-TAP 75.0 21.0 01.0 TAP 97.0 19.0 00.0 CH 11.1 19.0 00.0 m-TAP TAP 21.0 22.0 87.0 CH 36.0 22.0 51.0 TAP CH 14.0 21.0 96.0 A. naeslundii colony count at 400 µm depth : Table 3 presents the mean colony count (log 10 weight adjusted CFUs) of A. naeslundii at 400 µm depth. Due to normal distribution of data and homogeneity of the variances, one-way ANOVA was applied which showed a significant difference in bacterial count among the 5 groups (P < 0.001). Pairwise comparisons (Table 4 ) by the Tukey’s HSD test showed that the propolis and control groups did not have a statistically significant difference (P = 0.998), and both propolis and control groups had a significantly higher bacterial count than all three other groups (P 0.05). Comparison of A. naeslundii count at 200 and 400 µm depths after the application of different medicaments : The results of two-way ANOVA showed that the penetration depth significantly affected the bacterial count of A. naeslundii (P < 0.001). In all groups, the bacterial count at 400 µm depth was significantly lower than that at 200 µm depth (P < 0.001). In addition, a significant difference was observed among the study groups (P < 0.001). The control and propolis groups showed the highest bacterial count at both depths. The difference between these two groups was not statistically significant (P = 0.327); however, their bacterial count was significantly higher than the other three groups, namely CH, TAP, and mTAP. The interaction between group and depth was also significant (P = 0.048). It means that the rate of bacterial count reduction from 200 to 400 µm depth varied depending on the group. Within-group comparison of bacterial count at different depths: E. faecalis : Paired t-test was used to compare the E. faecalis count between 200 and 400 µm depths in the control, propolis, CH, and TAP groups, given the normal distribution of the data. In the control group, the colony count at 400 µm depth was significantly lower than that at 200 µm depth (P = 0.000). However, in the propolis group, there was no statistically significant difference between the two depths (P = 0.202). In the CH group, a significant decrease in colony count was observed at 400 µm compared to 200 µm (P = 0.04). In the TAP group, this decrease was also significant (P = 0.03). In the mTAP group, since the assumption of normality was not met, the Wilcoxon test was used. The results showed that colony count at 400 µm depth was significantly lower than that at 200 µm depth (P = 0.021). A. naeslundii : Paired t-test was used to analyze the difference in A. naeslundii colony count at 200 and 400 µm depths, given the normal distribution of data in all groups. In the control group, the colony count at 400 µm depth was significantly lower than that at 200 µm depth (P = 0.00). In the propolis group, a significant decrease was observed at 400 µm compared to 200 µm depth (P = 0.047). In the CH group, this difference was also significant (P = 0.009). In the TAP group, the decrease in colony count at 400 µm was significantly lower than that at 200 µm (P = 0.001). In the mTAP group, the colony count at 400 µm depth was significantly lower than that at 200 µm depth (P = 0.002). Comparison of microorganism-free specimens: Microbial growth was observed at 200 µm depth in all groups inoculated with E. faecalis . Thus, no comparison was made among the groups for this microorganism at 200 µm depth. Table 5 shows the growth status of E. faecalis at 400 µm and A. naeslundii at 200 and 400 µm depths in different groups. The Fisher's Exact test showed no statistically significant difference among the groups in terms of growth of A. naeslundii at 200 µm (P = 1.00) or 400 µm (P = 0.252) depth, or E. faecalis at 400 nm depth (P = 1.00). Table 5 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 10٪ 90٪ 100٪ propolis 0 10 10 0٪ 100٪ 100٪ m-TAP 1 9 10 10٪ 90٪ 100٪ TAP 1 9 10 10٪ 90٪ 100٪ CH 1 9 10 10٪ 90٪ 100٪ Total 4 46 50 8٪ 92٪ 100٪ A. naeslundii at 400 µm control 1 9 10 10٪ 90٪ 100٪ propolis 0 10 10 0٪ 100٪ 100٪ m-TAP 4 6 10 40٪ 60٪ 100٪ TAP 2 8 10 20٪ 80٪ 100٪ CH 2 8 10 20٪ 80٪ 100٪ Total 9 41 50 18٪ 82٪ 100٪ E. faecalis at 400 µm control 0 10 10 0٪ 100٪ 100٪ propolis 0 10 10 0٪ 100٪ 100٪ m-TAP 1 9 10 10٪ 90٪ 100٪ TAP 1 9 10 10٪ 90٪ 100٪ CH 1 9 10 10٪ 90٪ 100٪ Total 3 47 50 6٪ 94٪ 100٪ Effect size (partial eta squared): The effect size for A. naeslundii colony count for the group variable was 0.570 at 200 µm and 0.598 at 400 µm depth. The effect size for E. faecalis colony count for the group variable was 0.570 at 200 µm and 0.676 at 400 µm depth. Results of antibiogram test: The diameter of the growth inhibition zone produced by TAP and mTAP in both media was significantly larger than that of other medicaments. Unlike TAP and mTAP, no distinct inhibition zone was observed around propolis. This is not necessarily due to lack of antimicrobial activity, but rather to the inability of propolis to be released and diffused into the agar medium. CH also does not dissolve well in agar medium and does not diffuse or spread effectively; as a result, its inhibition zone is usually small or indistinct; sometimes it is not even visible at all. Discussion This study compared the antimicrobial effects of propolis, CH, TAP, and mTAP as intracanal medicaments on tubular dentin inoculated with a dual-species biofilm. The results clearly showed that, at 200 μm depth, all the tested medicaments had inhibitory effects on E. faecalis and A. naeslundii, which was consistent with other studies that investigated the antimicrobial effects of medicaments at 200 μm depth [16,32]. Therefore, the null hypothesis of the study was rejected. At 200 μm depth, CH, TAP, and mTAP caused the greatest reduction in bacterial count compared to the control group. While propolis was less effective, it was still able to significantly reduce the bacterial count compared to the control group. These results were consistent with previous studies showing the high efficacy of TAP in removing multi-species biofilms [1,7,12,32-34,]. Also, Zargar et al. [22] showed comparable efficacy of TAP and mTAP in inhibiting biofilms, which was consistent with the present results. Furthermore, Zargar et al. [16] confirmed the antimicrobial efficacy of TAP and CH at the same depth, although CH was unable to completely remove the biofilm. At 400 μm depth, the antimicrobial effect of propolis was further reduced and its statistical difference with the control group was not significant, while the other three medicaments still had a significant difference with the control group, and were able to reduce the microbial load at this depth. This finding is inconsistent with the results reported by BhanDari et al, [32] who demonstrated that propolis exhibited significant antibacterial activity against E. faecalis at 400 μm depth. Parhizkar [15] examined the antimicrobial effect of mTAP and showed that this compound was effective on a wide range of microorganisms. Zargar et al. [16] showed that TAP had a significant antimicrobial effect at 400 μm depth, while CH did not show a significant difference with the control group at this depth. Most studies have also reported higher antimicrobial efficacy of TAP, especially against E. faecalis, than CH. However, the current study showed optimal antibiofilm effect of CH at both 200 and 400 μm depths, which was different from previous findings. This difference could be related to the size of CH particles and the altered proton pump function in multi-species biofilms, wihich may augment its antimicrobial biofilms efficacy [16, 21], which could justify the present results. It should be noted that some previous studies reported relative resistance of E. faecalis and A. naeslundii to CH [35,36], which was in contrast to the present findings. In addition, it has been reported that dentin powder can reduce the effectiveness of CH in vitro [37]. However, no such effect was observed in the present study. Such differences may be due to variations in study designs, disinfection methods, duration of medicament application, sampling method, or sample transfer conditions [38]. The statistically significant difference between the efficacy of propolis and CH at both depths indicated limited antibacterial activity of propolis against E. faecalis and A. naeslundii and higher antimicrobial activity of CH. This result was in contrast to some previous studies that reported greater efficacy of propolis [32,34,39]. This difference is probably related to the limited penetration power of propolis in mature biofilms; propolis is more effective on planktonic form of bacteria, and has a weaker effect against mature multispecies biofilms due to the adhesive and protective structure of the biofilm matrix [40]. Also, factors such as the composition, formulation, time and area of collection, the concentration of waxy contaminants, the concentration of flavonoids, and the extraction method of propolis can affect its effectiveness against microorganisms inside the canal [32,33]. An in vivo study by Ahangari et al, [33] also showed that a 30% propolis extract collected from Azerbaijan had a similar antimicrobial activity to CH against E. faecalis, although its antimicrobial properties were slightly weaker. These findings confirm the role of geographical region in the properties of propolis. Some studies, including those by Awawdeh et al, [41] and Madhubala et al. [25] reported lower antimicrobial efficacy of CH than propolis, which was inconsistent with the results of the present study. This difference may be related to the short duration of CH use (1-2 days) in those studies, which did not allow enough time for the therapeutic effects of CH to occur. On the other hand, other studies, such as those by Shamma et al, [42] and Arun et al. [39], showed that the antibacterial effect of CH was better than that of propolis after 1 to 2 weeks of use, although in some cases the differences were not significant. This emphasizes the importance of the appropriate timing and conditions of use of CH [33,41]. CH, due to its bactericidal properties, can lead to bacterial cell death within a suitable period of time (1 week) [33,39,42-45]. However, the minimum time required to achieve maximum effectiveness is still unknown [33]. Some studies, such as Edara Lillygrace et al, [24] and Madhubala et al. [25] reported the efficacy of propolis in certain conditions, similar to TAP. Also, El-Tayeb [45] found no significant difference between the antibacterial activity of propolis and TAP, and introduced propolis as a comparable option to TAP in REPs [45,46]. However, most of these studies were designed based on single-species models [22,33], while, the present study used a dual-species biofilm model that better simulates the clinical environment. In addition, the Iranian propolis used in this study (Bahareh) may differ from other samples in terms of effective compounds and may be less effective. The bacterial count of both E. faecalis and A. naeslundii at 400 μm depth was significantly lower than that at 200 μm depth in all medicament groups. Evidence shows that biofilm accumulation is greater in more superficial layers of dentinal tubules, and mature biofilms less commonly penetrate into deeper areas. Furthermore, nutritional limitations at depth make it more difficult for the bacteria to survive. Therefore, the microbial load in these areas is inherently lower. Arias-Moliz et al. [47] suggested that the reduction in microbial load at greater depths may not be due solely to the higher drug efficacy, but also to a reduced ability of bacteria to colonize these areas. On the other hand, medicaments such as CH, TAP, and mTAP, especially if they have sufficient contact time, can penetrate to greater depths and have higher efficacy [48]. These findings are largely consistent with the results of Zargar et al. [16]. On the other hand, the reduced