Killing effect of antibacterial photodynamic therapy - aPDT with long-term exposure against young and old Enterococcus faecalis biofilms in dentin

Killing effect of antibacterial photodynamic therapy - aPDT with long-term exposure against young and old Enterococcus faecalis biofilms in dentin | 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 Killing effect of antibacterial photodynamic therapy - aPDT with long-term exposure against young and old Enterococcus faecalis biofilms in dentin Tianfeng Du, Yiting Wang, Xiaoke Liu, Bowen Yang, Kang Gan, Juanfang Zhu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4643951/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Feb, 2025 Read the published version in BMC Oral Health → Version 1 posted 14 You are reading this latest preprint version Abstract Background: Antibacterial photodynamic therapy - aPDT is a medical method that utilizes the activation of a nontoxic photoactive agent or photosensitizer by exposure to visible light of a specific wave-length in the presence of oxygen. The present study aimed to evaluate the killing in vitro effect of aPDT with 0.01% methylene blue (MB) against young and old Enterococcus faecalis ( E. faecalis ) biofilms in bovine dentin with the long-term exposure using confocal laser scanning microscopy (CLSM). Methods: Semicylindrical bovine dentin blocks were inoculated with E. faecalis and incubated in air to form 1- and 3-week-old biofilms. The biofilms in dentin were subjected to aPDT with 0.01% MB, 5% NaOCl and saline with the exposure of 3, 12 and 30 minutes. The dead portions of bacterial cells in E. faecalis biofilms were analyzed with using LIVE/DEAD bacteria viability staining and CLSM. Results: The visible changes in dentin structure caused by aPDT were verified with scanning electron microscopy. Significantly more bacteria were dead when aPDT with MB and 5% NaOCl were used with the long exposure time (12 and 30 minutes) than with 3 minutes (P < 0.05). The speed of killing was fastest during the first 3 minutes, and few more bacterial cells were killed after 12 minutes in the disinfection groups. Five percent NaOCl exhibited the highest effectiveness of bacterial killing in dentin at each time point than aPDT with MB groups (P < 0.05). The proportion of killed bacteria was higher in young biofilms than in mature biofilms in aPDT with MB and NaOCl groups (P < 0.05). Moreover, there were no clearly visible changes in structure of dentin surfaces subjected to aPDT with MB for 30 minutes. Conclusion: aPDT with 0.01% MB has the capability to kill bacterial cells in E. faecalis biofilms on bovine dentin, and does not result in visible changes of dentin structures. The antibacterial effect was time-dependent, but little additional killing was obtained after the first 12 minutes of exposure. Bacterial biofilms photodynamic therapy Enterococcus faecalis confocal laser scanning microscopy root canal disinfection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background It has been firmly established that the predominant etiological role of intracanal micro-organisms and their by-products in the development and progression of pulpal and periapical diseases [ 1 – 3 ]. Because bacteria in the necrotic root canal systems grow mostly in sessile biofilms, the success of root canal treatment is to eliminate such existing biofilms and prevent future microbial invasion of the disinfected root canal systems. Many studies have shown that even with a thorough cleaning and shaping technique and complete sealing, it is hard to obtain a bacteria-free root canal systems [ 4 , 5 ]. Hence, numerous novel materials and devices have been introduced as supplements to endodontic disinfection to destroy the residual bacterial load in root canals after chemo-mechanical instrumentation [ 6 – 9 ]. Enterococcus faecalis ( E. faecalis ) is a gram-positive, facultatively anaerobic coccus, and a frequently isolated species in root filled teeth with persistent periapical lesions. Thus, it is usually chosen to infect bacterial biofilms in vitro in root canals. Antibacterial Photodynamic Therapy - aPDT is a medical method that utilizes the activation of a nontoxic photoactive agent or photosensitizer by exposure to visible light of a specific wave-length in the presence of oxygen [ 10 ]. The transfer of energy from the activated photosensitizer to available oxygen results in the formation of toxic reactive oxygen species (ROS), such as singlet oxygen and free radicals. The ROS can kill microorganisms by destroying the essential cellular molecules, including membrane lipids, nucleic acids and proteins [ 11 ]. Studies have reported that aPDT remarkably reduces the number of bacterial cells in infected root canals compared to traditional endodontic instrumentation and irrigation treatments. Tennert et al. established the E. faecalis biofilm model in experimentally infected human root canals in primary infections and endodontic retreatments, analyzed the killing activity of aPDT against bacterial biofilms by calculating survival fractions of the samples with the use of counting-forming units, and considered that aPDT was an effective supplement in root canal disinfection, especially in endodontic retreatments [ 12 ]. Sampaio et al. showed that the use of aPDT with 3.32 ηg/mL of 1,9-dimethylmethylene blue at 18 J/cm 2 of LED light (λ 632 ± 2 nm) killed 99.999998% of E. faecalis counts in planktonic culture [ 13 ]. In recent years, several advances have been made in the study of bacterial biofilms in dentin using confocal laser scanning microscopy (CLSM), which more closely mimics clinical conditions. Zaura-Arite et al. combined the advantage of CLSM to visualize plaque non-destructively with a vitality staining technique to assess the immediate bacterial effect of chlorhexidine on biofilms, and confirmed that it was possible to visualize and quantitate the killing effect on bacterial biofilms under CLSM [ 14 ]. Du et al. established a noninvasive model of infected root canals for evaluating the antimicrobial effectiveness of disinfecting irrigation solutions against bacterial biofilms by using CLSM and 3D reconstructions [ 15 ]. The portion of dead E. faecalis cells subjected to disinfecting approaches was meaning of the volume ratio of red fluorescence to green-and-red fluorescence in three-dimensional reconstructions. López-Jiménez et al. also introduced E. faecalis suspensions onto coverslips for 24-hours incubation to allow biofilm formation, and used CLSM to enumerate live and dead bacterial cells in size of 0.23 × 0.23 × 0.4 µm and evaluated the killing effects of aPDT against biofilms [ 16 ]. However, no available data has focused on the killing activity of aPDT against young and old bacterial biofilms in root canal disinfection with long-term exposure by viability staining and CLSM. Therefore, the purpose of this study was to test the antibacterial effect of aPDT against E. faecalis biofilms in bovine dentin blocks by calculating the portion of dead bacterial cells using CLSM. Methods Bovine Dentin Discs Preparation In the present study, freshly extracted caries-free bovine incisors from