Evaluation of antimicrobial activity of a fast-setting bioceramic endodontic material

Evaluation of antimicrobial activity of a fast-setting bioceramic endodontic material | 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 Article Evaluation of antimicrobial activity of a fast-setting bioceramic endodontic material Mengzhen JI, Yaqi CHI, Ye WANG, Kaixin XIONG, Xuan CHEN, Ling ZOU This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1100100/v2 This work is licensed under a CC BY 4.0 License Status: Under Review Version 2 posted 16 You are reading this latest preprint version Show more versions Abstract Purpose To evaluate the antimicrobial activity of the fast-setting bioceramic iRoot Fast Set Root Repair Material (iRoot FS) and two other calcium silicate cements. Methods The antimicrobial activity of iRoot FS, ProRoot MTA and Biodentine against E. faecalis and P. gingivalis were evaluated in this study. The materials were freshly mixed or set for 1 and 7 days to conduct the agar diffusion test, direct contact test and carry-over effect test, and the pH values were also measured. The data were analyzed by an analysis of variance and two-way ANOVA (α = 0.05). Results In the direct contact test, all three materials showed good antibacterial activity after setting for 20 minutes. The antibacterial properties of the three materials decreased with the increase of setting time. The suspension of all the three materials showed high pH values (11–12). With the extension of setting time, the pH of iRoot FS and Biodentine slightly decreased. Conclusion Fresh iRoot FS, Biodentine, and MTA killed E. faecalis and P. gingivalis effectively, and the antimicrobial effect of all the three materials decreased over 1 and 7 days after mixing. All three materials showed a tendency of alkalinity which last for at least 7 days after setting. Biodentine iRoot FS MTA direct contact test retrograde filling material Figures Figure 1 Figure 2 1. Introduction Endodontic surgery is a future treatment of non-healing apical periodontitis when root canal retreatment has failed or is not possible 1 . The root-end filling materials should possess a good sealing ability and be nontoxic, non-carcinogenic, non-genotoxic, biocompatible, insoluble and show dimensional stability 2 . Also, an ideal root-end filling material should possess antimicrobial activity to inhibit bacteria growth to prevent endodontic surgical failure caused by further microleakage 3 . Mineral trioxide aggregate (MTA), which is the first family of calcium silicate cements, has shown favorable clinical performance and was called “gold standard” for root-end-filling material 4 ; however, The long setting time is a major drawback, hence, several kinds of materials with a shorter setting time than MTA have been introduced in recent years 2 . Biodentine is a new version of calcium-silicate based inorganic cement and is commercially claimed to be a ‘bioactive dentine substitute’ 5 . Further, a novel premixed calcium phosphate silicate cement with a short setting time, iRoot Fast Set Root Repair Material (iRoot FS, Innovative Bioceramix) was introduced to the market recently and has been used as a permanent root canal repair material in endodontic treatments and apical surgery 6 . The literature results were variable regarding the antimicrobial activity of these retrograde filling materials. MTA products were investigated in many articles with different methodologies 7 – 9 , while few studies were conducted on Biodentine 10 ; 11 , and none were found for iRoot FS. According to a previous study, the microbiota of persistent periapical infection is polymicrobial with predominance of E. faecalis and P. gingivalis , regardless of the method used for microbial identification 12 . Hence, E. faecalis and P. gingivalis were used in this study to appraise the antimicrobial property of these materials. Therefore, the aim of this study was to evaluate the antimicrobial activity of three retrograde filling materials: MTA, Biodentine, and iRoot FS. 2. Materials And Methods 2.1. Specimen preparation Five microgram of the ProRoot MTA (Dentsply, York County, PA, USA), Biodentine™ (Septodont, Saint Maur des Fosses, France) and iRoot FS (Innovative Bioceramix, BC, Canada) were prepared according to the manufacturers’ instructions and placed on 5mm diameter sterile filter papers. The test samples were divided into three groups as described by Damlar et al . 3 . Briefly, samples tested at 20 min after mixing were designated as ‘fresh samples’, those tested on the first day after mixing were designated as ‘1-day samples’, and those tested on the seventh day after mixing were designated ‘7-day samples’. All the materials were allowed to set in a 100% moist atmosphere at 37°C before experimenting. For the control groups, 5mm diameter sterile filter papers were immersed in 0.12% chlorhexidine (CHX) and sterile saline respectively for 5 second before each test. 2.2. Bacterial strains and culture conditions Oral microorganism strains, E. faecalis (ATCC 19433) and P. gingivalis (ATCC 33277) were obtained from State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, China. E. faecalis were cultivated in brain heart infusion broth (BHI broth, Becton, Dickinson and Company, US) and BHI agar plate, while P. gingivalis were cultivated in BHI broth and blood agar plate supplemented with 0.0005% hemin, 0.0001% vitamin K. Both strains were incubated anaerobically (N 2 80%; H 2 10%;CO 2 10%) at 37℃. 2.3. Agar diffusion test Bacterial suspension was prepared for each bacterial strain and the turbidity was adjusted to 0.1 OD, which corresponds to approximately 10 8 colony-forming units (CFU)/mL. Then 100µL of P. gingivalis suspension was streaked on blood agar plates, while E. faecalis was streaked on BHI agar plates. A sterile scratcher was used to inoculate the bacterial suspension onto the agar plate to achieve a lawn of growth. The plates were dried for 5s in room temperature before the filter papers coated with the materials, sterile saline or CHX were placed on each plate. The plates were cultured anaerobically (N 2 80%; H 2 10%;CO 2 10%) at 37℃ for 48h before the diameter of the halo formed around the materials (inhibition zone) was observed. The tests were conducted in triplicate. 