antibacterial efficacy of propolis at 400 μm depth may be attributed to its limited permeability into dentinal tubules. However, when these compounds are prepared as nanoparticles, their drug stability, therapeutic efficacy, and penetration power are significantly increased compared to the pure drug solution [32]. At both depths and for both microorganisms, the effect size of the medicament group was very high. This indicates that the type of treatment was a determining factor in the difference in colony count and played a significant role in reducing the microbial load. Despite the reduction in bacterial count at different depths and the effect of medicaments on bacterial count, there was no significant difference between the groups in terms of the degree of complete bacterial clearance (i.e., samples completely free of microbial growth). In other words, even effective drugs were unable to completely eliminate biofilms in all specimens. This may be due to the inherent resistance of biofilms and their complex structure, which limits the penetration of medicaments. This finding was consistent with studies that showed that biofilms, unlike single-microbial colonies, are difficult to completely eliminate [50]. Similarly, Zargar et al. [22] emphasized that although TAP and mTAP were effective in eliminating the planktonic form, complete inhibition of biofilm was not achieved. In addition, the antibiogram test with the agar diffusion method was used in the current study to confirm the antimicrobial properties of the medicaments and to initially assess their diffusion ability in the culture medium and also to investigate the resistance pattern of the bacteria. As a complementary method, this test provides valuable qualitative information about the susceptibility or resistance of bacteria to drugs and helps to better understand the clinical potential of antimicrobial compounds [50]. In the obtained results, TAP and mTAP showed the largest diameter of the growth inhibition zone against E. faecalis and A. naeslundii , indicating good diffusion ability and high inhibitory effect against these bacteria. This finding was consistent with previous studies showing strong inhibitory effect of TAP even at low concentrations [51,52]. In contrast, propolis did not produce any distinct growth inhibition zone. This does not necessarily mean that propolis is ineffective, but is likely due to the limited ability of its active compounds to diffuse into agar media, which is related to its physical properties, such as high stickiness, high viscosity, and resinous nature. The low solubility of propolis in aqueous media has also been reported to affect the accuracy of the assessment in agar diffusion tests [53,54]. CH also produced a small, pale, and indistinct halo, which was consistent with previous findings. Studies have shown that it does not dissolve well in gelatinous media and that its active ions (OH-) do not have the ability to diffuse well into agar [55,56]. As a result, its antimicrobial activity in agar diffusion tests is often lower than the clinical reality. Overall, these differences are more likely to be due to their physicochemical properties and their ability to diffuse in the agar medium than to the extent of their antimicrobial effect. Therefore, propolis and CH, due to their low solubility and high viscosity, require the use of direct contact models that do not rely on diffusion in agar or liquid for more accurate evaluation [54,56]. The main limitations of this study were as follows: (I) Limitations in completely simulating the clinical conditions: Despite the careful design of this in vitro study and the use of human dentin cylinders to most closely simulate intraoral conditions, the dynamic conditions of the oral cavity cannot be completely simulated in vitro. (II) Variability of propolis based on geographical area and harvest time: The active components of propolis vary depending on the season, geographical location, and type of dominant plants. (III) Evaluation of a dual-species biofilm and limited assessment time points: Further studies are required especially focusing on multi-species biofilms and in conditions close to the clinical environment, to confirm the present results. Also, the effects of medicaments should be evaluated at different time points of 1, 3, 7, 14 and 21 days. The effects of different doses and forms of medicaments (gel, paste, suspension) and their efficacy in combination with antibiotics should also be investigated. Furthermore, more advanced equipment and tests such as confocal laser microscopy and polymerase chain reaction may be used for a more accurate analysis of biofilm structure and bacterial viability, and high-performance liquid chromatography may be used to standardize the active ingredients of propolis. Finally, clinical trials are required to obtain more generalizable results. Conclusion According to the obtained results, it may be stated that although all the tested medicaments led to a significant reduction in bacterial count, their effectiveness varied in depth and against resistant biofilm. Among them, TAP and mTAP, as antibiotic compounds, showed the most effective performance in reducing the bacterial count at different depths and against resistant microorganisms. CH, with its biological properties and favorable antimicrobial effect, is also considered a suitable option if non-antibiotic treatment is needed. Although propolis has significant biological benefits, its clinical effectiveness, especially against complex human biofilms, has not yet been definitively determined. 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 Declarations Ethics approval : The study protocol was approved by the university ethics committee (IR.SBMU.DRC.REC.1403.064). Consent for publication : Not applicable Availability of data and materials : The datasets used and analyzed during the current study are available from the corresponding author on reasonable request. Competing interests : The authors declare that they have no competing interests. Funding : Not applicable 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. 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Cite Share Download PDF Status: Published Journal Publication published 16 Feb, 2026 Read the published version in BMC Oral Health → Version 1 posted Editorial decision: Revision requested 21 Oct, 2025 Reviews received at journal 19 Oct, 2025 Reviews received at journal 07 Oct, 2025 Reviews received at journal 06 Oct, 2025 Reviewers agreed at journal 02 Oct, 2025 Reviewers agreed at journal 28 Sep, 2025 Reviewers agreed at journal 26 Sep, 2025 Reviewers invited by journal 24 Sep, 2025 Editor invited by journal 12 Sep, 2025 Editor assigned by journal 11 Sep, 2025 Submission checks completed at journal 10 Sep, 2025 First submitted to journal 10 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Sciences","correspondingAuthor":false,"prefix":"","firstName":"Nazanin","middleName":"","lastName":"Zargar","suffix":""},{"id":525190963,"identity":"92e88930-726c-4b76-a020-2cb66d84d5b0","order_by":2,"name":"Maryam Pourhajibagher","email":"","orcid":"","institution":"Tehran University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Maryam","middleName":"","lastName":"Pourhajibagher","suffix":""},{"id":525190964,"identity":"2941dc5d-972e-40dd-b6da-2e9197983c30","order_by":3,"name":"Rezvan Shahhosseini","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8ElEQVRIiWNgGAWjYBAC+wYw9Z+HgZn54AMGhgOEtRhA1DDLMbCzJRuQpMWYgZ/HTII4Lcfbn0n83MGW2MDMYFbNU3NHjp+B+eGjG3i02PccSJPsPcMD0pJ2m+fYM2PJBjZj4xw8WuwkEg4b8LZJgLQcu83DdjhxwwEeNml8WozlHzYb/m0zAGphbCvm+UeEFsMZzIyPedsSjIGBzMbM20aEFoMzaYyPZdsOyDEAdUjO7TtsLNlMwC8Gx48/OPi27QAPA//5jx/efDssx8/e/PAxPi1wYH+AgYGJB8RiJkY5DDD+IEX1KBgFo2AUjBgAAN+iS7iCLCaKAAAAAElFTkSuQmCC","orcid":"","institution":"Shahid Beheshti University Of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Rezvan","middleName":"","lastName":"Shahhosseini","suffix":""}],"badges":[],"createdAt":"2025-08-31 21:23:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7502815/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7502815/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12903-026-07901-x","type":"published","date":"2026-02-16T15:57:11+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":92932918,"identity":"2be594a5-9024-4d9f-9992-b8623aa05975","added_by":"auto","created_at":"2025-10-07 09:26:57","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1733701,"visible":true,"origin":"","legend":"","description":"","filename":"MAINARTICLE.docx","url":"https://assets-eu.researchsquare.com/files/rs-7502815/v1/793cf5b1c24f176c76b1798a.docx"},{"id":92932051,"identity":"b648c752-b8be-42ab-bce7-8fecad91915b","added_by":"auto","created_at":"2025-10-07 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09:18:57","extension":"xml","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":185738,"visible":true,"origin":"","legend":"","description":"","filename":"9b0106d94f9a4f54a1376a997ca536a31structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7502815/v1/bdbe948e56806e6298682ecb.xml"},{"id":92931592,"identity":"9d96e8f0-1cf2-4cf3-9a7d-8025527ba2bf","added_by":"auto","created_at":"2025-10-07 09:10:57","extension":"html","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":198465,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7502815/v1/a7202cfb52a0d89e414b2afd.html"},{"id":92931579,"identity":"63cea5c3-9b3d-4c87-ba13-8672d116d2dd","added_by":"auto","created_at":"2025-10-07 09:10:57","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":180991,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrograph indicating smear layer removal; open dentinal tubules can be observed at different magnifications (×2000, ×5000, and ×12000)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7502815/v1/bba215690467feda22a49221.png"},{"id":92931577,"identity":"f1134227-145f-4d32-9631-3a8203289efe","added_by":"auto","created_at":"2025-10-07 09:10:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":226765,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrograph of bacterial biofilm formed within the dentinal tubules after 1 week\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7502815/v1/b8093369e7ebf33f75abc5ae.png"},{"id":92932050,"identity":"040d794e-ff47-41a7-a3dd-23a1a52712a3","added_by":"auto","created_at":"2025-10-07 09:18:57","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":205630,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrograph of bacterial biofilm formed at the opening of dentinal tubules after 3 weeks at different magnifications (×2000, ×5000, and ×12000)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7502815/v1/de74ed8af137ff3821ed8250.png"},{"id":92932919,"identity":"fa0ac344-a2dd-4c7a-b502-c3e3f889a6a3","added_by":"auto","created_at":"2025-10-07 09:26:57","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":348623,"visible":true,"origin":"","legend":"\u003cp\u003eSEM micrograph of biofilm growth in dentinal tubules after 3 weeks (both \u003cem\u003eA. naeslundii \u003c/em\u003eand \u003cem\u003eE. faecalis\u003c/em\u003e) at different magnifications (×2000, ×5000, and ×12000)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7502815/v1/0e65752b46f5db75573e13bf.png"},{"id":103251278,"identity":"29b639c5-c25f-4f68-9103-6ced5fc6488c","added_by":"auto","created_at":"2026-02-23 16:07:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2214132,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7502815/v1/507133dc-12ce-4d39-9a65-4832c65cf067.