animals killed for commercial reasons kept in 0.01% sodium hypochlorite were used as bacterial biofilm culturing substrates, as described previously [ 17 ]. After soft tissues and calculus deposits were removed with a periodontal curette, these bovine teeth were horizontally cut at the cement-enamel junction with a 0.6-mm-thick (Isomet, Buehler Ltd, Lake Bluff, IL, USA) under water cooling. A thin coronal groove was prepared in the middle of each crown using a low-speed handpiece with a bur, which was then fractured with a blade and a hammer into two pieces. To obtain uniform substrate to culture bacterial biofilms, the enamel of each piece was discarded, and the dentin surface was ground by 320-grit, 600-grit and 1500-grit silicon carbide paper in sequence and shaped by a fine carbide bur (Tulsa Dentsply, Tulsa, OK, USA) with a low-speed handpiece to standardize thickness of 1.5 mm and diameter of 12 mm. Using 5.25% NaOCl and 6% citric acid (pH 4.0) in an ultrasonic bath for 5 minutes to remove the smear layer, each dentin disc was irrigated and then rinsed in distilled water for 5 minutes. These samples placed in a glass bottle containing distilled water were autoclaved at 121 o C for 20 minutes, and then kept at 4 o C before any subsequent experiments. Two specimens were fixed in 4% glutaraldehyde in 0.1 M phosphate-buffered saline (pH 7.2) at 4 o C for 24 hours, dehydrated by increasing concentrations of ethanol solutions (30%, 50%, 70%, 90% and 100%), and dried using a critical point drier. Then the samples were sputter-coated with gold-palladium in a vacuum evaporator. The opening of dentinal tubules in bovine dentin blocks was observed by SEM (JEOL JSM-7500F, JEOL, Japan) at a magnification of 2000×-10000× operating at 10 kV. E. faecalis Biofilms in Dentin Canal Samples In the present study, E. faecalis (American Type Culture Collection 29212) stored at -80 o C was used as the test organism and grown overnight on brain-heart infusion (BHI) agar (Becton-Dickinson, sparks, MD, USA) plates at 37 o C in air. A single colony from one BHI agar plate was inoculated onto another sterile BHI agar plate for incubation at 37 o C in air overnight. The pure bacteria were harvested and suspended in BHI broth. Cell density was spectrophotometrically standardized to 3×10 6 colony-forming units (CFU)/mL in BHI broth. Each bovine disc was kept in a well of 24-well tissue culture plate (Costar, Corning, NY, USA) in order to make sure the dentin surface of each sample was infected by E. faecalis suspensions. Eighteen hundred µL of sterile BHI broth and 200 µL of bacterial suspension were transferred to the dentin surface of each bovine sample for 1- and 3-week-old incubation at 37 o C in air. Fresh BHI medium was changed once a week for the 3-week-old specimens. The 1- and 3-week-old E. faecalis biofilms on four samples were examined by SEM as described previously. Disinfection of Dentin Samples with aPDT After incubation for 1 and 3 weeks, the dentin discs with E. faecalis biofilms were taken out of the wells and rinsed with sterile saline for 1 minute to remove unattached bacteria and culture broth. The 1- and 3-week-old bacterial biofilms on dentin discs were randomly subjected to the following three disinfecting treatments for 3, 12 and 30 minutes with nine specimens in each group: aPDT with 0.01% MB (Sigma, St. Louis, MO, USA), 2 mL of 5% NaOCl (Tianjin Dengke Ltd, Tianjin, China) and 2 mL of 0.85% sterile saline (control). In the aPDT with 0.01% MB group, each sample was treated with 300 µL of 0.01% MB for 5 minutes in dark incubation, and then with aPDT (LaserHF, Hager & Werken, Tubingen, Germany) with a wavelength of 660 nm for 3, 12 and 30 minutes according to the manufacturer's instructions. The maximum output energy was 50 mW, and the outer diameter of optical fiber in the needle tip of the handpiece was 320 µm. The needle tip of aPDT was placed 1 mm above the top of the biofilms, and moved like letter "Z" at the same level (Fig. 1 ). In 12- and 30-minute aPDT groups, the LaserHF handpiece was activated every 3 minutes and powered off for 1 minute. Two mL of 5% NaOCl or saline was added every three minutes in the 12- and 30-minute groups. After exposure, each specimen was gently washed with saline for 1 minute and then examined with viability staining and CLSM as described previously [ 17 , 18 ]. Confocal Laser Scanning Microscopy Examination The LIVE/DEAD BacLight Bacterial Viability kit L-7012 (Molecular Probes, Eugene, OR, USA) containing SYTO 9 and propidium iodide (PI) was used to stain the E. faecalis biofilm cells in bovine dentin discs according to the manufacturer's instructions. The excitation/emission maxima for the two dyes are approximately 480/500 nm for SYTO 9 and 490/635 nm for PI. Bacteria with intact cell membranes stain fluorescent green by SYTO 9, whereas bacteria with damaged membranes stain red by PI. All of the specimens were rinsed with phosphate-buffered saline for 1 minute, and then viewed immediately by CLSM (Olympus FV1200, Olympus, Japan) using a 40× lens. Five randomly selected areas (0.3 mm × 0.3 mm for each area) on bacterial biofilms were examined by CLSM and 3D reconstruction analysis in each bovine disc per time point. The CLSM images were captured using Fluoview version 4.3 software (Olympus, Melville, NY, USA) at a resolution of 512 × 512 pixels. A stack of 15 slices (1-µm step size) was scanned at each randomly chosen area on 1-week-old biofilms, and the scanning depth for 3-week-old biofilms was set at 50 µm. The dead cell volume (red fluorescence) and live cell volume (green fluorescence) were reconstructed into a 3D model and the portion of dead cell volume was analyzed by using the Imaris 7.2 software (Bitplane Inc, St Paul, MN). Visible Changes in Structure of Bovine Dentin Discs Six uninfected bovine dentin discs were randomly subjected to aPDT with MB for 12 and 30 minutes as described previously. There were three samples in each group at the same exposure time. After 1-day-old incubation, the dentin specimens were washed for 1 minutes using saline. The visible structural changes in three uninfected bovine dentin blocks and six aPDT-treated specimens were observed by SEM. Statistical Analysis The proportions of dead cell volume in E. faecalis biofilms were presented as the means and standard deviations (means ± SDs). Differences between the proportions of dead cell volume were subjected to univariate analysis of variance using SPSS 22.0 software (SPSS Inc, Chicago, IL, USA). Post hoc multiple comparisons were used to isolate and compare the results at a significance level of P < 0.05. Results SEM Images of E. faecalis Biofilms on Dentin Blocks SEM images of sterile dentin blocks showed a clean dentin surface, removal of the smear layer, the opening of dentinal tubules, and absence of microbial and debris attachment (Fig. 2 A). A large volume of bacterial cells was observed by SEM, with the presence of E. faecalis biofilms on the dentin surface and dentinal tubule openings, and even inside tubules. A heavier infection and more small clusters of E. faecalis cells on 3-week-old biofilms were found than that on 