2.4. Direct contact test (DCT) The filter papers coated with the materials or sterile saline were placed at the bottom of 96-well plates, followed by 200µL of the bacteria suspension (10 7 CFU/mL) being added in each well in direct contact with the materials. After being cultured at 37℃ anaerobically for 1h, the bacteria suspension transferred from each well were serially diluted. The survival of the microorganisms was determined by culturing 100 µL aliquots on BHI agar plates after they were serially diluted 10 3 -10 5 fold. Then the colonies on the plates were counted and the CFU/mL value was calculated. The loss of viability was calculated by the following formula: loss of viability = (CFU control–CFU sample)/CFU control. The tests were conducted in triplicate. 2.5. Carry-over effect test The carry-over effects of the retrograde filling materials were assessed with procedures described by Ozcan et al. 13 with some modifications. The filter papers coated with the materials or sterile saline were placed at the bottom of 96-well plates, and sterile saline (20 µL) was placed in direct contact with the materials. After incubation at 37°C for 1 h, 230 µL of culture broth was added to each well. After mixing gently with a pipette, 20µL of the broth was transferred to a tube containing 960µL of culture broth. Then 20µL of the bacteria suspension (1.5×10 8 CFU/ml) was added to the tube. Ten-fold serial dilutions were prepared and plated onto BHI agar plates for colony forming. After incubation at 37°C for 48 h, survival of the bacteria was compared between experimental groups and control group to investigate the antimicrobial activity of the materials. The tests were conducted in triplicate. 2.6. The pH value measurement For the pH value measurement, 25mg of each endodontic material were mixed and evenly spread on the bottom of the 24-well plate and were allowed to set in a 100% moist atmosphere at 37°C before experimenting, then 1ml of distilled water (pH = 7.4) were added to each well after 20 minutes, 1 day and 7 days, respectively. After 1 hour, the solution was drawn from the wells and centrifuged at 10,000 rpm for 10 min, and the pH measurement was performed with a Five Easy PluspHFEP20 pH meter (METTLER TOLEDO, Zurich, Switzerland). 2.7. Statistical Analysis The data was analysed with two-way ANOVA and the Tukey’s post hoc test for multiple comparisons between the antimicrobial effects of the three retrograde filling materials against each bacterial strain tested. Statistical analysis was performed using SPSS 21.0 (SPSS Inc., Chicago, IL, USA), and P < 0.05 was considered statistically significant. 3. Results 3.1 Antimicrobial activity No inhibition zone was observed in the agar diffusion test except for the positive control. The results of the DCT with E. faecalis and P. gingivalis are shown in Fig. 1 a and 1 b. The negative controls exhibited bacteria growth in all test periods. All three materials presented highest antimicrobial effect against E. faecalis and P. gingivalis when freshly mixed. Fresh ProRootMTA, iRoot FS and Biodentine inhibited most E. faecalis (ProRootMTA 77.5%, iRoot FS 91.2%, Biodentine 80.7%), However, the antimicrobial activity of iRoot FS against E. faecalis were lower than the other two materials after setting for 1 or 7 days. As for the antimicrobial activity against P. gingivalis , Fresh ProRootMTA and Biodentine inhibited almost all P. gingivalis (ProRootMTA 97.9%, Biodentine 98.9%) while iRoot FS inhibited the growth of all P. gingivalis (100%). iRoot FS and Biodentine produced almost complete inhibition after setting for 1 day, while the effect of MTA was relatively lower (p < 0.05). The 7-day samples of the three test materials showed significantly lower growth inhibition of P. gingivalis when compared with the other time interval groups (p < 0.05), while Biodentine showed relatively highest antimicrobial effect and that of MTA was lowest (p < 0.05). Carry-over of the antimicrobial effect from the materials was not observed (Fig. 2 ). 3.2 The pH values measurements pH values of the leachate of the materials are shown in Table 1 . All three endodontic materials showed significantly strong alkaline effect in all observed time intervals. No significant difference was noticed when freshly mixed among the materials, and Biodentine presented highest pH values after setting for 1 or 7 days. Table 1 The PH values of MTA, Biodentine, and iRoot FS leachates at different time intervals (mean ± S.D.;n = 3) MTA Biodentine iRoot FS Fresh 1d 7d 11.85(±0.040) A,a 12.01(±0.009) A,b 11.46(±0.062) A,c 12.11(±0.012) B,a 12.07(±0.026) A,a 11.66(±0.038) B,b 11.87(±0.027) A,a 11.81(±0.030) B,a 11.00(±0.075) C,b Superscript capital letters represent statistically significant differences in the same row, and superscript lowercases indicate statistically significant differences in the same column. The same letters indicate no significant differences among the compared groups (P > 0.05) and vice versa. 4. Discussion The DCT used in the present study is a quantitative and reproducible method designed to simulate the contact of the microorganism with retrograde filling materials 14 . This procedure allows us to assess the antimicrobial effect of test materials at different stages of the setting reaction. In 2009, Zhang et al . reported a modified DCT 15 , in which the suspension of MTA was obtained to contact the bacteria suspension. However, the retrograde filling materials were in direct contact with the microorganisms inside the root canals, the DCT applied in the present study might better mimic the clinical situation. The agar diffusion test (ADT) is another method to evaluate the antimicrobial activity of root-end filling materials. Since the outcome of ADT depend on the material diffusibility in the medium 3 , the solid root-end filling materials may not be diffusible, which could be a possible explanation for the negative outcome of ADT in the present study. Therefore, DCT seems more appropriate in evaluating the antimicrobial activity of solidified materials. MTA was introduced innovatively as a root-filling material by Dr. Torabinejad in 1995 16 . According to previous studies 17 ; 18 , the antibacterial and antifungal properties of MTA were associated with elevated pH value. In the present study, no inhibition zone was observed in agar diffusion test, while direct contact test revealed antimicrobial effect of MTA against