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"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","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRegenerative endodontic procedures (REPs) are a novel therapeutic approach for treatment of immature developing teeth that have been traumatized by infection, trauma, or caries, and have developed irreversible pulpitis or pulp necrosis [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The main goal of REPs is to strengthen the root structure and facilitate its development in width and length. In REPs, presence of residual bacteria is significantly associated with impaired root development and incomplete closure of the apex [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Therefore, effective root canal disinfection plays a key role in supporting the proliferation and regeneration of pulp tissue [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRoot canal disinfection, which involves the use of irrigating solutions and intracanal medicaments, is one of the main steps in endodontic treatment, especially REPs [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Also, preservation of viable cells during the treatment process is of great importance considering the possible toxic effects of irrigating solutions and intracanal medications, as the survival of these cells is essential for the successful regeneration of pulpal tissue [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eA wide diversity has been identified in the root canal system microbiota [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (\u003cem\u003eE. faecalis\u003c/em\u003e) and \u003cem\u003eActinomyces naeslundii\u003c/em\u003e (\u003cem\u003eA. naeslundii\u003c/em\u003e) are among the bacterial species that have been frequently isolated from chronic and persistent root canal infections [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. \u003cem\u003eE. faecalis\u003c/em\u003e is the most common resistant bacterial species associated with dental infections, which has been abundantly observed and isolated from the root canals of permanent and primary teeth with failed endodontic treatment [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The high resistance of this microorganism to irrigating solutions and intracanal medicaments, the ability to grow in a wide range of pH, tolerance in nutritionally-deprived conditions, the ability to penetrate into dentinal tubules, and strong adhesion to dentin collagen are among the main reasons for its survival in chronic apical periodontitis. In addition to superficial penetration into dentinal tubules to a depth of 1000 \u0026micro;m [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], \u003cem\u003eE. faecalis\u003c/em\u003e uses the root cementum as a long-term refuge for reinfection. Thus, this microorganism is commonly used for research purposes in endodontics [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOn the other hand, \u003cem\u003eA naeslundii\u003c/em\u003e is a Gram-positive, facultative rod-shaped anaerobic that has been increasingly recognized for its ability to form biofilm and its association with endodontic treatment failures [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Adhesion \u003cem\u003eof A naeslundii\u003c/em\u003e to dentin collagen is mediated by its unique thin, hair-like surface structures (fimbriae) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Recently, this microorganism was identified as the most common microorganism in immature permanent teeth with pulp necrosis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], suggesting a possible association with failure of REPs.\u003c/p\u003e\u003cp\u003eBoth \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eA. naeslundii\u003c/em\u003e ​​ have the ability to form biofilm and penetrate into dentinal tubules, which allows them to withstand the adverse conditions of the root canal environment [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In addition, they are easily cultured in vitro, which makes them suitable for in vitro studies [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCalcium hydroxide (CH) has been widely used as an intracanal medicament, and has been recommended for REPs due to its favorable antibacterial properties. However, some concerns exist regarding its effectiveness as a potent and durable antibacterial agent in some cases of REPs. Also, canal debridement and cleaning after its application are challenging [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWith the development of antibiotics, various intracanal medicaments are used in modern dentistry to reduce the microbial load of root canals [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Triple antibiotic paste (TAP) is the most commonly used intracanal medicament in REPs, which is a combination of ciprofloxacin, metronidazole, and minocycline [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, the use of TAP is associated with problems such as coronal discoloration, changes in dentin structure, and cytotoxicity at high concentrations [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Using an antibiotic with comprehensive antibacterial effects against root canal pathogens with minimal discoloration potential and minimal cytotoxic effects on various stem cells may lead to successful clinical outcomes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. High concentrations of TAP are toxic for stem cells from the apical papilla [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and negatively affect their adhesion and proliferation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Therefore, a recent study recommended low concentrations of TAP to reduce its toxicity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. According to recent findings, a concentration of 20 mg/mL TAP can be effective for eliminating a 3-week mature \u003cem\u003eE. faecalis\u003c/em\u003e biofilm up to 400 \u0026micro;m deep from the internal canal wall, while the antibacterial effect of CH is limited to only 200 \u0026micro;m depth from the internal canal wall with less efficacy [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTooth discoloration is a major drawback of TAP. Therefore, in some cases, the use of dual antibiotic paste, which consists of only ciprofloxacin and metronidazole, is suggested [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, a systematic review reported significantly lower antimicrobial efficacy of dual antibiotic paste than TAP [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Therefore, some modifications were made in the TAP combination. In one of these modified combinations, which consists of penicillin G, metronidazole, and ciprofloxacin, it was observed that replacing minocycline with penicillin G probably results in a higher efficacy of the drug against \u003cem\u003eE. faecalis\u003c/em\u003e biofilm formation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In addition, this combination was also effective against a diverse group of root canal microbiota [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In vitro studies have shown that Actinomyces species are sensitive to moxifloxacin (a fluoroquinolone) and amoxicillin [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCurrently, evidence-based dentistry has attracted attention to the use of herbal products in endodontics, which are mainly recommended due to their high biocompatibility and fewer side effects than chemical and synthetic antimicrobial agents [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Propolis is a natural resinous substance with high biocompatibility, which is extracted from plant resins collected by honeybees. Due to the presence of flavonoids, propolis has significant antimicrobial and anti-inflammatory properties [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The toxicity of propolis has been reported to be approximately 10 times lower than that of CH [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. It has been shown that propolis is more effective against resistant microorganisms and is more biocompatible with periradicular tissues than the conventional root canal medicaments [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eDespite extensive efforts in this field, complete elimination of microorganisms from the root canal system remains challenging. Therefore, it is imperative to find a more effective medicament to remove the residual microorganisms from dentinal tubules. Most previous studies on this topic used single-species biofilms (mainly E. faecalis) to evaluate antimicrobial activity, and studies on multi-species biofilms formed on human dentin are limited in number [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Since single-species biofilms do not accurately reflect the clinical conditions, the present study focused on a dual-species biofilm of microorganisms that have a higher prevalence in immature teeth. Therefore, this study aimed to compare the antimicrobial effects of propolis, CH, TAP, and modified TAP (mTAP) as intracanal medicaments on tubular dentin inoculated with a dual-species biofilm. The null hypothesis of the study was that no significant difference would be found in the efficacy of different medicaments for inhibition of the dual-species biofilm.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eThis ex vivo study was conducted on dentin blocks obtained from the root canals of 56 single-rooted, single-canal teeth extracted as part of prosthodontic treatment or due to poor periodontal prognosis. All extractions were performed with the patient\u0026rsquo;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).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eEligibility criteria:\u003c/h2\u003e\u003cp\u003eSingle-rooted single-canal extracted teeth with a circular-shaped canal and no history of endodontic treatment were included after ensuring absence of root caries, cracks, fractures, intracanal calcifications, and internal/external root resorption by their inspection under a microscope [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Teeth with gradual curvature extended to the apical region, and those with ribbon-shaped or short roots were excluded.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSample size:\u003c/h3\u003e\n\u003cp\u003eThe sample size was calculated to be 10 in each group using one-way ANOVA menu of PASS 21 (due to quantitative nature of the dependent variable and presence of 5 groups), assuming alpha\u0026thinsp;=\u0026thinsp;0.05, beta\u0026thinsp;=\u0026thinsp;0.2 (study power of 80%), and mean and standard deviation values obtained from a study by Edara Lillygrace et al [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Also, 6 specimens were used for scanning electron microscopy (SEM) assessment.\u003c/p\u003e\n\u003ch3\u003eSpecimen preparation:\u003c/h3\u003e\n\u003cp\u003eA modified model of the method described by Haapasalo and Qrstavik [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] was used in this study. Eligible teeth (n\u0026thinsp;=\u0026thinsp;56) were disinfected by immersion in 5.25% sodium hypochlorite (Chamin, Tehran, Iran) for 48 hours. They were then stored in 0.09% sterile saline (Darupakhsh, Tehran, Iran) at room temperature until use. All crowns were cut at the cementoenamel junction by a rotary diamond disc operating at 700 rpm (Dorsa, Tehran, Iran) under copious water irrigation perpendicular to the longitudinal tooth axis. Root end was also cut such that a 6-mm cylinder was obtained from the middle-third of the root. The root cementum was also removed by a diamond fissure bur (Mani, Takanezawa, Japan) and highspeed handpiece under water coolant. Gates Glidden drills #1, 2 and 3 were then used to dilate the canals. In all phases of specimen preparation, copious irrigation was performed with 5.25% sodium hypochlorite and saline. Finally, cylinders with 6 mm length, 4 mm external diameter and equal internal diameter corresponding to the diameter of a #3 Gates Glidden drill were obtained. To eliminate the smear layer, the specimens were placed in an ultrasonic bath (Vector 55; Jeltraft, Jelenko) containing 17% EDTA (pH\u0026thinsp;=\u0026thinsp;7.8) for 5 minutes, followed by immersion in 5.25% sodium hypochlorite for 5 minutes, and a final 10-minute rinse with distilled water [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eBiofilm formation:\u003c/h3\u003e\n\u003cp\u003eThe dentin cylinders were autoclave-sterilized in separate test tubes containing saline. Next, they were incubated at 37\u0026deg;C under aerobic conditions for one week to ensure absence of microbial colonies and accuracy of sterilization.