1-week-old biofilms (Fig. 2 B, 2 C). CLSM Images of E. faecalis Biofilms Treated with aPDT After 1-week-old and 3-week-old incubation with E. faecalis young and old biofilm cells grown well in the bovine dentin blocks were verified by CLSM and 3D reconstructions (Fig. 3 , 4 ). There were many dead cells (red fluorescence) in E. faecalis biofilms on dentin discs subjected to aPDT with MB and 5% NaOCl at each time point, while the majority of live cells (green fluorescence) were presented on bacterial biofilms after saline irrigation. aPDT with MB and 5% NaOCl killed about 35%-81% of bacteria cells in dentin samples, and approximately 7%-10% of the cells were dead in the control group (saline) in 1- and 3-week-old biofilms. The proportion of dead E. faecalis cell volume depended on the exposure time, treatment, and age of the bacterial biofilm (Table 1 ). Significantly more bacteria were dead when aPDT with MB and 5% NaOCl were used with long exposure time (12 and 30 minutes) than after 3 minutes (P < 0.05). The speed of killing was fastest during the first 3 minutes of exposure (about 11.65%-20.76% killing per minute), slowed down strongly from 3 minutes to 12 minutes (1.81%-2.92% killing per minute), and was negligible from 12 minutes to 30 minutes (0.14%-0.25% killing per minute) in the disinfection groups. Five percent NaOCl exhibited the highest effectiveness by killing 50.40%-81.38% of bacteria in dentin at each time-point compared to 34.96%-70.96% killing in the aPDT with MB groups (P < 0.05). The amount of killing was 7.13–11.88% higher in 1-week-old bacterial cells (young biofilms) than in 3-week-old cells (old biofilms) (P < 0.05). Table 1 Proportion of Dead E. faecalis Cell Volume in Bovine Dentin Blocks (1- and 3-week-old Biofilms) Exposed to Different Disinfecting Solutions for 3, 12, or 30 Minutes Time PDT 5% NaOCl Saline 1 wk 3 wk 1 wk 3 wk 1 wk 3 wk 3 min 43.07 ± 0.86 a 34.96 ± 4.18 c 62.28 ± 0.78 d 50.40 ± 3.89 g 7.37 ± 3.35 h 9.50 ± 0.57 h 12 min 68.36 ± 5.70 be 61.23 ± 2.52 d 78.58 ± 2.59 f 68.35 ± 3.73 be 7.43 ± 1.70 h 9.00 ± 0.71 h 30 min 70.96 ± 1.32 b 63.73 ± 2.51 de 81.38 ± 1.64 f 72.85 ± 2.85 b 8.80 ± 3.94 h 9.80 ± 1.80 h Different superscript letters indicate statistically significant differences between groups ( P < 0.05). SEM Images of Visible Changes in Dentin Surfaces Figure 5 shows the homogenous dentinal tubules, but no residual microorganisms and smear layer in the dentin blocks in aPDT with MB groups and control group, which indicated that no visible changes in the structure of dentin surfaces were found after 12- and 30-minute exposure to aPDT with MB. Discussion Endodontic diseases are an inflammatory reaction of pulpal and periradicular tissues caused by bacteria and their by-products in the root canal systems. Actually, micro-organisms in the infected root canal systems grow mostly in sessile biofilms, which are more resistant to various antibacterial approaches in endodontic therapies [ 19 ]. E. faecalis was chosen as the test organism in many preliminary studies because of its frequent presence in root-filled teeth with persistent periapical lesions, and its ability to attach itself to dentin, invade dentinal tubules and form biofilm communities [ 20 ]. Therefore, many researchers have established E. faecalis biofilms in different growing environment in vitro in order to evaluate the killing activity of irrigating solutions during root canal disinfection [ 9 , 21 ]. Haapasalo and Orstavik inoculated E. faecalis suspensions onto cylindrical bovine dentin spencimens for 3-week-old culturing, and found a heavy infection that reached 400 microns from the canal lumen [ 22 ]. Kishen et al. showed the maximum reduction of bacterial cells in E. faecalis biofilms after instrumentation-syringe irrigation analyzed using SEM, quantitative reverse transcriptase real-time PCR (qRT-PCT) and CLSM [ 23 ]. Accordingly, E. faecalis biofilms were introduced on bovine dentin to assess the killing efficacy of aPDT with 0.01% MB in the present study. To correlate well with the actual clinic setting, this study is the first time that E. faecalis biofilms in dentin canals have been established after 1- and 3-week incubation periods to test the killing activity of aPDT with MB with long-term exposure as a newer disinfection strategy with using CLSM and analyzing dead portions of bacterial biofilms. In recent decades, many researchers have considered aPDT as an alternative supplemental approach to improve root canal disinfection because conventional chemo-mechanical procedures may not be able to remove all of the bacterial cells and obtain a complete cleaning of the root canal systems [ 13 , 24 ]. The results regarding the antibacterial effect of aPDT against E. faecalis biofilms in dentin blocks in vitro were obtained in the present study are consistent with those of previous studies. According to the proportions of dead bacterial cell volume in 3D reconstruction images, the aPDT with 0.01% MB treatment killed 43.07%-70.96% of the bacterial cells on 1-week-old biofilms and 34.96%-63.73% of cells on 3-week-old E. faecalis biofilms, although this was a little lower than that seen with 5% NaOCl for the same exposure time. It has been reported that MB (a non-toxic dye) as a photosensitizer has the ability to absorb energy from a light source and transfer this energy to another molecule producing reactive oxygen species [ 25 ]. When aPDT was present, these cytotoxic singlet oxygen species induced damage of the cellular plasma membrane and/or the cell DNA to play a key role in the bactericidal action. The time elapsed between the photosensitizer reaching into dentinal tubules and the actual photo-activation is called "pre-irradiation time" ranging from 5 to 15 minutes, which allows the photosensitizer to penetrate inside bacterial biofilms and obtain more light absorption [ 26 , 27 ]. Hence, five minutes as a pre-irradiation time was set in this study to help keep 0.01% MB inside E. faecalis biofilms and exert its antibacterial effect. Results from the present study showed that the killing activity of aPDT and 5% NaOCl depended on exposure time. NaOCl was selected as a disinfection agent because these are the most commonly used antimicrobial irrigating solutions during root canal disinfection. During the first 3 minutes of exposure, the killing speed of bacterial cells was approximately 11.65%-20.76% per minute, while after the first 3 minutes to 12 minutes, the killing effect against bacterial biofilms continued but at a slower speed (1.81%-2.92% per minute). After 12 minutes, the killing activity of both aPDT and NaOCl was poor and only small amounts of new bacteria were killed between 12 and 30 minutes. Limited penetration depth and persisted cells of bacteria in the biofilm may be reasons for the incomplete killing even after 30 minutes of exposure [ 15 , 28 ]. In accordance with previous studies, bacteria in young biofilms in dentin were more susceptible to disinfecting approaches than cells in old biofilms. Wang et al. indicated that the physical barrier created by the extracellular polymeric matrix (EPS), the viable but nonculturable (VBNC) state of old biofilm bacteria, and persister cells all contributed to the higher resistance of bacteria in mature biofilms [ 29 ]. One of the concerns about antibacterial photodynamic therapy inside the root canals is the energy transformation during the interaction with dentinal surfaces, which could lead to changes of dentinal structures and a temperature increase in the root canal systems. In the present study, the structure of dentine surfaces subjected to aPDT with 0.01% MB up to 30 min was not visibly different from samples from the untreated control group using SEM observation. These results were in agreement with those described by Alfredo et al. in their study, in which they showed application of laser in the root at 1.5 W in all operating modes, and 3.0 W in the pulsed mode for 20s could be safely used in endodontic treatment [ 30 ]. Although the studies used a different type of light source, the total obtained energies were very close to 30 J to 60 J. Conclusion The results of this study indicated that aPDT with 0.01% MB has the capability to kill bacterial cells in E. faecalis biofilms on bovine dentin, and does not result in visible changes of dentin structures. The antibacterial effect was time dependent, but no remarkable additional killing was obtained after the first 12 minutes of exposure. Five percent NaOCl was more effective against E. faecalis biofilms in dentin than aPDT at the same exposure time. Young E. faecalis biofilms in dentin canals were more susceptible to disinfection approaches than mature biofilms. Abbreviations E. faecalis : Enterococcus faecalis ; aPDT: antibacterial photodynamic therapy； ROS: reactive oxygen species；CLSM: confocal laser scanning microscopy；BHI: brain-heart infusion；PI: propidium iodide；qRT-PCT: real-time PCR；EPS extracellular polymeric matrix；VBNC: viable but nonculturable Declarations Ethics approval and consent to participate Ethical approval The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Science and Ethics Commission of the First Affiliated Hospital of Zhengzhou University. Informed consent Not applicable. Consent for publication Not applicable. Availability of data and materials The datasets used for the current study are available from the corresponding author on reasonable request Competing interests The authors declare that they have no conflicts of interests. Funding This research was funded by the National Natural Science Foundation of China (No. 81500847 to Tianfeng Du), and the Medical Science and Technology Project of Henan Province (No. 201702042 to Juanfang Zhu). Acknowledgements Not Applicable. Authors’ contributions Tianfeng Du and Yiting Wang contributed equally to this study. Conceptualization, Tianfeng Du and Yiting Wang; Funding acquisition, Tianfeng Du and Juanfang Zhu; Methodology, Tianfeng Du, Yiting Wang and Xiaoke Liu; Software, Bowen Yang and Kang Gan; Supervision, Juanfang Zhu; Writing-original draft, Tianfeng Du; Writing-review and editing, Tianfeng Du. All authors have read and agreede to the published version of the manuscript. References Kakehashi S, Stanley HR, Fitzgerald RJ: The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats . Oral Surg Oral Med Oral Pathol 1965, 20 :340-349. 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Cite Share Download PDF Status: Published Journal Publication published 22 Feb, 2025 Read the published version in BMC Oral Health → Version 1 posted Editorial decision: Revision requested 22 Jul, 2024 Reviews received at journal 18 Jul, 2024 Reviews received at journal 15 Jul, 2024 Reviews received at journal 12 Jul, 2024 Reviews received at journal 10 Jul, 2024 Reviewers agreed at journal 09 Jul, 2024 Reviewers agreed at journal 07 Jul, 2024 Reviewers agreed at journal 05 Jul, 2024 Reviewers agreed at journal 05 Jul, 2024 Reviewers invited by journal 05 Jul, 2024 Editor invited by journal 04 Jul, 2024 Editor assigned by journal 28 Jun, 2024 Submission checks completed at journal 28 Jun, 2024 First submitted to journal 26 Jun, 2024 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. 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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-4643951","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":328932960,"identity":"7e7242af-b7c9-4848-9028-dd9d9ce7bc90","order_by":0,"name":"Tianfeng Du","email":"","orcid":"","institution":"First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Tianfeng","middleName":"","lastName":"Du","suffix":""},{"id":328932961,"identity":"6110972d-bf60-4934-9ecd-505588a13cd5","order_by":1,"name":"Yiting Wang","email":"","orcid":"","institution":"Zhengzhou People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yiting","middleName":"","lastName":"Wang","suffix":""},{"id":328932962,"identity":"8a2801a0-e5b5-47a8-a4f0-950dce2df3a9","order_by":2,"name":"Xiaoke Liu","email":"","orcid":"","institution":"Luohe Central Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaoke","middleName":"","lastName":"Liu","suffix":""},{"id":328932963,"identity":"1ae70de5-3a8c-4bc5-9e06-cbdb5997d58b","order_by":3,"name":"Bowen Yang","email":"","orcid":"","institution":"First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Bowen","middleName":"","lastName":"Yang","suffix":""},{"id":328932964,"identity":"8c567762-5885-477a-bc0f-bd513bcb6bd3","order_by":4,"name":"Kang Gan","email":"","orcid":"","institution":"First Affiliated Hospital of Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Kang","middleName":"","lastName":"Gan","suffix":""},{"id":328932965,"identity":"76064b98-eccf-4649-8dc3-c373e8c0c223","order_by":5,"name":"Juanfang Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIie3QMQrCQBCF4QmB2CxiOTbJCYQHAct4FUNAG4uUqcQgxMYDCHqO1BGLNDmAwSa5QU4g7irWu6Xg/tUU87HMEtlsvxgTIVPDKM/7wZg0ahC3fcimhN6E18VEmIjgfERa0XaBti+kj/zZTkOcSwNU5Ll4xEWXUhLOKw1xeYNwIOFJcgBTFZc64kkiX2GB9lqwMCHiQ8C4O4aEeZVKssS0ieUnw+CW4JSUUD82ruu+H7LI1xJ1Dpznd4Z+XeV2Zns2m832t70AlKc8sXclSYsAAAAASUVORK5CYII=","orcid":"","institution":"First Affiliated Hospital of Zhengzhou University","correspondingAuthor":true,"prefix":"","firstName":"Juanfang","middleName":"","lastName":"Zhu","suffix":""}],"badges":[],"createdAt":"2024-06-26 16:06:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4643951/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4643951/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12903-025-05657-4","type":"published","date":"2025-02-22T15:57:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60806773,"identity":"e781237a-e855-41b6-a3e5-ab307e297bc7","added_by":"auto","created_at":"2024-07-22 10:04:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":66259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiagram of the irradiation protocol used for the aPDT.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4643951/v1/a34e64cd7428681ad57af183.png"},{"id":60807332,"identity":"9d6d5af9-42e6-47f4-9f85-0773a2108c86","added_by":"auto","created_at":"2024-07-22 10:12:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":261269,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM images of sterile dentin blocks \u003c/strong\u003e(A) 1-week-old \u003cem\u003eE. faecalis\u003c/em\u003e biofilms, (B) 3-week-old \u003cem\u003eE. faecalis\u003c/em\u003e biofilms and (C) on bovine dentin blocks.