both E. faecalis and P. gingivalis . An in vitro study 2 showed that MTA exerts antibacterial effects against some facultative bacteria but not on any species of absolute anaerobes, however, another study by Kim et al. 19 found that freshly mixed ProRoot MTA formed a bacterial growth inhibition zone against P. gingivalis in disk diffusion test. Since MTA has been tested in many researches but with contradictory results 17 such difference may be attributed to the usage of different methodologies, bacterial strains, aerobic and anaerobic conditions. Biodentine was developed as dentin replacement material. In addition to shorter setting time and satisfactory strength, it was also reported to be less porosity and less leakage 20 , less tooth discolors 21 – 23 and excellent biocompatibility 24 compared with MTA. In the present study, the antimicrobial effect of Biodentine against E. faecalis was similar to that of MTA, and the effect was lower when tested 7 days after setting, which is in accordance with a previous study by Koruyucu et al 14 . iRoot FS (Innovative Bioceramix, Vancouver, BC, Canada) was introduced as a root canal repair material. As a premixed material, iRoot FS solidifies only when exposed to a moist environment. Previous studies have reported that iRoot FS has similar apical sealing ability and mechanical properties to MTA 25 and that iRoot FS has a shorter setting time (initial 18 min and final 57 min) than MTA. There are great potentials for the clinical application of iRoot FS as the material is cytocompatible while facilitating cell adhesion, proliferation, differentiation and maintenance of normal cell function 26 . However, the antimicrobial effect of iRoot FS is unknown. In the present study, iRoot FS showed satisfactory antimicrobial effect when tested 20 min or 1 day after setting, and the effect became relatively lower than MTA and Biodentine when tested 7 days after setting, which might be attribute to its shorter setting time. The pH values measured in this study were between 11 and 12, all the three materials showed strong alkaline pH, which is in accordance with previous studies 17 ; 27 . However, though Biodentine exhibited the highest pH value at all time intervals, which might explain its superior antimicrobial effect 7 days after setting, it did not show the strongest antibacterial activity against E. faecalis . Therefore, as Zhang et al . mentioned in a previous study 28 , the antibacterial action cannot be rationally explained by pH alone. Moreover, in clinical situations, a desirable high pH after MTA application cannot be maintained due to the buffering capacity of dentin 19 . The selection of the used bacterial species in this in vitro study was intended to represent the poly-micro flora in the periapical lesions. However, the real situation in vivo is far more complex and hard to simulate in vitro . Further studies against biofilms 29 or in vivo studies are required to better understand the various properties of the retrograde filling materials. 5. Conclusions Within the limitations of this study, fresh iRoot FS, Biodentine, and MTA killed E. faecalis and P. gingivalis effectively, and the antimicrobial effect of all the three materials decreased one and seven days after mixing. All three materials showed a tendency of alkalinity 7days of the study. Declarations Author contributions M.J. and L. Z. designed the study Y. C., M. J., Y. W.and K. X. conducted the study and prepared the figures and the table. M. J., Y. C. and Y. W. analyzed the data. M. J., Y. C. and X. C. drafted the manuscript. X. C. and L. Z. revised the manuscript critically for important intellectual content. Data availability statement The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. All data generated or analyzed during this study are included in this published article. Competing interests The author(s) declare no competing interests. References Kohli, M. R., Berenji, H., Setzer, F. C., Lee, S. M. & Karabucak, B. Outcome of Endodontic Surgery: A Meta-Analysis of the Literature-Part 3: Comparison of Endodontic Microsurgical Techniques with 2 Different Root-End Filling Materials. J Endod. 44 , 923–931 (2018). Parirokh, M. & Torabinejad, M. 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J Endod. 41 , 1862–1866 (2015). Shokouhinejad, N., Nekoofar, M. H., Pirmoazen, S., Shamshiri, A. R. & Dummer, P. M. Evaluation and Comparison of Occurrence of Tooth Discoloration after the Application of Various Calcium Silicate-Based Cements: An Ex Vivo Study. J Endod. 42 , 140–144 (2016). Marconyak, L. J. et al. A Comparison of Coronal Tooth Discoloration Elicited by Various Endodontic Reparative Materials. J Endod. 42 , 470–473 (2016). Ghilotti, J. et al. Comparative Surface Morphology, Chemical Composition, and Cytocompatibility of Bio-C Repair, Biodentine, and ProRoot MTA on hDPCs. Materials (Basel). 13 , (2020). Shi, S., Zhang, D. D., Chen, X., Bao, Z. F. & Guo, Y. J. Apical Sealing Ability of Bioceramic Paste and Mineral Trioxide Aggregate Retrofillings: A Dye Leakage Study. Iran Endod J. 10 , 99–103 (2015). Luo, T., Liu, J., Sun, Y., Shen, Y. & Zou, L. Cytocompatibility of Biodentine and iRoot FS with Human Periodontal Ligament Cells: An in Vitro Study. Int. Endod. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1100100","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[{"code":1,"date":"2021-11-30 21:07:08","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research 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2","display":"","copyAsset":false,"role":"figure","size":517510,"visible":true,"origin":"","legend":"\u003cp\u003eOutcome of carry-over effect test against (a) \u003cem\u003eE. faecalis \u003c/em\u003eand (b) \u003cem\u003eP. gingivalis\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-1100100/v2/3bb17e07c42130fa596505db.jpg"},{"id":21976077,"identity":"1079f919-9b63-47ca-a854-9e30c0a8be0f","added_by":"auto","created_at":"2022-05-27 18:45:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":346896,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1100100/v2/bebccf2b-cc0b-4c96-9248-7d841293e37c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of antimicrobial activity of a fast-setting bioceramic endodontic material","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEndodontic surgery is a future treatment of