\u003c/p\u003e\u003cp\u003eSubsequently, sterile specimens were randomly placed in microtubes containing 300 mL \u003cem\u003eA. naeslundii\u003c/em\u003e (PTCC1201) suspension with a final concentration of 10\u003csup\u003e6\u003c/sup\u003e colony forming units/milliliter (CFUs/mL) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and 300 mL of \u003cem\u003eE. faecalis\u003c/em\u003e (ATCC29212) with a final concentration of 10\u003csup\u003e8\u003c/sup\u003e CFUs/mL. The concentrations were adjusted initially by visual comparison to 0.5 McFarland standard and then by reading their optical density by a spectrophotometer (Epoch, Germany) at 600 nm wavelength [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Optical density values between 0.08\u0026ndash;0.13 correspond to 0.5 McFarland standard. To enhance bacterial penetration, the specimens were centrifuged in four consecutive phases at 1400 x g, 2000 x g, 3600 x g, and 5600 x g each for 5 minutes. After each cycle, the bacterial suspension was refreshed [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Inoculated specimens were incubated in microtubes containing 1 mL of fresh sterile brain heart infusion (BHI) broth (Merck, Germany) with 1% sucrose at 37\u0026deg;C for 21 days to allow biofilm formation. BHI was refreshed once every 2 days to ensure bacterial viability [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. After incubation, two specimens were randomly selected for SEM assessment of biofilm growth.\u003c/p\u003e\n\u003ch3\u003eSEM assessment:\u003c/h3\u003e\n\u003cp\u003eSix specimens were randomly selected and prepared for SEM assessment; of which, two were assessed to ensure elimination of smear layer and opening of dentinal tubules. Two other specimens were removed from the incubator after one week, and the remaining two after 3 weeks of incubation to ensure biofilm formation within the dentinal tubules by SEM assessment. Next, a diamond fissure bur was used to create to longitudinal grooves on the buccal and lingual surfaces of the specimens such that they did not reach the root canal lumen [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The specimens were then split in half at the site of grooves by using a sterile chisel and a hammer. To fix the biofilm, the specimens were immersed in ethanol for 10 minutes.\u003c/p\u003e\u003cp\u003eTo prepare samples for SEM, the specimens were immersed in 2.5% glutaraldehyde solution (Merck, Germany) at 4\u0026deg;C for 30 minutes. Next, to remove glutaraldehyde, the specimens were entirely immersed in 1.5% sodium cacodylate buffer (Merck, Germany). Subsequently, they were immersed in 25% ethanol for 5 minutes (Merck, Germany), 50% ethanol for 5 minutes, 70% ethanol for 5 minutes, and 90% ethanol also for 5 minutes. Finally, they were immersed in 100% ethanol 3 times, each time for 5 minutes for dehydration. Next, they were exposed to air to dry and were then gold sputter coated and underwent SEM assessment (FEIESEM QUANTA 200; EDAX SILICON DRIFT 2017) to ensure biofilm formation within the dentinal tubules.\u003c/p\u003e\u003cp\u003eThe 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.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of medicaments:\u003c/h2\u003e\u003cp\u003eTo prepare TAP and mTAP in the form of gel with equal standard concentration, 6% methyl cellulose powder and distilled water were used to synthesize a neutral carrier. The medicament powder and methyl cellulose were weighed by a digital scale (Sartorius, Germany) with 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e g accuracy. To prepare 20 mL of TAP with 0.1% concentration (equal to 1 mg/mL), 6.66 mg of metronidazole (Merck, Germany), ciprofloxacin (Merck, Germany), and minocycline (GALENpharma GmbH, Wittland13, Germany) in 1:1:1 ratio (a total of 20 mg of antibiotic powder) along with 1.2 g of 6% methyl cellulose powder were mixed in a sterile container. Sterile distilled water was then added to reach the final volume to 20 mL. The obtained mixture was homogenized with a magnetic stirrer (IKA, Germany) to obtain a suspension with 1 mg/mL final concentration of TAP [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. The same was performed to prepare mTAP with the difference that ciprofloxacin, metronidazole, and penicillin G (Merck, Germany) were used in the abovementioned amounts and ratios.\u003c/p\u003e\u003cp\u003eCH paste was prepared in a creamy consistency by mixing 1 g of CH powder (Merck, Germany) with 1 mL of saline on a shaker (Heidolph, Germany) for 1 minute to obtain a homogenous paste [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe propolis used in this study was dried powdered extract (Gyah Kala, Tehran, Iran) that was identified and purified using high-pressure liquid chromatography. The extract was first extracted using the Maceration method at a 1:10 ratio with an ethanol-water solvent and then dried and powdered using a spray dryer (Labplant SD-06, UK). The resulting powder was brown in color with a bulk density ranging from 40 to 70 g/100ml. To prepare propolis paste, 20 mg of propolis powder was mixed with sterile saline in 1:1.5 (w/v) ratio and placed in a shaker for 1 minute to obtain a paste with a creamy consistency [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe fifth group was the negative control group, in which biofilm was developed on the specimens, but no medicament was applied in the canal.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eAntibiogram by the agar well diffusion method:\u003c/h3\u003e\n\u003cp\u003eTo examine bacterial susceptibility to certain concentration of medicaments, an antibiogram test was performed. For this purpose, plates were prepared from Mueller Hinton agar (Merck, Germany) and after cooling, \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eA. naeslundii\u003c/em\u003e ​​were cultured on different plates by the spread culture method. Immediately after that, a well was created by removing a small agar cylinder from the middle of each plate to place the medicaments, and 20 \u0026micro;L of the medicaments at the desired concentration were poured into each well. The plates were incubated at 37\u0026deg;C for 24 hours. After incubation, the diameter of the growth inhibition zone of each drug was measured and recorded separately [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Based on the results of the antibiogram test of the study groups, TAP and mTAP with 0.1% concentration, propolis 90%, and CH 100% were selected for this study.\u003c/p\u003e\n\u003ch3\u003eApplication of medicaments:\u003c/h3\u003e\n\u003cp\u003eThe experiments had to be performed on mature bacterial biofilm, defined as a 3-week-incubated biofilm in which the bacteria develop resistance to disinfectants [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. After a 21-day period, the specimens were transferred to aseptic conditions for injection of the prepared medicaments next to the flame. The specimens were removed from the tube, and each specimen was individually rinsed with 10 mL of sterile saline to remove residual culture medium. The canal of each dentin cylinder was then dried with paper points #60 and #70 (MetaBiomed, Tehran, Iran). After drying of each specimen separately with sterile gauze aseptically, their external surface was covered with two layers of nail varnish to create conditions similar to the clinical environment and to allow the irrigating solutions to affect only the inside of the canal. The specimens were then randomly divided into 5 groups (n\u0026thinsp;=\u0026thinsp;10).\u003c/p\u003e\u003cp\u003eUnder aseptic conditions, a certain amount of the medicaments was delivered into the canal of dentin cylinders with a size 40 Lentulo (Mani, Takanezawa, Japan) using the ENDO IT device (NSK, NAKANISHI Inc, Japan) such that it completely filled the specimen lumen from the apex to the coronal opening. The opening and the end of dentin cylinders were sealed with paraffin. Next, the specimens were placed upright in separate microtubes, and incubated at 37\u0026deg;C under humid conditions for 7 days (similar to the clinical environment).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eIntracanal sampling:\u003c/h2\u003e\u003cp\u003eDentin cylinders were removed from the incubator and, in order to take samples under aseptic conditions, the following steps were performed next to the flame: First, each specimen was removed from the microtube, paraffin was removed from both ends, and the intracanal medicament was rinsed with 10 mL of sterile saline under pressure. Next, each specimen was held with sterile forceps, and the canal wall was first trimmed with #4 Gates Glidden drill (200 \u0026micro;m depth from inside the canal) followed by #5 Gates Glidden drill (400 \u0026micro;m depth from inside the canal). The dentin powder obtained from each depth was poured separately into microtubes that had been previously weighed with an accuracy of 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e g and autoclave-sterilized. Then, the weight of the microtube plus the dentin chips was measured again with an accuracy of 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e g, and by calculating the difference between these two values, the weight of the dentin chips was obtained. Next, 1 mL of BHI broth (Merck, Germany) was added to each microtube, and the contents of each microtube were serially diluted 3 times (1:10); 10 \u0026micro;L of the diluted solution was inoculated onto bile esculin agar (Merck, Germany) for \u003cem\u003eE. faecalis\u003c/em\u003e growth, and CFAT (Merck, Germany) for \u003cem\u003eA. naeslundii\u003c/em\u003e growth and spread-cultured. After 24 hours of incubation at 37\u0026deg;C, the formed colony units were visually counted [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. 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.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis:\u003c/h2\u003e\u003cp\u003eThe Shapiro-Wilk test was applied to assess the normality of data distribution in the study groups. Since the colony count data distribution was normal, and homogeneity of the variances was confirmed by the Levene test, a two-way repeated measures ANOVA was used to analyze the main effects of antibiotic type, depth, and their interaction effect on colony count. Pairwise comparisons were performed by the Bonferroni and Tukey tests. The nonparametric Wilcoxon test and the parametric Paired t-test were used for intra-group comparisons. To assess bacterial growth versus non-growth, and to determine the frequency of sterile samples, Fisher\u0026rsquo;s Exact Test was employed. It should be noted that in order to adjust the effect of sample weight, the colony count was first divided by the weight of the samples, and the logarithm to base 10 of the adjusted variables was used as the comparison criterion. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003eSEM results:\u003c/h2\u003e\n \u003cp\u003eSmear layer removal from the dentinal canal wall of the specimens was confirmed by SEM micrographs. The opening of dentinal tubules was clearly visible on the micrographs at three different magnifications (\u0026times;2000, \u0026times;5000, and \u0026times;12000) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Observation of open dentinal tubules at different magnifications not only confirmed the success of the preparation method, but also indicated that the tubules served as pathways for bacterial penetration and colonization.