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4643951/v1/87adc27097cd13f7150ea812.png"},{"id":60806774,"identity":"7340d30e-9c2d-4f20-96a9-7db4be690685","added_by":"auto","created_at":"2024-07-22 10:04:26","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":686581,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCLSM images of 1-week-old \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. faecalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ebiofilms in infected bovine dentin blocks after being subjected to\u003c/strong\u003e: (A1) 3-minute PDT with 0.01% MB; (A2) 12-minute PDT with 0.01% MB; (A3) 30-minute PDT with 0.01% MB; (B1) 3-minute 5% NaOCl; (B2) 12-minute 5% NaOCl; (B3) 30-minute 5% NaOCl; (C1) 3-minute sterile saline; (C2) 12-minute sterile saline; (C3) 30-minute sterile saline.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4643951/v1/5b65458c6150bebd6c640746.png"},{"id":60806776,"identity":"9500d863-e3a3-4fcc-ac25-5ad5558961a7","added_by":"auto","created_at":"2024-07-22 10:04:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":725454,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCLSM images of 3-week-old \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. faecalis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ebiofilms in infected bovine dentin blocks after being subjected to: \u003c/strong\u003e(A1) 3-minute PDT with 0.01% MB; (A2) 12-minute PDT with 0.01% MB; (A3) 30-minute PDT with 0.01% MB; (B1) 3-minute 5% NaOCl; (B2) 12-minute 5% NaOCl; (B3) 30-minute 5% NaOCl; (C1) 3-minute sterile saline; (C2) 12-minute sterile saline; (C3) 30-minute sterile saline.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4643951/v1/11f947af1bb33915c4aeb2e0.png"},{"id":60806778,"identity":"95f935cd-9485-4088-9fe8-700049fd41d1","added_by":"auto","created_at":"2024-07-22 10:04:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":223539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM images of bovine dentinal tubules subjected to: \u003c/strong\u003e(A) 12-minute PDT with 0.01% MB; (B) 30-minute PDT with 0.01% MB; (C) no treatment (control).\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4643951/v1/f3d9fbafcd1303ef556b41ea.png"},{"id":77052727,"identity":"3f20d1e9-4084-47ea-8a3f-8b1a31b88298","added_by":"auto","created_at":"2025-02-24 16:24:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4683300,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4643951/v1/4d48136f-5400-4720-b363-97bb9d5c6bef.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Killing effect of antibacterial photodynamic therapy - aPDT with long-term exposure against young and old Enterococcus faecalis biofilms in dentin","fulltext":[{"header":"Background","content":"\u003cp\u003eIt has been firmly established that the predominant etiological role of intracanal micro-organisms and their by-products in the development and progression of pulpal and periapical diseases [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Because bacteria in the necrotic root canal systems grow mostly in sessile biofilms, the success of root canal treatment is to eliminate such existing biofilms and prevent future microbial invasion of the disinfected root canal systems. Many studies have shown that even with a thorough cleaning and shaping technique and complete sealing, it is hard to obtain a bacteria-free root canal systems [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Hence, numerous novel materials and devices have been introduced as supplements to endodontic disinfection to destroy the residual bacterial load in root canals after chemo-mechanical instrumentation [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (\u003cem\u003eE. faecalis\u003c/em\u003e) is a gram-positive, facultatively anaerobic coccus, and a frequently isolated species in root filled teeth with persistent periapical lesions. Thus, it is usually chosen to infect bacterial biofilms in vitro in root canals.\u003c/p\u003e \u003cp\u003eAntibacterial Photodynamic Therapy - aPDT is a medical method that utilizes the activation of a nontoxic photoactive agent or photosensitizer by exposure to visible light of a specific wave-length in the presence of oxygen [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The transfer of energy from the activated photosensitizer to available oxygen results in the formation of toxic reactive oxygen species (ROS), such as singlet oxygen and free radicals. The ROS can kill microorganisms by destroying the essential cellular molecules, including membrane lipids, nucleic acids and proteins [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Studies have reported that aPDT remarkably reduces the number of bacterial cells in infected root canals compared to traditional endodontic instrumentation and irrigation treatments. Tennert et al. established the \u003cem\u003eE. faecalis\u003c/em\u003e biofilm model in experimentally infected human root canals in primary infections and endodontic retreatments, analyzed the killing activity of aPDT against bacterial biofilms by calculating survival fractions of the samples with the use of counting-forming units, and considered that aPDT was an effective supplement in root canal disinfection, especially in endodontic retreatments [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Sampaio et al. showed that the use of aPDT with 3.32 ηg/mL of 1,9-dimethylmethylene blue at 18 J/cm\u003csup\u003e2\u003c/sup\u003e of LED light (λ 632\u0026thinsp;\u0026plusmn;\u0026thinsp;2 nm) killed 99.999998% of \u003cem\u003eE. faecalis\u003c/em\u003e counts in planktonic culture [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn recent years, several advances have been made in the study of bacterial biofilms in dentin using confocal laser scanning microscopy (CLSM), which more closely mimics clinical conditions. Zaura-Arite et al. combined the advantage of CLSM to visualize plaque non-destructively with a vitality staining technique to assess the immediate bacterial effect of chlorhexidine on biofilms, and confirmed that it was possible to visualize and quantitate the killing effect on bacterial biofilms under CLSM [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Du et al. established a noninvasive model of infected root canals for evaluating the antimicrobial effectiveness of disinfecting irrigation solutions against bacterial biofilms by using CLSM and 3D reconstructions [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The portion of dead \u003cem\u003eE. faecalis\u003c/em\u003e cells subjected to disinfecting approaches was meaning of the volume ratio of red fluorescence to green-and-red fluorescence in three-dimensional reconstructions. L\u0026oacute;pez-Jim\u0026eacute;nez et al. also introduced \u003cem\u003eE. faecalis\u003c/em\u003e suspensions onto coverslips for 24-hours incubation to allow biofilm formation, and used CLSM to enumerate live and dead bacterial cells in size of 0.23 \u0026times; 0.23 \u0026times; 0.4 \u0026micro;m and evaluated the killing effects of aPDT against biofilms [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, no available data has focused on the killing activity of aPDT against young and old bacterial biofilms in root canal disinfection with long-term exposure by viability staining and CLSM. Therefore, the purpose of this study was to test the antibacterial effect of aPDT against \u003cem\u003eE. faecalis\u003c/em\u003e biofilms in bovine dentin blocks by calculating the portion of dead bacterial cells using CLSM.