non-healing apical periodontitis when root canal retreatment has failed or is not possible \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The root-end filling materials should possess a good sealing ability and be nontoxic, non-carcinogenic, non-genotoxic, biocompatible, insoluble and show dimensional stability \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Also, an ideal root-end filling material should possess antimicrobial activity to inhibit bacteria growth to prevent endodontic surgical failure caused by further microleakage \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMineral trioxide aggregate (MTA), which is the first family of calcium silicate cements, has shown favorable clinical performance and was called \u0026ldquo;gold standard\u0026rdquo; for root-end-filling material \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e; however, The long setting time is a major drawback, hence, several kinds of materials with a shorter setting time than MTA have been introduced in recent years \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Biodentine is a new version of calcium-silicate based inorganic cement and is commercially claimed to be a \u0026lsquo;bioactive dentine substitute\u0026rsquo; \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Further, a novel premixed calcium phosphate silicate cement with a short setting time, iRoot Fast Set Root Repair Material (iRoot FS, Innovative Bioceramix) was introduced to the market recently and has been used as a permanent root canal repair material in endodontic treatments and apical surgery \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe literature results were variable regarding the antimicrobial activity of these retrograde filling materials. MTA products were investigated in many articles with different methodologies \u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e, while few studies were conducted on Biodentine \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e; \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, and none were found for iRoot FS. According to a previous study, the microbiota of persistent periapical infection is polymicrobial with predominance of \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eP. gingivalis\u003c/em\u003e, regardless of the method used for microbial identification \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Hence, \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eP. gingivalis\u003c/em\u003e were used in this study to appraise the antimicrobial property of these materials. Therefore, the aim of this study was to evaluate the antimicrobial activity of three retrograde filling materials: MTA, Biodentine, and iRoot FS.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Specimen preparation\u003c/h2\u003e \u003cp\u003eFive microgram of the ProRoot MTA (Dentsply, York County, PA, USA), Biodentine\u0026trade; (Septodont, Saint Maur des Fosses, France) and iRoot FS (Innovative Bioceramix, BC, Canada) were prepared according to the manufacturers\u0026rsquo; instructions and placed on 5mm diameter sterile filter papers. The test samples were divided into three groups as described by Damlar \u003cem\u003eet al\u003c/em\u003e.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Briefly, samples tested at 20 min after mixing were designated as \u0026lsquo;fresh samples\u0026rsquo;, those tested on the first day after mixing were designated as \u0026lsquo;1-day samples\u0026rsquo;, and those tested on the seventh day after mixing were designated \u0026lsquo;7-day samples\u0026rsquo;. All the materials were allowed to set in a 100% moist atmosphere at 37\u0026deg;C before experimenting. For the control groups, 5mm diameter sterile filter papers were immersed in 0.12% chlorhexidine (CHX) and sterile saline respectively for 5 second before each test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Bacterial strains and culture conditions\u003c/h2\u003e \u003cp\u003eOral microorganism strains, \u003cem\u003eE. faecalis\u003c/em\u003e (ATCC 19433) and \u003cem\u003eP. gingivalis\u003c/em\u003e (ATCC 33277) were obtained from State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, China. \u003cem\u003eE. faecalis\u003c/em\u003e were cultivated in brain heart infusion broth (BHI broth, Becton, Dickinson and Company, US) and BHI agar plate, while \u003cem\u003eP. gingivalis\u003c/em\u003e were cultivated in BHI broth and blood agar plate supplemented with 0.0005% hemin, 0.0001% vitamin K. Both strains were incubated anaerobically (N\u003csub\u003e2\u003c/sub\u003e 80%; H\u003csub\u003e2\u003c/sub\u003e 10%;CO\u003csub\u003e2\u003c/sub\u003e 10%) at 37℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Agar diffusion test\u003c/h2\u003e \u003cp\u003eBacterial suspension was prepared for each bacterial strain and the turbidity was adjusted to 0.1 OD, which corresponds to approximately 10\u003csup\u003e8\u003c/sup\u003e colony-forming units (CFU)/mL. Then 100\u0026micro;L of \u003cem\u003eP. gingivalis\u003c/em\u003e suspension was streaked on blood agar plates, while \u003cem\u003eE. faecalis\u003c/em\u003e was streaked on BHI agar plates. A sterile scratcher was used to inoculate the bacterial suspension onto the agar plate to achieve a lawn of growth. The plates were dried for 5s in room temperature before the filter papers coated with the materials, sterile saline or CHX were placed on each plate. The plates were cultured anaerobically (N\u003csub\u003e2\u003c/sub\u003e 80%; H\u003csub\u003e2\u003c/sub\u003e 10%;CO\u003csub\u003e2\u003c/sub\u003e 10%) at 37℃ for 48h before the diameter of the halo formed around the materials (inhibition zone) was observed. The tests were conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Direct contact test (DCT)\u003c/h2\u003e \u003cp\u003eThe filter papers coated with the materials or sterile saline were placed at the bottom of 96-well plates, followed by 200\u0026micro;L of the bacteria suspension (10\u003csup\u003e7\u003c/sup\u003e CFU/mL) being added in each well in direct contact with the materials. After being cultured at 37℃ anaerobically for 1h, the bacteria suspension transferred from each well were serially diluted. The survival of the microorganisms was determined by culturing 100 \u0026micro;L aliquots on BHI agar plates after they were serially diluted 