\u003c/p\u003e\n \u003cp\u003eAfter one week of bacterial inoculation, formation of a dual-species biofilm consisting of \u003cem\u003eA naeslundii\u003c/em\u003e (rod-shaped) and \u003cem\u003eE. faecalis\u003c/em\u003e (cocci-shaped) was clearly observed within the dentinal tubules (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThree weeks after culture, the specimens underwent SEM to assess the growth and maturity of the biofilm. The images obtained showed extensive bacterial colonization as a dual-species biofilm at the opening of dentinal tubules at three different magnifications (\u0026times;2000, \u0026times;5000 and \u0026times;12000) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). High-magnification SEM micrographs showed simultaneous presence of two bacterial species, \u003cem\u003eA. naeslundii\u003c/em\u003e (rod-shaped) and \u003cem\u003eE. faecalis\u003c/em\u003e (cocci-shaped), on the dentin surface and within the dentinal tubules (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). After 3 weeks, the association of these two species as a mature dual-species biofilm could be observed on different areas of the dentin surface and also within the dentinal tubules (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Morphological differences between the two species, including differences in cell morphology and their arrangement and attachment within the biofilm structure, were clearly discernible and indicated simultaneous participation of both species in biofilm formation [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eColonies formed on specific culture medium after inoculation of the medicaments:\u003c/h2\u003e\n \u003cp\u003eAssessment of colony growth in the specific culture medium clearly showed the difference in the efficacy of the tested medicaments. In the control group, extensive and unrestricted growth of \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eA. naeslundii\u003c/em\u003e indicated absence of any inhibitory factor and eligibility of this group for comparison with other groups. In contrast, the images of the medicament groups indicated a significant difference in the degree of inhibition of colony growth.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eE. faecalis colony count at 200 \u0026micro;m depth:\u003c/h2\u003e\n \u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e presents the mean colony count (log 10 weight adjusted CFUs) of \u003cem\u003eE. faecalis\u003c/em\u003e at 200 \u0026micro;m depth. The colony count data had a normal distribution in all groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) except in the mTAP group (P\u0026thinsp;=\u0026thinsp;0.01). Homogeneity of the variances was also confirmed by the Levene test (P\u0026thinsp;=\u0026thinsp;0.237). Thus, one-way ANOVA was applied, which showed a significant difference in colony count among the 5 groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Pairwise comparisons (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) by the Tukey\u0026rsquo;s HSD test showed that the control group had a significantly higher mean \u003cem\u003eE. faecalis\u003c/em\u003e colony count than the other four groups at 200 \u0026micro;m depth. The propolis group had a higher colony count than the CH group (P\u0026thinsp;=\u0026thinsp;0.041). Other comparisons were not statistically significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMean colony count (log 10 weight adjusted CFUs) of \u003cem\u003eE. faecalis\u003c/em\u003e at 200 and 400 \u0026micro;m depths in the study groups (n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDepth\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStd. Deviation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMinimum\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMaximum\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e200 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003econtrol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e88.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epropolis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e72.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e80.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e37.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e57.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e55.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e27.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e65.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e06.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e46.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e38.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e90.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e400 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003econtrol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e94.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e44.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epropolis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e82.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e38.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e44.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e08.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e56.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e63.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e81.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e25.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e72.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePairwise comparison of the groups regarding the mean \u003cem\u003eE. faecalis\u003c/em\u003e count at 200 and 400 \u0026micro;m depths\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDepth\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCompared groups\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean Difference (|I-J|)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStd. Error\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSig.\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"10\"\u003e\n \u003cp\u003e200 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003econtrol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epropolis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e77.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e009.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e31.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e41.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003epropolis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e54.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e58.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e075.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e04.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e04.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e05.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"10\"\u003e\n \u003cp\u003e400 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003econtrol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epropolis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e91.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e86.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e01.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003epropolis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e73.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e03.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e70.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e48.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e09.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n \u003ch2\u003eE. faecalis colony count at 400 \u0026micro;m depth:\u003c/h2\u003e\n \u003cp\u003eTable\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e presents the mean colony count (log 10 weight adjusted CFUs) of \u003cem\u003eE. faecalis\u003c/em\u003e at 400 \u0026micro;m depth. Due to non-homogeneity of the variances as shown by the Levene test, the robust Welch test was applied to compare the 5 groups, which revealed a significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Pairwise comparisons (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e) by the Games-Howell test showed that there was no statistically significant difference between the propolis and control groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05), and both the control and propolis groups had a significantly higher bacterial count than the other three groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No other significant differences were found (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eComparison of E. faecalis count at 200 and 400 \u0026micro;m depths after the application of different medicaments\u003c/em\u003e:\u003c/p\u003e\n \u003cp\u003eTwo-way ANOVA showed that penetration depth significantly affected the bacterial count of \u003cem\u003eE. faecalis\u003c/em\u003e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The mean bacterial count at 400 \u0026micro;m depth was significantly lower than that at 200 \u0026micro;m depth. This reduction in bacterial count was observed in all groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). On the other hand, a statistically significant difference was also observed among the study groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The control and propolis groups showed the highest bacterial count at both depths, and the difference between these two groups was not statistically significant (P\u0026thinsp;=\u0026thinsp;0.082); however, both had a significantly higher bacterial count than the CH, TAP, and mTAP groups. The interaction between depth and group was not significant (P\u0026thinsp;=\u0026thinsp;0.082), indicating that the pattern of bacterial count reduction from 200 to 400 \u0026micro;m depth was similar in all groups, and the intensity of the effect of depth was almost the same, regardless of the type of medicament.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cem\u003eA. naeslundii colony count at 200 \u0026micro;m depth\u003c/em\u003e:\u003c/h2\u003e\n \u003cp\u003eTable \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e presents the mean colony count (log 10 weight adjusted CFUs) of \u003cem\u003eA. naeslundii\u003c/em\u003e at 200 \u0026micro;m depth. The colony count data had a normal distribution in all groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Due to normal data distribution and homogeneity of the variances, one-way ANOVA was applied, which revealed a significant difference in colony count among the 5 groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Pairwise comparisons (Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) by the Tukey\u0026rsquo;s HSD test showed that the control group had a significantly higher mean count than the other four groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The propolis group also had a higher bacterial count than the CH group (P\u0026thinsp;=\u0026thinsp;0.016). Other pairwise comparisons were not statistically significant (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMean colony count (log 10 weight adjusted CFUs) of \u003cem\u003eA. naeslundii\u003c/em\u003e at 200 \u0026micro;m and 400 \u0026micro;m depths in the study groups\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDepth\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStd. Deviation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMinimum\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMaximum\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e200 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003econtrol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e83.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e43.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e40.