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBovine Dentin Discs Preparation\u003c/h2\u003e \u003cp\u003eIn the present study, freshly extracted caries-free bovine incisors from animals killed for commercial reasons kept in 0.01% sodium hypochlorite were used as bacterial biofilm culturing substrates, as described previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. After soft tissues and calculus deposits were removed with a periodontal curette, these bovine teeth were horizontally cut at the cement-enamel junction with a 0.6-mm-thick (Isomet, Buehler Ltd, Lake Bluff, IL, USA) under water cooling. A thin coronal groove was prepared in the middle of each crown using a low-speed handpiece with a bur, which was then fractured with a blade and a hammer into two pieces. To obtain uniform substrate to culture bacterial biofilms, the enamel of each piece was discarded, and the dentin surface was ground by 320-grit, 600-grit and 1500-grit silicon carbide paper in sequence and shaped by a fine carbide bur (Tulsa Dentsply, Tulsa, OK, USA) with a low-speed handpiece to standardize thickness of 1.5 mm and diameter of 12 mm. Using 5.25% NaOCl and 6% citric acid (pH 4.0) in an ultrasonic bath for 5 minutes to remove the smear layer, each dentin disc was irrigated and then rinsed in distilled water for 5 minutes. These samples placed in a glass bottle containing distilled water were autoclaved at 121 \u003csup\u003eo\u003c/sup\u003eC for 20 minutes, and then kept at 4 \u003csup\u003eo\u003c/sup\u003eC before any subsequent experiments.\u003c/p\u003e \u003cp\u003eTwo specimens were fixed in 4% glutaraldehyde in 0.1 M phosphate-buffered saline (pH 7.2) at 4 \u003csup\u003eo\u003c/sup\u003eC for 24 hours, dehydrated by increasing concentrations of ethanol solutions (30%, 50%, 70%, 90% and 100%), and dried using a critical point drier. Then the samples were sputter-coated with gold-palladium in a vacuum evaporator. The opening of dentinal tubules in bovine dentin blocks was observed by SEM (JEOL JSM-7500F, JEOL, Japan) at a magnification of 2000\u0026times;-10000\u0026times; operating at 10 kV.\u003c/p\u003e \u003cp\u003e \u003cb\u003eE. faecalis\u003c/b\u003e \u003cb\u003eBiofilms in Dentin Canal Samples\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn the present study, \u003cem\u003eE. faecalis\u003c/em\u003e (American Type Culture Collection 29212) stored at -80 \u003csup\u003eo\u003c/sup\u003eC was used as the test organism and grown overnight on brain-heart infusion (BHI) agar (Becton-Dickinson, sparks, MD, USA) plates at 37 \u003csup\u003eo\u003c/sup\u003eC in air. A single colony from one BHI agar plate was inoculated onto another sterile BHI agar plate for incubation at 37 \u003csup\u003eo\u003c/sup\u003eC in air overnight. The pure bacteria were harvested and suspended in BHI broth. Cell density was spectrophotometrically standardized to 3\u0026times;10\u003csup\u003e6\u003c/sup\u003e colony-forming units (CFU)/mL in BHI broth.\u003c/p\u003e \u003cp\u003eEach bovine disc was kept in a well of 24-well tissue culture plate (Costar, Corning, NY, USA) in order to make sure the dentin surface of each sample was infected by \u003cem\u003eE. faecalis\u003c/em\u003e suspensions. Eighteen hundred \u0026micro;L of sterile BHI broth and 200 \u0026micro;L of bacterial suspension were transferred to the dentin surface of each bovine sample for 1- and 3-week-old incubation at 37 \u003csup\u003eo\u003c/sup\u003eC in air. Fresh BHI medium was changed once a week for the 3-week-old specimens. The 1- and 3-week-old \u003cem\u003eE. faecalis\u003c/em\u003e biofilms on four samples were examined by SEM as described previously.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDisinfection of Dentin Samples with aPDT\u003c/h2\u003e \u003cp\u003eAfter incubation for 1 and 3 weeks, the dentin discs with \u003cem\u003eE. faecalis\u003c/em\u003e biofilms were taken out of the wells and rinsed with sterile saline for 1 minute to remove unattached bacteria and culture broth. The 1- and 3-week-old bacterial biofilms on dentin discs were randomly subjected to the following three disinfecting treatments for 3, 12 and 30 minutes with nine specimens in each group: aPDT with 0.01% MB (Sigma, St. Louis, MO, USA), 2 mL of 5% NaOCl (Tianjin Dengke Ltd, Tianjin, China) and 2 mL of 0.85% sterile saline (control).\u003c/p\u003e \u003cp\u003eIn the aPDT with 0.01% MB group, each sample was treated with 300 \u0026micro;L of 0.01% MB for 5 minutes in dark incubation, and then with aPDT (LaserHF, Hager \u0026amp; Werken, Tubingen, Germany) with a wavelength of 660 nm for 3, 12 and 30 minutes according to the manufacturer's instructions. The maximum output energy was 50 mW, and the outer diameter of optical fiber in the needle tip of the handpiece was 320 \u0026micro;m. The needle tip of aPDT was placed 1 mm above the top of the biofilms, and moved like letter \"Z\" at the same level (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In 12- and 30-minute aPDT groups, the LaserHF handpiece was activated every 3 minutes and powered off for 1 minute. Two mL of 5% NaOCl or saline was added every three minutes in the 12- and 30-minute groups. After exposure, each specimen was gently washed with saline for 1 minute and then examined with viability staining and CLSM as described previously [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eConfocal Laser Scanning Microscopy Examination\u003c/h2\u003e \u003cp\u003eThe LIVE/DEAD BacLight Bacterial Viability kit L-7012 (Molecular Probes, Eugene, OR, USA) containing SYTO 9 and propidium iodide (PI) was used to stain the \u003cem\u003eE. faecalis\u003c/em\u003e biofilm cells in bovine dentin discs according to the manufacturer's instructions. The excitation/emission maxima for the two dyes are approximately 480/500 nm for SYTO 9 and 490/635 nm for PI. Bacteria with intact cell membranes stain fluorescent green by SYTO 9, whereas bacteria with damaged membranes stain red by PI. All of the specimens were rinsed with phosphate-buffered saline for 1 minute, and then viewed immediately by CLSM (Olympus FV1200, Olympus, Japan) using a 40\u0026times; lens. Five randomly selected areas (0.3 mm \u0026times; 0.3 mm for each area) on bacterial biofilms were examined by CLSM and 3D reconstruction analysis in each bovine disc per time point.\u003c/p\u003e \u003cp\u003eThe CLSM images were captured using Fluoview version 4.3 software (Olympus, Melville, NY, USA) at a resolution of 512 \u0026times; 512 pixels. A stack of 15 slices (1-\u0026micro;m step size) was scanned at each randomly chosen area on 1-week-old biofilms, and the scanning depth for 3-week-old biofilms was set at 50 \u0026micro;m. The dead cell volume (red fluorescence) and live cell volume (green fluorescence) were reconstructed into a 3D model and the portion of dead cell volume was analyzed by using the Imaris 7.2 software (Bitplane Inc, St Paul, MN).