10\u003csup\u003e3\u003c/sup\u003e-10\u003csup\u003e5\u003c/sup\u003e fold. Then the colonies on the plates were counted and the CFU/mL value was calculated. The loss of viability was calculated by the following formula: loss of viability = (CFU control\u0026ndash;CFU sample)/CFU control. The tests were conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Carry-over effect test\u003c/h2\u003e \u003cp\u003eThe carry-over effects of the retrograde filling materials were assessed with procedures described by Ozcan et al. \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e with some modifications. The filter papers coated with the materials or sterile saline were placed at the bottom of 96-well plates, and sterile saline (20 \u0026micro;L) was placed in direct contact with the materials. After incubation at 37\u0026deg;C for 1 h, 230 \u0026micro;L of culture broth was added to each well. After mixing gently with a pipette, 20\u0026micro;L of the broth was transferred to a tube containing 960\u0026micro;L of culture broth. Then 20\u0026micro;L of the bacteria suspension (1.5\u0026times;10\u003csup\u003e8\u003c/sup\u003e CFU/ml) was added to the tube. Ten-fold serial dilutions were prepared and plated onto BHI agar plates for colony forming. After incubation at 37\u0026deg;C for 48 h, survival of the bacteria was compared between experimental groups and control group to investigate the antimicrobial activity of the materials. The tests were conducted in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. The pH value measurement\u003c/h2\u003e \u003cp\u003eFor the pH value measurement, 25mg of each endodontic material were mixed and evenly spread on the bottom of the 24-well plate and were allowed to set in a 100% moist atmosphere at 37\u0026deg;C before experimenting, then 1ml of distilled water (pH\u0026thinsp;=\u0026thinsp;7.4) were added to each well after 20 minutes, 1 day and 7 days, respectively. After 1 hour, the solution was drawn from the wells and centrifuged at 10,000 rpm for 10 min, and the pH measurement was performed with a Five Easy PluspHFEP20 pH meter (METTLER TOLEDO, Zurich, Switzerland).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical Analysis\u003c/h2\u003e \u003cp\u003eThe data was analysed with two-way ANOVA and the Tukey\u0026rsquo;s post hoc test for multiple comparisons between the antimicrobial effects of the three retrograde filling materials against each bacterial strain tested. Statistical analysis was performed using SPSS 21.0 (SPSS Inc., Chicago, IL, USA), and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Antimicrobial activity\u003c/h2\u003e \u003cp\u003eNo inhibition zone was observed in the agar diffusion test except for the positive control.\u003c/p\u003e \u003cp\u003eThe results of the DCT with \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eP. gingivalis\u003c/em\u003e are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb. The negative controls exhibited bacteria growth in all test periods. All three materials presented highest antimicrobial effect against \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eP. gingivalis\u003c/em\u003e when freshly mixed. Fresh ProRootMTA, iRoot FS and Biodentine inhibited most \u003cem\u003eE. faecalis\u003c/em\u003e (ProRootMTA 77.5%, iRoot FS 91.2%, Biodentine 80.7%), However, the antimicrobial activity of iRoot FS against \u003cem\u003eE. faecalis\u003c/em\u003e were lower than the other two materials after setting for 1 or 7 days. As for the antimicrobial activity against \u003cem\u003eP. gingivalis\u003c/em\u003e, Fresh ProRootMTA and Biodentine inhibited almost all \u003cem\u003eP. gingivalis\u003c/em\u003e (ProRootMTA 97.9%, Biodentine 98.9%) while iRoot FS inhibited the growth of all \u003cem\u003eP. gingivalis\u003c/em\u003e (100%). iRoot FS and Biodentine produced almost complete inhibition after setting for 1 day, while the effect of MTA was relatively lower (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The 7-day samples of the three test materials showed significantly lower growth inhibition of \u003cem\u003eP. gingivalis\u003c/em\u003e when compared with the other time interval groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while Biodentine showed relatively highest antimicrobial effect and that of MTA was lowest (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCarry-over of the antimicrobial effect from the materials was not observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2 The pH values measurements\u003c/h2\u003e \u003cp\u003epH values of the leachate of the materials are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. All three endodontic materials showed significantly strong alkaline effect in all observed time intervals. No significant difference was noticed when freshly mixed among the materials, and Biodentine presented highest pH values after setting for 1 or 7 days.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe PH values of MTA, Biodentine, and iRoot FS leachates at different time intervals (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D.;n\u0026thinsp;=\u0026thinsp;3)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMTA\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBiodentine\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eiRoot FS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFresh\u003c/p\u003e \u003cp\u003e1d\u003c/p\u003e \u003cp\u003e7d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.85(\u0026plusmn;0.040)\u003csup\u003eA,a\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e12.01(\u0026plusmn;0.009)\u003csup\u003eA,b\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e11.46(\u0026plusmn;0.062)\u003csup\u003eA,c\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.11(\u0026plusmn;0.012)\u003csup\u003eB,a\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e12.07(\u0026plusmn;0.026)\u003csup\u003eA,a\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e11.66(\u0026plusmn;0.038)\u003csup\u003eB,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11.87(\u0026plusmn;0.027)\u003csup\u003eA,a\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e11.81(\u0026plusmn;0.030)\u003csup\u003eB,a\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e11.00(\u0026plusmn;0.075)\u003csup\u003eC,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eSuperscript capital letters represent statistically significant differences in the same row, and superscript lowercases indicate statistically significant differences in the same column. The same letters indicate no significant differences among the compared groups (P\u0026thinsp;\u0026gt;\u0026thinsp;0.05) and vice versa.