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e71.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epropolis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e09.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e69.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e70.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e42.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e63.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e41.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e67.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e08.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e50.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e10.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e51.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e05.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e400 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003econtrol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e55.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e33.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e05.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epropolis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e61.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e44.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e71.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e85.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e64.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e47.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e95.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e63.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e49.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e46.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e79.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e17.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"char\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePairwise comparison of the groups regarding the mean \u003cem\u003eA. naeslundii\u003c/em\u003e count at 200 and 400 \u0026micro;m depths\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDepth\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCompared groups\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean Difference (|I-J|)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eStd. Error\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSig.\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"10\"\u003e\n \u003cp\u003e200 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003econtrol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epropolis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e73.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e01.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e48.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003epropolis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e46.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e26.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e60.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e07.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e74.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e01.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e97.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e74.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e23.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e97.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"10\"\u003e\n \u003cp\u003e400 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003econtrol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003epropolis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e06.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e99.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e70.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e02.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e91.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e001.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e05.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003epropolis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e75.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e01.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e97.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e00.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e87.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e36.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e22.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e51.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e14.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e96.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e\u003cem\u003eA. naeslundii colony count at 400 \u0026micro;m depth\u003c/em\u003e:\u003c/h2\u003e\n \u003cp\u003eTable\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e presents the mean colony count (log 10 weight adjusted CFUs) of \u003cem\u003eA. naeslundii\u003c/em\u003e at 400 \u0026micro;m depth. Due to normal distribution of data and homogeneity of the variances, one-way ANOVA was applied which showed a significant difference in bacterial count among the 5 groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Pairwise comparisons (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) by the Tukey\u0026rsquo;s HSD test showed that the propolis and control groups did not have a statistically significant difference (P\u0026thinsp;=\u0026thinsp;0.998), and both propolis and control groups had a significantly higher bacterial count than all three other groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No other significant differences were found (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eComparison of A. naeslundii count at 200 and 400 \u0026micro;m depths after the application of different medicaments\u003c/em\u003e:\u003c/p\u003e\n \u003cp\u003eThe results of two-way ANOVA showed that the penetration depth significantly affected the bacterial count of \u003cem\u003eA. naeslundii\u003c/em\u003e (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In all groups, the bacterial count at 400 \u0026micro;m depth was significantly lower than that at 200 \u0026micro;m depth (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In addition, a significant difference was observed among the study groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The control and propolis groups showed the highest bacterial count at both depths. The difference between these two groups was not statistically significant (P\u0026thinsp;=\u0026thinsp;0.327); however, their bacterial count was significantly higher than the other three groups, namely CH, TAP, and mTAP. The interaction between group and depth was also significant (P\u0026thinsp;=\u0026thinsp;0.048). It means that the rate of bacterial count reduction from 200 to 400 \u0026micro;m depth varied depending on the group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003eWithin-group comparison of bacterial count at different depths:\u003c/h2\u003e\u003cspan\u003e\n \u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eE. faecalis\u003c/span\u003e: Paired t-test was used to compare the \u003cem\u003eE. faecalis\u003c/em\u003e count between 200 and 400 \u0026micro;m depths in the control, propolis, CH, and TAP groups, given the normal distribution of the data. In the control group, the colony count at 400 \u0026micro;m depth was significantly lower than that at 200 \u0026micro;m depth (P\u0026thinsp;=\u0026thinsp;0.000). However, in the propolis group, there was no statistically significant difference between the two depths (P\u0026thinsp;=\u0026thinsp;0.202). In the CH group, a significant decrease in colony count was observed at 400 \u0026micro;m compared to 200 \u0026micro;m (P\u0026thinsp;=\u0026thinsp;0.04). In the TAP group, this decrease was also significant (P\u0026thinsp;=\u0026thinsp;0.03). In the mTAP group, since the assumption of normality was not met, the Wilcoxon test was used. The results showed that colony count at 400 \u0026micro;m depth was significantly lower than that at 200 \u0026micro;m depth (P\u0026thinsp;=\u0026thinsp;0.021).\u003c/p\u003e\n \u003c/span\u003e\u003cspan\u003e\n \u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eA. naeslundii\u003c/span\u003e: Paired t-test was used to analyze the difference in \u003cem\u003eA. naeslundii\u003c/em\u003e colony count at 200 and 400 \u0026micro;m depths, given the normal distribution of data in all groups. In the control group, the colony count at 400 \u0026micro;m depth was significantly lower than that at 200 \u0026micro;m depth (P\u0026thinsp;=\u0026thinsp;0.00). In the propolis group, a significant decrease was observed at 400 \u0026micro;m compared to 200 \u0026micro;m depth (P\u0026thinsp;=\u0026thinsp;0.047). In the CH group, this difference was also significant (P\u0026thinsp;=\u0026thinsp;0.009). In the TAP group, the decrease in colony count at 400 \u0026micro;m was significantly lower than that at 200 \u0026micro;m (P\u0026thinsp;=\u0026thinsp;0.001). In the mTAP group, the colony count at 400 \u0026micro;m depth was significantly lower than that at 200 \u0026micro;m depth (P\u0026thinsp;=\u0026thinsp;0.002).\u003c/p\u003e\n \u003c/span\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003eComparison of microorganism-free specimens:\u003c/h2\u003e\n \u003cp\u003eMicrobial growth was observed at 200 \u0026micro;m depth in all groups inoculated with \u003cem\u003eE. faecalis\u003c/em\u003e. Thus, no comparison was made among the groups for this microorganism at 200 \u0026micro;m depth. Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e shows the growth status of \u003cem\u003eE. faecalis\u003c/em\u003e at 400 \u0026micro;m and \u003cem\u003eA. naeslundii\u003c/em\u003e at 200 and 400 \u0026micro;m depths in different groups. The Fisher\u0026apos;s Exact test showed no statistically significant difference among the groups in terms of growth of \u003cem\u003eA. naeslundii\u003c/em\u003e at 200 \u0026micro;m (P\u0026thinsp;=\u0026thinsp;1.00) or 400 \u0026micro;m (P\u0026thinsp;=\u0026thinsp;0.252) depth, or \u003cem\u003eE. faecalis\u003c/em\u003e at 400 nm depth (P\u0026thinsp;=\u0026thinsp;1.00).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eGrowth status of \u003cem\u003eE. faecalis\u003c/em\u003e at 400 \u0026micro;m and \u003cem\u003eA. naeslundii\u003c/em\u003e at 200 and 400 \u0026micro;m depths in different groups\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eno growth\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003egrowth\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"12\"\u003e\n \u003cp\u003e\u003cem\u003eA. naeslundii\u003c/em\u003e at 200 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003econtrol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003epropolis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e92٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"12\"\u003e\n \u003cp\u003e\u003cem\u003eA. naeslundii\u003c/em\u003e at 400 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003econtrol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003epropolis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e80٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e82٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"12\"\u003e\n \u003cp\u003e\u003cem\u003eE. faecalis\u003c/em\u003e at 400 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003econtrol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003epropolis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003em-TAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eTAP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e90٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94٪\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100٪\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003eEffect size (partial eta squared):\u003c/h2\u003e\n \u003cp\u003eThe effect size for \u003cem\u003eA. naeslundii\u003c/em\u003e colony count for the group variable was 0.570 at 200 \u0026micro;m and 0.598 at 400 \u0026micro;m depth. The effect size for \u003cem\u003eE. faecalis\u003c/em\u003e colony count for the group variable was 0.570 at 200 \u0026micro;m and 0.676 at 400 \u0026micro;m depth.