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eVisible Changes in Structure of Bovine Dentin Discs\u003c/h2\u003e \u003cp\u003eSix uninfected bovine dentin discs were randomly subjected to aPDT with MB for 12 and 30 minutes as described previously. There were three samples in each group at the same exposure time. After 1-day-old incubation, the dentin specimens were washed for 1 minutes using saline. The visible structural changes in three uninfected bovine dentin blocks and six aPDT-treated specimens were observed by SEM.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe proportions of dead cell volume in \u003cem\u003eE. faecalis\u003c/em\u003e biofilms were presented as the means and standard deviations (means\u0026thinsp;\u0026plusmn;\u0026thinsp;SDs). Differences between the proportions of dead cell volume were subjected to univariate analysis of variance using SPSS 22.0 software (SPSS Inc, Chicago, IL, USA). Post hoc multiple comparisons were used to isolate and compare the results at a significance level of P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSEM Images of\u003c/b\u003e \u003cb\u003eE. faecalis\u003c/b\u003e \u003cb\u003eBiofilms on Dentin Blocks\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSEM images of sterile dentin blocks showed a clean dentin surface, removal of the smear layer, the opening of dentinal tubules, and absence of microbial and debris attachment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). A large volume of bacterial cells was observed by SEM, with the presence of \u003cem\u003eE. faecalis\u003c/em\u003e biofilms on the dentin surface and dentinal tubule openings, and even inside tubules. A heavier infection and more small clusters of \u003cem\u003eE. faecalis\u003c/em\u003e cells on 3-week-old biofilms were found than that on 1-week-old biofilms (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCLSM Images of\u003c/b\u003e \u003cb\u003eE. faecalis\u003c/b\u003e \u003cb\u003eBiofilms Treated with aPDT\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAfter 1-week-old and 3-week-old incubation with \u003cem\u003eE. faecalis\u003c/em\u003e young and old biofilm cells grown well in the bovine dentin blocks were verified by CLSM and 3D reconstructions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). There were many dead cells (red fluorescence) in \u003cem\u003eE. faecalis\u003c/em\u003e biofilms on dentin discs subjected to aPDT with MB and 5% NaOCl at each time point, while the majority of live cells (green fluorescence) were presented on bacterial biofilms after saline irrigation. aPDT with MB and 5% NaOCl killed about 35%-81% of bacteria cells in dentin samples, and approximately 7%-10% of the cells were dead in the control group (saline) in 1- and 3-week-old biofilms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe proportion of dead \u003cem\u003eE. faecalis\u003c/em\u003e cell volume depended on the exposure time, treatment, and age of the bacterial biofilm (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Significantly more bacteria were dead when aPDT with MB and 5% NaOCl were used with long exposure time (12 and 30 minutes) than after 3 minutes (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The speed of killing was fastest during the first 3 minutes of exposure (about 11.65%-20.76% killing per minute), slowed down strongly from 3 minutes to 12 minutes (1.81%-2.92% killing per minute), and was negligible from 12 minutes to 30 minutes (0.14%-0.25% killing per minute) in the disinfection groups. Five percent NaOCl exhibited the highest effectiveness by killing 50.40%-81.38% of bacteria in dentin at each time-point compared to 34.96%-70.96% killing in the aPDT with MB groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The amount of killing was 7.13\u0026ndash;11.88% higher in 1-week-old bacterial cells (young biofilms) than in 3-week-old cells (old biofilms) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\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\u003eProportion of Dead \u003cem\u003eE. faecalis\u003c/em\u003e Cell Volume in Bovine Dentin Blocks (1- and 3-week-old Biofilms) Exposed to Different Disinfecting Solutions for 3, 12, or 30 Minutes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePDT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e5% NaOCl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e \u003cp\u003eSaline\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1 wk\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 wk\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1 wk\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3 wk\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1 wk\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003e3 wk\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.96\u0026thinsp;\u0026plusmn;\u0026thinsp;4.18\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e62.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50.40\u0026thinsp;\u0026plusmn;\u0026thinsp;3.89\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7.37\u0026thinsp;\u0026plusmn;\u0026thinsp;3.35\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e9.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e12 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68.36\u0026thinsp;\u0026plusmn;\u0026thinsp;5.70\u003csup\u003ebe\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e61.23\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e78.58\u0026thinsp;\u0026plusmn;\u0026thinsp;2.59\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68.35\u0026thinsp;\u0026plusmn;\u0026thinsp;3.73\u003csup\u003ebe\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e9.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e30 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70.96\u0026thinsp;\u0026plusmn;\u0026thinsp;1.32\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e63.73\u0026thinsp;\u0026plusmn;\u0026thinsp;2.51\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e81.38\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e72.85\u0026thinsp;\u0026plusmn;\u0026thinsp;2.85\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8.80\u0026thinsp;\u0026plusmn;\u0026thinsp;3.94\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e9.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.80\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003eDifferent superscript letters indicate statistically significant differences between groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSEM Images of Visible Changes in Dentin Surfaces\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the homogenous dentinal tubules, but no residual microorganisms and smear layer in the dentin blocks in aPDT with MB groups and control group, which indicated that no visible changes in the structure of dentin surfaces were found after 12- and 30-minute exposure to aPDT with MB.