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe DCT used in the present study is a quantitative and reproducible method designed to simulate the contact of the microorganism with retrograde filling materials \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. This procedure allows us to assess the antimicrobial effect of test materials at different stages of the setting reaction. In 2009, Zhang \u003cem\u003eet al\u003c/em\u003e. reported a modified DCT \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, in which the suspension of MTA was obtained to contact the bacteria suspension. However, the retrograde filling materials were in direct contact with the microorganisms inside the root canals, the DCT applied in the present study might better mimic the clinical situation. The agar diffusion test (ADT) is another method to evaluate the antimicrobial activity of root-end filling materials. Since the outcome of ADT depend on the material diffusibility in the medium \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, the solid root-end filling materials may not be diffusible, which could be a possible explanation for the negative outcome of ADT in the present study. Therefore, DCT seems more appropriate in evaluating the antimicrobial activity of solidified materials.\u003c/p\u003e \u003cp\u003eMTA was introduced innovatively as a root-filling material by Dr. Torabinejad in 1995 \u003csup\u003e16\u003c/sup\u003e. According to previous studies \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e; \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, the antibacterial and antifungal properties of MTA were associated with elevated pH value. In the present study, no inhibition zone was observed in agar diffusion test, while direct contact test revealed antimicrobial effect of MTA against both \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eP. gingivalis\u003c/em\u003e. An \u003cem\u003ein vitro\u003c/em\u003e study \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e showed that MTA exerts antibacterial effects against some facultative bacteria but not on any species of absolute anaerobes, however, another study by Kim \u003cem\u003eet al.\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e found that freshly mixed ProRoot MTA formed a bacterial growth inhibition zone against \u003cem\u003eP. gingivalis\u003c/em\u003e in disk diffusion test. Since MTA has been tested in many researches but with contradictory results \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e such difference may be attributed to the usage of different methodologies, bacterial strains, aerobic and anaerobic conditions.\u003c/p\u003e \u003cp\u003eBiodentine was developed as dentin replacement material. In addition to shorter setting time and satisfactory strength, it was also reported to be less porosity and less leakage \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, less tooth discolors \u003csup\u003e\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and excellent biocompatibility \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e compared with MTA. In the present study, the antimicrobial effect of Biodentine against \u003cem\u003eE. faecalis\u003c/em\u003e was similar to that of MTA, and the effect was lower when tested 7 days after setting, which is in accordance with a previous study by Koruyucu \u003cem\u003eet al\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eiRoot FS (Innovative Bioceramix, Vancouver, BC, Canada) was introduced as a root canal repair material. As a premixed material, iRoot FS solidifies only when exposed to a moist environment. Previous studies have reported that iRoot FS has similar apical sealing ability and mechanical properties to MTA \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e and that iRoot FS has a shorter setting time (initial 18 min and final 57 min) than MTA. There are great potentials for the clinical application of iRoot FS as the material is cytocompatible while facilitating cell adhesion, proliferation, differentiation and maintenance of normal cell function \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. However, the antimicrobial effect of iRoot FS is unknown. In the present study, iRoot FS showed satisfactory antimicrobial effect when tested 20 min or 1 day after setting, and the effect became relatively lower than MTA and Biodentine when tested 7 days after setting, which might be attribute to its shorter setting time.\u003c/p\u003e \u003cp\u003eThe pH values measured in this study were between 11 and 12, all the three materials showed strong alkaline pH, which is in accordance with previous studies \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e; \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. However, though Biodentine exhibited the highest pH value at all time intervals, which might explain its superior antimicrobial effect 7 days after setting, it did not show the strongest antibacterial activity against \u003cem\u003eE. faecalis\u003c/em\u003e. Therefore, as Zhang \u003cem\u003eet al\u003c/em\u003e. mentioned in a previous study \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, the antibacterial action cannot be rationally explained by pH alone. Moreover, in clinical situations, a desirable high pH after MTA application cannot be maintained due to the buffering capacity of dentin \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe selection of the used bacterial species in this \u003cem\u003ein vitro\u003c/em\u003e study was intended to represent the poly-micro flora in the periapical lesions. However, the real situation \u003cem\u003ein vivo\u003c/em\u003e is far more complex and hard to simulate \u003cem\u003ein vitro\u003c/em\u003e. Further studies against biofilms \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e or \u003cem\u003ein vivo\u003c/em\u003e studies are required to better understand the various properties of the retrograde filling materials.