\u003c/p\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003eResults of antibiogram test:\u003c/h2\u003e\n \u003cp\u003eThe diameter of the growth inhibition zone produced by TAP and mTAP in both media was significantly larger than that of other medicaments. Unlike TAP and mTAP, no distinct inhibition zone was observed around propolis. This is not necessarily due to lack of antimicrobial activity, but rather to the inability of propolis to be released and diffused into the agar medium. CH also does not dissolve well in agar medium and does not diffuse or spread effectively; as a result, its inhibition zone is usually small or indistinct; sometimes it is not even visible at all.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study compared the antimicrobial effects of propolis, CH, TAP, and mTAP as intracanal medicaments on tubular dentin inoculated with a dual-species biofilm. The results clearly showed that, at 200 μm depth, all the tested medicaments had inhibitory effects on \u003cem\u003eE. faecalis\u0026nbsp;\u003c/em\u003eand A. naeslundii, which was consistent with other studies that investigated the antimicrobial effects of medicaments at 200 μm depth [16,32]. Therefore, the null hypothesis of the study was rejected. At 200 μm depth, CH, TAP, and mTAP caused the greatest reduction in bacterial count compared to the control group. While propolis was less effective, it was still able to significantly reduce the bacterial count compared to the control group. These results were consistent with previous studies showing the high efficacy of TAP in removing multi-species biofilms [1,7,12,32-34,]. Also, Zargar et al. [22] showed comparable efficacy of TAP and mTAP in inhibiting biofilms, which was consistent with the present results. Furthermore, Zargar et al. [16] confirmed the antimicrobial efficacy of TAP and CH at the same depth, although CH was unable to completely remove the biofilm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAt 400 μm depth, the antimicrobial effect of propolis was further reduced and its statistical difference with the control group was not significant, while the other three medicaments still had a significant difference with the control group, and were able to reduce the microbial load at this depth. This finding is inconsistent with the results reported by BhanDari et al, [32] who demonstrated that propolis exhibited significant antibacterial activity against \u003cem\u003eE. faecalis\u0026nbsp;\u003c/em\u003eat 400 μm depth. Parhizkar [15] examined the antimicrobial effect of mTAP and showed that this compound was effective on a wide range of microorganisms.\u003c/p\u003e\n\u003cp\u003eZargar et al. [16] showed that TAP had a significant antimicrobial effect at 400 μm depth, while CH did not show a significant difference with the control group at this depth. Most studies have also reported higher antimicrobial efficacy of TAP, especially against E. faecalis, than CH. However, the current study showed optimal antibiofilm effect of CH at both 200 and 400 μm depths, which was different from previous findings. This difference could be related to the size of CH particles and the altered proton pump function in multi-species biofilms, wihich may augment its antimicrobial biofilms efficacy [16, 21], which could justify the present results.\u003c/p\u003e\n\u003cp\u003eIt should be noted that some previous studies reported relative resistance of \u003cem\u003eE. faecalis\u0026nbsp;\u003c/em\u003eand \u003cem\u003eA. naeslundii\u0026nbsp;\u003c/em\u003eto CH [35,36], which was in contrast to the present findings. In addition, it has been reported that dentin powder can reduce the effectiveness of CH in vitro [37]. However, no such effect was observed in the present study. Such differences may be due to variations in study designs, disinfection methods, duration of medicament application, sampling method, or sample transfer conditions [38].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe statistically significant difference between the efficacy of propolis and CH at both depths indicated limited antibacterial activity of propolis against \u003cem\u003eE. faecalis\u0026nbsp;\u003c/em\u003eand \u003cem\u003eA. naeslundii\u0026nbsp;\u003c/em\u003eand higher antimicrobial activity of CH. This result was in contrast to some previous studies that reported greater efficacy of propolis [32,34,39]. This difference is probably related to the limited penetration power of propolis in mature biofilms; propolis is more effective on planktonic form of bacteria, and has a weaker effect against mature multispecies biofilms due to the adhesive and protective structure of the biofilm matrix [40]. Also, factors such as the composition, formulation, time and area of collection, the concentration of waxy contaminants, the concentration of flavonoids, and the extraction method of propolis can affect its effectiveness against microorganisms inside the canal [32,33]. An in vivo study by Ahangari et al, [33] also showed that a 30% propolis extract collected from Azerbaijan had a similar antimicrobial activity to CH against E. faecalis, although its antimicrobial properties were slightly weaker. These findings confirm the role of geographical region in the properties of propolis.\u003c/p\u003e\n\u003cp\u003eSome studies, including those by Awawdeh et al, [41] and Madhubala et al. [25] reported lower antimicrobial efficacy of CH than propolis, which was inconsistent with the results of the present study. This difference may be related to the short duration of CH use (1-2 days) in those studies, which did not allow enough time for the therapeutic effects of CH to occur. On the other hand, other studies, such as those by Shamma et al, [42] and Arun et al. [39], showed that the antibacterial effect of CH was better than that of propolis after 1 to 2 weeks of use, although in some cases the differences were not significant. This emphasizes the importance of the appropriate timing and conditions of use of CH [33,41]. CH, due to its bactericidal properties, can lead to bacterial cell death within a suitable period of time (1 week) [33,39,42-45]. However, the minimum time required to achieve maximum effectiveness is still unknown [33].\u003c/p\u003e\n\u003cp\u003eSome studies, such as Edara Lillygrace et al, [24] and Madhubala et al. [25] reported the efficacy of propolis in certain conditions, similar to TAP. Also, El-Tayeb [45] found no significant difference between the antibacterial activity of propolis and TAP, and introduced propolis as a comparable option to TAP in REPs [45,46]. However, most of these studies were designed based on single-species models [22,33], while, the present study used a dual-species biofilm model that better simulates the clinical environment. In addition, the Iranian propolis used in this study (Bahareh) may differ from other samples in terms of effective compounds and may be less effective.\u003c/p\u003e\n\u003cp\u003eThe bacterial count of both \u003cem\u003eE. faecalis\u0026nbsp;\u003c/em\u003eand \u003cem\u003eA. naeslundii\u0026nbsp;\u003c/em\u003eat 400 μm depth was significantly lower than that at 200 μm depth in all medicament groups. Evidence shows that biofilm accumulation is greater in more superficial layers of dentinal tubules, and mature biofilms less commonly penetrate into deeper areas. Furthermore, nutritional limitations at depth make it more difficult for the bacteria to survive. Therefore, the microbial load in these areas is inherently lower. Arias-Moliz et al. [47] suggested that the reduction in microbial load at greater depths may not be due solely to the higher drug efficacy, but also to a reduced ability of bacteria to colonize these areas. On the other hand, medicaments such as CH, TAP, and mTAP, especially if they have sufficient contact time, can penetrate to greater depths and have higher efficacy [48]. These findings are largely consistent with the results of Zargar et al. [16]. On the other hand, the reduced antibacterial efficacy of propolis at 400 μm depth may be attributed to its limited permeability into dentinal tubules. However, when these compounds are prepared as nanoparticles, their drug stability, therapeutic efficacy, and penetration power are significantly increased compared to the pure drug solution [32]. At both depths and for both microorganisms, the effect size of the medicament group was very high. This indicates that the type of treatment was a determining factor in the difference in colony count and played a significant role in reducing the microbial load.\u003c/p\u003e\n\u003cp\u003eDespite the reduction in bacterial count at different depths and the effect of medicaments on bacterial count, there was no significant difference between the groups in terms of the degree of complete bacterial clearance (i.e., samples completely free of microbial growth). In other words, even effective drugs were unable to completely eliminate biofilms in all specimens. This may be due to the inherent resistance of biofilms and their complex structure, which limits the penetration of medicaments. This finding was consistent with studies that showed that biofilms, unlike single-microbial colonies, are difficult to completely eliminate [50]. Similarly, Zargar et al. [22] emphasized that although TAP and mTAP were effective in eliminating the planktonic form, complete inhibition of biofilm was not achieved.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, the antibiogram test with the agar diffusion method was used in the current study to confirm the antimicrobial properties of the medicaments and to initially assess their diffusion ability in the culture medium and also to investigate the resistance pattern of the bacteria. As a complementary method, this test provides valuable qualitative information about the susceptibility or resistance of bacteria to drugs and helps to better understand the clinical potential of antimicrobial compounds [50]. In the obtained results, TAP and mTAP showed the largest diameter of the growth inhibition zone against \u003cem\u003eE. faecalis\u0026nbsp;\u003c/em\u003eand \u003cem\u003eA. naeslundii\u003c/em\u003e, indicating good diffusion ability and high inhibitory effect against these bacteria. This finding was consistent with previous studies showing strong inhibitory effect of TAP even at low concentrations [51,52]. In contrast, propolis did not produce any distinct growth inhibition zone. This does not necessarily mean that propolis is ineffective, but is likely due to the limited ability of its active compounds to diffuse into agar media, which is related to its physical properties, such as high stickiness, high viscosity, and resinous nature. The low solubility of propolis in aqueous media has also been reported to affect the accuracy of the assessment in agar diffusion tests [53,54]. CH also produced a small, pale, and indistinct halo, which was consistent with previous findings. Studies have shown that it does not dissolve well in gelatinous media and that its active ions (OH-) do not have the ability to diffuse well into agar [55,56]. As a result, its antimicrobial activity in agar diffusion tests is often lower than the clinical reality. Overall, these differences are more likely to be due to their physicochemical properties and their ability to diffuse in the agar medium than to the extent of their antimicrobial effect. Therefore, propolis and CH, due to their low solubility and high viscosity, require the use of direct contact models that do not rely on diffusion in agar or liquid for more accurate evaluation [54,56].