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eWithin the limitations of this study, fresh iRoot FS, Biodentine, and MTA killed E. faecalis and P. gingivalis effectively, and the antimicrobial effect of all the three materials decreased one and seven days after mixing. All three materials showed a tendency of alkalinity 7days of the study.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.J. and L. Z. designed the study\u003c/p\u003e\n\u003cp\u003eY. C., M. J., Y. W.and K. X. conducted the study and prepared the figures and the table.\u003c/p\u003e\n\u003cp\u003eM. J., Y. C. and Y. W. analyzed the data.\u003c/p\u003e\n\u003cp\u003eM. J., Y. C. and X. C. drafted the manuscript.\u003c/p\u003e\n\u003cp\u003eX. C. and L. Z. revised the manuscript critically for important intellectual content.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. All data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKohli, M. R., Berenji, H., Setzer, F. C., Lee, S. M. \u0026amp; Karabucak, B. Outcome of Endodontic Surgery: A Meta-Analysis of the Literature-Part 3: Comparison of Endodontic Microsurgical Techniques with 2 Different Root-End Filling Materials. J Endod. \u003cb\u003e44\u003c/b\u003e, 923\u0026ndash;931 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParirokh, M. \u0026amp; Torabinejad, M. Mineral Trioxide Aggregate: A Comprehensive Literature review\u0026ndash;Part I: Chemical, Physical, and Antibacterial Properties. J Endod. \u003cb\u003e36\u003c/b\u003e, 16\u0026ndash;27 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDamlar, I., Ozcan, E., Yula, E., Yalcin, M. \u0026amp; Celik, S. Antimicrobial Effects of Several Calcium Silicate-Based Root-End Filling Materials. Dent. Mater. J. \u003cb\u003e33\u003c/b\u003e, 453\u0026ndash;457 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKollmuss, M., Preis, C. E., Kist, S., Hickel, R. \u0026amp; Huth, K. C. Differences in Physical Characteristics and Sealing Ability of Three Tricalcium Silicate-Based Cements Used as Root-End-Filling Materials. Am. J. Dent. \u003cb\u003e30\u003c/b\u003e, 185\u0026ndash;189 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalkondu, \u0026Ouml;., Karapinar, K. M. \u0026amp; Kazazoğlu, E. A Review On Biodentine, a Contemporary Dentine Replacement and Repair Material. \u003cem\u003eBiomed Res. Int.\u003c/em\u003e 2014, 160951 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Y. et al. Cell Migration and Osteo/Odontogenesis Stimulation of iRoot FS as a Potential Apical Barrier Material in Apexification. Int. Endod. J. \u003cb\u003e53\u003c/b\u003e, 467\u0026ndash;477 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorita, M. et al. Antibacterial Activities and Mineral Induction Abilities of Proprietary MTA Cements. Dent. Mater. J. \u003cb\u003e40\u003c/b\u003e, 297\u0026ndash;303 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhedmat, S., Aminipor, M., Pourhajibagher, M., Kharazifar, M. J. \u0026amp; Bahador, A. Comparison of Antibacterial Activities of ProRoot MTA, OrthoMTA, and RetroMTA Against Three Anaerobic Endodontic Bacteria. J Dent (Tehran). \u003cb\u003e15\u003c/b\u003e, 294\u0026ndash;299 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQueiroz, M. B. et al. Physicochemical, Biological, and Antibacterial Evaluation of Tricalcium Silicate-Based Reparative Cements with Different Radiopacifiers. Dent. Mater. \u003cb\u003e37\u003c/b\u003e, 311\u0026ndash;320 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNikhil, V., Madan, M., Agarwal, C. \u0026amp; Suri, N. Effect of Addition of 2% Chlorhexidine Or 10% Doxycycline On Antimicrobial Activity of Biodentine. J Conserv Dent. \u003cb\u003e17\u003c/b\u003e, 271\u0026ndash;275 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeveci, C., Tuzuner, T., Cinar, C., Odabas, M. E. \u0026amp; Buruk, C. K. Short-Term Antibacterial Activity and Compressive Strength of Biodentine Containing Chlorhexidine/Cetirimide Mixtures. Niger. J. Clin. Pract. \u003cb\u003e22\u003c/b\u003e, 227\u0026ndash;231 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarbosa-Ribeiro, M. et al. Microbiological Analysis of Endodontically Treated Teeth with Apical Periodontitis Before and After Endodontic Retreatment. Clin Oral Investig. \u003cb\u003e25\u003c/b\u003e, 2017\u0026ndash;2027 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOzcan, E., Yula, E., Arslanoğlu, Z. \u0026amp; Inci, M. Antifungal Activity of Several Root Canal Sealers Against Candida Albicans. Acta Odontol. Scand. \u003cb\u003e71\u003c/b\u003e, 1481\u0026ndash;1485 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoruyucu, M. et al. An Assessment of Antibacterial Activity of Three Pulp Capping Materials On Enterococcus Faecalis by a Direct Contact Test: An in Vitro Study. Eur J Dent. \u003cb\u003e9\u003c/b\u003e, 240\u0026ndash;245 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, H., Pappen, F. G. \u0026amp; Haapasalo, M. Dentin Enhances the Antibacterial Effect of Mineral Trioxide Aggregate and Bioaggregate. J Endod. \u003cb\u003e35\u003c/b\u003e, 221\u0026ndash;224 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTorabinejad, M., Rastegar, A. F., Kettering, J. D. \u0026amp; Pitt, F. T. Bacterial Leakage of Mineral Trioxide Aggregate as a Root-End Filling Material. J Endod. \u003cb\u003e21\u003c/b\u003e, 109\u0026ndash;112 (1995).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElReash, A. A. et al. Antimicrobial Activity and pH Measurement of Calcium Silicate Cements Versus New Bioactive Resin Composite Restorative Material. BMC Oral Health. \u003cb\u003e19\u003c/b\u003e, 235 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhavana, V. et al. Evaluation of Antibacterial and Antifungal Activity of New Calcium-Based Cement (Biodentine) Compared to MTA and Glass Ionomer Cement. J Conserv Dent. \u003cb\u003e18\u003c/b\u003e, 44\u0026ndash;46 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim, R. J., Kim, M. O., Lee, K. S., Lee, D. Y. \u0026amp; Shin, J. H. An in Vitro Evaluation of the Antibacterial Properties of Three Mineral Trioxide Aggregate (MTA) Against Five Oral Bacteria. Arch. Oral Biol. \u003cb\u003e60\u003c/b\u003e, 1497\u0026ndash;1502 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRefaei, P., Jahromi, M. Z. \u0026amp; Moughari, A. Comparison of the Microleakage of Mineral Trioxide Aggregate, Calcium-Enriched Mixture Cement, and Biodentine Orthograde Apical Plug. Dent Res J (Isfahan). \u003cb\u003e17\u003c/b\u003e, 66\u0026ndash;72 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKohli, M. R., Yamaguchi, M., Setzer, F. C. \u0026amp; Karabucak, B. Spectrophotometric Analysis of Coronal Tooth Discoloration Induced by Various Bioceramic Cements and Other Endodontic Materials. J Endod. \u003cb\u003e41\u003c/b\u003e, 1862\u0026ndash;1866 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShokouhinejad, N., Nekoofar, M. H., Pirmoazen, S., Shamshiri, A. R. \u0026amp; Dummer, P. M. Evaluation and Comparison of Occurrence of Tooth Discoloration after the Application of Various Calcium Silicate-Based Cements: An Ex Vivo Study. J Endod. \u003cb\u003e42\u003c/b\u003e, 140\u0026ndash;144 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarconyak, L. J. et al. A Comparison of Coronal Tooth Discoloration Elicited by Various Endodontic Reparative Materials. J Endod. \u003cb\u003e42\u003c/b\u003e, 470\u0026ndash;473 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGhilotti, J. et al. Comparative Surface Morphology, Chemical Composition, and Cytocompatibility of Bio-C Repair, Biodentine, and ProRoot MTA on hDPCs. Materials (Basel). \u003cb\u003e13\u003c/b\u003e, (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi, S., Zhang, D. D., Chen, X., Bao, Z. F. \u0026amp; Guo, Y. J. Apical Sealing Ability of Bioceramic Paste and Mineral Trioxide Aggregate Retrofillings: A Dye Leakage Study. Iran Endod J. \u003cb\u003e10\u003c/b\u003e, 99\u0026ndash;103 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLuo, T., Liu, J., Sun, Y., Shen, Y. \u0026amp; Zou, L. Cytocompatibility of Biodentine and iRoot FS with Human Periodontal Ligament Cells: An in Vitro Study. Int. Endod. J. \u003cb\u003e51\u003c/b\u003e, 779\u0026ndash;788 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQuintana, R. M. et al. Bone Tissue Reaction, Setting Time, Solubility, and pH of Root Repair Materials. Clin Oral Investig. \u003cb\u003e23\u003c/b\u003e, 1359\u0026ndash;1366 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, H., Shen, Y., Ruse, N. D. \u0026amp; Haapasalo, M. Antibacterial Activity of Endodontic Sealers by Modified Direct Contact Test Against Enterococcus Faecalis. J Endod. \u003cb\u003e35\u003c/b\u003e, 1051\u0026ndash;1055 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuiz-Linares, M., de Oliveira, F. J., Solana, C., Baca, P. \u0026amp; Ferrer-Luque, C. M. Current Status On Antimicrobial Activity of a Tricalcium Silicate Cement. J Oral Sci. \u003cb\u003e64\u003c/b\u003e, 113\u0026ndash;117 (2022).\u003c/span\u003e\u003c/li\u003e\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Biodentine, iRoot FS, MTA, direct contact test, retrograde filling material","lastPublishedDoi":"10.21203/rs.3.rs-1100100/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1100100/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eTo evaluate the antimicrobial activity of the fast-setting bioceramic iRoot Fast Set Root Repair Material (iRoot FS) and two other calcium silicate cements.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe antimicrobial activity of iRoot FS, ProRoot MTA and Biodentine against \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eP. gingivalis\u003c/em\u003e were evaluated in this study. The materials were freshly mixed or set for 1 and 7 days to conduct the agar diffusion test, direct contact test and carry-over effect test, and the pH values were also measured. The data were analyzed by an analysis of variance and two-way ANOVA (α\u0026thinsp;=\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn the direct contact test, all three materials showed good antibacterial activity after setting for 20 minutes. The antibacterial properties of the three materials decreased with the increase of setting time. The suspension of all the three materials showed high pH values (11\u0026ndash;12). With the extension of setting time, the pH of iRoot FS and Biodentine slightly decreased.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eFresh iRoot FS, Biodentine, and MTA killed \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eP. gingivalis\u003c/em\u003e effectively, and the antimicrobial effect of all the three materials decreased over 1 and 7 days after mixing. All three materials showed a tendency of alkalinity which last for at least 7 days after setting.\u003c/p\u003e","manuscriptTitle":"Evaluation of antimicrobial activity of a fast-setting bioceramic endodontic material","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2022-05-27 18:45:23","doi":"10.21203/rs.3.rs-1100100/v2","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-08-03T17:30:59+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-06-29T01:01:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-06-18T15:09:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-06-03T14:32:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16a79b64-1113-4522-9d47-ec1339a92ac6","date":"2022-06-02T10:57:28+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"f1b5bfb4-a8bd-4066-8a3e-547780904777","date":"2022-06-01T23:17:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7e8af4ff-7a21-4652-944b-cfc73fe5c1d9","date":"2022-06-01T19:22:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-05-23T06:22:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-05-23T03:46:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"192a2b70-7be9-4bc5-ab3f-fa5faa568947","date":"2022-05-16T12:27:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"a89d48de-967b-4117-8c6b-af255b963adb","date":"2022-05-16T12:12:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-05-16T12:11:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-05-16T11:57:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-05-16T10:55:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-05-16T10:50:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2022-05-12T10:08:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4a9540b5-72ff-4425-a620-55794fb249e1","owner":[],"postedDate":"May 27th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-09-13T11:44:27+00:00","versionOfRecord":[],"versionCreatedAt":"2022-05-27 18:45:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-1100100","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1100100","identity":"rs-1100100","version":["v2"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}