\u003c/p\u003e\n\u003cp\u003eThe main limitations of this study were as follows:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(I) Limitations in completely simulating the clinical conditions: Despite the careful design of this in vitro study and the use of human dentin cylinders to most closely simulate intraoral conditions, the dynamic conditions of the oral cavity cannot be completely simulated in vitro.\u003c/p\u003e\n\u003cp\u003e(II) Variability of propolis based on geographical area and harvest time: The active components of propolis vary depending on the season, geographical location, and type of dominant plants.\u003c/p\u003e\n\u003cp\u003e(III) Evaluation of a dual-species biofilm and limited assessment time points: Further studies are required especially focusing on multi-species biofilms and in conditions close to the clinical environment, to confirm the present results. \u0026nbsp;Also, the effects of medicaments should be evaluated at different time points of 1, 3, 7, 14 and 21 days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe effects of different doses and forms of medicaments (gel, paste, suspension) and their efficacy in combination with antibiotics should also be investigated. Furthermore, more advanced equipment and tests such as confocal laser microscopy and polymerase chain reaction may be used for a more accurate analysis of biofilm structure and bacterial viability, and high-performance liquid chromatography may be used to standardize the active ingredients of propolis. Finally, clinical trials are required to obtain more generalizable results. \u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eAccording to the obtained results, it may be stated that although all the tested medicaments led to a significant reduction in bacterial count, their effectiveness varied in depth and against resistant biofilm. Among them, TAP and mTAP, as antibiotic compounds, showed the most effective performance in reducing the bacterial count at different depths and against resistant microorganisms. CH, with its biological properties and favorable antimicrobial effect, is also considered a suitable option if non-antibiotic treatment is needed. Although propolis has significant biological benefits, its clinical effectiveness, especially against complex human biofilms, has not yet been definitively determined.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTAP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTriple Antibiotic Paste\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003emTAP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eModified Triple Antibiotic Paste\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCH\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecalcium hydroxide\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCFU\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eColony Forming Unit\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eStandard Deviation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eREP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRegenerative endodontic procedures\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eScanning electron microscopy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBHI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBrain Heart Infusion\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e: The study protocol was approved by the university ethics committee (IR.SBMU.DRC.REC.1403.064).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eDr. Zohreh Ahangari: designed the study and contributed to the language editing of the manuscript.\u003c/p\u003e\n\u003cp\u003eDr. Nazanin Zargar: contributed to the study design, data analysis, interpretation of results, and critical revision of the manuscript.\u003c/p\u003e\n\u003cp\u003eDr. Maryam\u0026nbsp;Pourhajibagher: performed the experiments, assisted with microbiological procedures, and contributed to the manuscript’s language editing.\u003c/p\u003e\n\u003cp\u003eDr. Rezvan Shahhosseini: conducted the experiments, wrote the initial draft of the manuscript, and was responsible for sample preparation.\u003c/p\u003e\n\u003cp\u003eAll authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgemants\u003c/strong\u003e: Not applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMoradi S, Moushekhian S, Najafi E, Sedigh HS, Navabi S. 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Effect of novel and traditional intracanal medicaments on biofilm viability in infected root canals. J Endod. 2024;50(10):1412-9.\u003c/li\u003e\n \u003cli\u003eTawde MM, Mehta R, Mehta A, Nair P. Antimicrobial efficacy of triple antibiotic paste herbal combination and calcium hydroxide against resistant biofilms. Int J Clin Pediatr Dent. 2024;17(3):243-54.\u003c/li\u003e\n \u003cli\u003eEl-Tayeb MM, Aboelsaad NM, Hassan RA. Evaluation of antibacterial activity of propolis and triple antibiotic paste against Enterococcus faecalis in root canal disinfection. BMC Oral Health. 2019;19:141.\u003c/li\u003e\n \u003cli\u003eAsgary S, Fazlyab M, Nosrat A. Regenerative Endodontic Treatment versus Apical Plug in Immature Teeth: Three-Year Follow-Up. J Clin Pediatr Dent. 2016;40(5):356-60.\u003c/li\u003e\n \u003cli\u003eArias-Moliz MT, Ordinola-Zapata R, Baca P, Ruiz-Linares M, Garc\u0026iacute;a Garc\u0026iacute;a E, Hungaro Duarte MA, Monteiro Bramante C, Ferrer-Luque CM. Antimicrobial activity of Chlorhexidine, Peracetic acid and Sodium hypochlorite/etidronate irrigant solutions against Enterococcus faecalis biofilms. Int Endod J. 2015;48(12):1188-93.\u003c/li\u003e\n \u003cli\u003eKhanvilkar U PS, Bandekar S, Dhok V, Arora S, Pawar AM, Pagnoni F, Reda R, Testarelli L. Scanning Electron Microscopy Analysis of the Intratubular Radicular Dentin Penetration of Calcium Hydroxide, Triple Antibiotic Paste, and Nitrofurantoin. J Pers Med. 2023;13(11):1554.\u003c/li\u003e\n \u003cli\u003eFlemming HC, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S, et al. Biofilms: an emergent form of bacterial life. Nat Rev Microbiol. 2016;14(9):563-75.\u003c/li\u003e\n \u003cli\u003eKarlowsky JA, Bouchillon SK, Benaouda A, Soraa N, Zerouali K, Mohamed N, Alami T, Sahm DF. Antimicrobial susceptibility testing of clinical isolates of Gram-negative bacilli collected in Morocco by the ATLAS Global Surveillance Program from 2018 to 2020. J Glob Antimicrob Resist. 2022;30:23-30.\u003c/li\u003e\n \u003cli\u003eGolla S, Gambhir N, Gupta N, Singh R, Singh D. A Comparative Evaluation of Herbal Extracts and Triple Antibiotic Paste as Intracanal Medicament against Enterococcus faecalis: A Microbiological Study. Int J Clin Pediatr Dent. 2024;17(3):285-290.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eChamorro-Petronacci CM, Torres BS, Guerrero-Nieves R, P\u0026eacute;rez-Say\u0026aacute;ns M, Carvalho-de Abreu Fantini M, Cides-da-Silva LC, Magari\u0026ntilde;os B, Rivas-Mundi\u0026ntilde;a B. Efficacy of Ciprofloxacin, Metronidazole and Minocycline in Ordered Mesoporous Silica against Enterococcus faecalis for Dental Pulp Revascularization: An In-Vitro Study. Materials (Basel). 2022;15(6):2266.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eGhaffari P, Zeighami H, Najdalizade M, Eftekhar L. \u003cem\u003eIn vitro\u003c/em\u003e antibacterial effect of a nano-zinc oxide eugenol sealer alone and in combination with chitosan, propolis, and nanosilver on \u003cem\u003eEnterococcus faecalis\u003c/em\u003e. Dent Res J (Isfahan). 2024;21:56.\u003c/li\u003e\n \u003cli\u003eBouzahouane H, Ayari A, Guehria I, Riah O. The Propolis: Antimicrobial Activity and chemicl composition Analysis: Properties of Propolis. J Microbiol Biotech Food Sci. 2021;10(6):e3211.\u003c/li\u003e\n \u003cli\u003eP Ravishanker, C Subba Rao.\u0026nbsp;Antimicrobial efficacy of four calcium hydroxide formulations and chlorhexidine gel using agar diffusion model. The Internet J Dent Sci. 2008;8(1).\u003c/li\u003e\n \u003cli\u003eGangwar A. Antimicrobial effectiveness of different preparations of calcium hydroxide. Indian J Dent Res. 2011;22(1):66-70. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Actinomyces naeslundii, Antibiotic, Biofilms, Calcium Hydroxide, Enterococcus faecalis, Propolis, Regeneration","lastPublishedDoi":"10.21203/rs.3.rs-7502815/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7502815/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives: \u003c/strong\u003eThis 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods: \u003c/strong\u003eIn this ex vivo study, dentin cylinders were obtained from the root canals of 56 single-rooted extracted teeth that were instrumented and inoculated with \u003cem\u003eEnterococcus faecalis \u003c/em\u003e(\u003cem\u003eE. faecalis\u003c/em\u003e) and \u003cem\u003eActinomyces naeslundii\u003c/em\u003e (\u003cem\u003eA. naeslundii\u003c/em\u003e) susentions in order to biofilms formation. After that, the cylinders were randomly assigned to 5 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 and 400 µm depths and analyzed by ANOVA, Shapiro-Wilk, Levene, Tukey, robust Welch, Games-Howell, Fisher’s exact, Wilcoxon, and paired t tests (alpha=0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAll medicaments significantly decreased the colony count (P\u0026lt;0.05). CH, TAP, and mTAP had equally optimal efficacy at both depths; while, propolis caused a significant reduction in bacterial count only at 200 µm (P=0.047). The 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\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eTAP and mTAP showed high efficacy, and CH should acceptable efficacy for elimination of \u003cem\u003eE. faecalis \u003c/em\u003eand \u003cem\u003eA. naeslundii \u003c/em\u003edouble-species biofilm. Propolis showed lower efficacy, highlighting the need for further modifications to enhance its penetration depth.\u003c/p\u003e","manuscriptTitle":"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","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-07 09:10:52","doi":"10.21203/rs.3.rs-7502815/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-21T10:30:31+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-19T07:41:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-07T15:57:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-06T07:23:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"192371086689214124576214011252625464440","date":"2025-10-02T13:45:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"233894982127679704566396451190267294527","date":"2025-09-29T02:50:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272531787974572550077848147833285479131","date":"2025-09-27T00:43:56+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-25T00:42:03+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-12T12:51:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-11T05:09:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-11T00:46:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2025-09-11T00:42:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a786849f-d5a4-4870-9fe8-3c74c3071a95","owner":[],"postedDate":"October 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:04:13+00:00","versionOfRecord":{"articleIdentity":"rs-7502815","link":"https://doi.org/10.1186/s12903-026-07901-x","journal":{"identity":"bmc-oral-health","isVorOnly":false,"title":"BMC Oral Health"},"publishedOn":"2026-02-16 15:57:11","publishedOnDateReadable":"February 16th, 2026"},"versionCreatedAt":"2025-10-07 09:10:52","video":"","vorDoi":"10.1186/s12903-026-07901-x","vorDoiUrl":"https://doi.org/10.1186/s12903-026-07901-x","workflowStages":[]},"version":"v1","identity":"rs-7502815","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7502815","identity":"rs-7502815","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}