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eEndodontic diseases are an inflammatory reaction of pulpal and periradicular tissues caused by bacteria and their by-products in the root canal systems. Actually, micro-organisms in the infected root canal systems grow mostly in sessile biofilms, which are more resistant to various antibacterial approaches in endodontic therapies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. \u003cem\u003eE. faecalis\u003c/em\u003e was chosen as the test organism in many preliminary studies because of its frequent presence in root-filled teeth with persistent periapical lesions, and its ability to attach itself to dentin, invade dentinal tubules and form biofilm communities [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Therefore, many researchers have established \u003cem\u003eE. faecalis\u003c/em\u003e biofilms in different growing environment in vitro in order to evaluate the killing activity of irrigating solutions during root canal disinfection [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Haapasalo and Orstavik inoculated \u003cem\u003eE. faecalis\u003c/em\u003e suspensions onto cylindrical bovine dentin spencimens for 3-week-old culturing, and found a heavy infection that reached 400 microns from the canal lumen [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Kishen et al. showed the maximum reduction of bacterial cells in \u003cem\u003eE. faecalis\u003c/em\u003e biofilms after instrumentation-syringe irrigation analyzed using SEM, quantitative reverse transcriptase real-time PCR (qRT-PCT) and CLSM [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Accordingly, \u003cem\u003eE. faecalis\u003c/em\u003e biofilms were introduced on bovine dentin to assess the killing efficacy of aPDT with 0.01% MB in the present study. To correlate well with the actual clinic setting, this study is the first time that \u003cem\u003eE. faecalis\u003c/em\u003e biofilms in dentin canals have been established after 1- and 3-week incubation periods to test the killing activity of aPDT with MB with long-term exposure as a newer disinfection strategy with using CLSM and analyzing dead portions of bacterial biofilms.\u003c/p\u003e \u003cp\u003eIn recent decades, many researchers have considered aPDT as an alternative supplemental approach to improve root canal disinfection because conventional chemo-mechanical procedures may not be able to remove all of the bacterial cells and obtain a complete cleaning of the root canal systems [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The results regarding the antibacterial effect of aPDT against \u003cem\u003eE. faecalis\u003c/em\u003e biofilms in dentin blocks in vitro were obtained in the present study are consistent with those of previous studies. According to the proportions of dead bacterial cell volume in 3D reconstruction images, the aPDT with 0.01% MB treatment killed 43.07%-70.96% of the bacterial cells on 1-week-old biofilms and 34.96%-63.73% of cells on 3-week-old \u003cem\u003eE. faecalis\u003c/em\u003e biofilms, although this was a little lower than that seen with 5% NaOCl for the same exposure time. It has been reported that MB (a non-toxic dye) as a photosensitizer has the ability to absorb energy from a light source and transfer this energy to another molecule producing reactive oxygen species [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. When aPDT was present, these cytotoxic singlet oxygen species induced damage of the cellular plasma membrane and/or the cell DNA to play a key role in the bactericidal action. The time elapsed between the photosensitizer reaching into dentinal tubules and the actual photo-activation is called \"pre-irradiation time\" ranging from 5 to 15 minutes, which allows the photosensitizer to penetrate inside bacterial biofilms and obtain more light absorption [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Hence, five minutes as a pre-irradiation time was set in this study to help keep 0.01% MB inside \u003cem\u003eE. faecalis\u003c/em\u003e biofilms and exert its antibacterial effect.\u003c/p\u003e \u003cp\u003eResults from the present study showed that the killing activity of aPDT and 5% NaOCl depended on exposure time. NaOCl was selected as a disinfection agent because these are the most commonly used antimicrobial irrigating solutions during root canal disinfection. During the first 3 minutes of exposure, the killing speed of bacterial cells was approximately 11.65%-20.76% per minute, while after the first 3 minutes to 12 minutes, the killing effect against bacterial biofilms continued but at a slower speed (1.81%-2.92% per minute). After 12 minutes, the killing activity of both aPDT and NaOCl was poor and only small amounts of new bacteria were killed between 12 and 30 minutes. Limited penetration depth and persisted cells of bacteria in the biofilm may be reasons for the incomplete killing even after 30 minutes of exposure [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In accordance with previous studies, bacteria in young biofilms in dentin were more susceptible to disinfecting approaches than cells in old biofilms. Wang et al. indicated that the physical barrier created by the extracellular polymeric matrix (EPS), the viable but nonculturable (VBNC) state of old biofilm bacteria, and persister cells all contributed to the higher resistance of bacteria in mature biofilms [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne of the concerns about antibacterial photodynamic therapy inside the root canals is the energy transformation during the interaction with dentinal surfaces, which could lead to changes of dentinal structures and a temperature increase in the root canal systems. In the present study, the structure of dentine surfaces subjected to aPDT with 0.01% MB up to 30 min was not visibly different from samples from the untreated control group using SEM observation. These results were in agreement with those described by Alfredo et al. in their study, in which they showed application of laser in the root at 1.5 W in all operating modes, and 3.0 W in the pulsed mode for 20s could be safely used in endodontic treatment [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Although the studies used a different type of light source, the total obtained energies were very close to 30 J to 60 J.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe results of this study indicated that aPDT with 0.01% MB has the capability to kill bacterial cells in \u003cem\u003eE. faecalis\u003c/em\u003e biofilms on bovine dentin, and does not result in visible changes of dentin structures. The antibacterial effect was time dependent, but no remarkable additional killing was obtained after the first 12 minutes of exposure. Five percent NaOCl was more effective against \u003cem\u003eE. faecalis\u003c/em\u003e biofilms in dentin than aPDT at the same exposure time. Young \u003cem\u003eE. faecalis\u003c/em\u003e biofilms in dentin canals were more susceptible to disinfection approaches than mature biofilms.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eE. faecalis\u003c/em\u003e:\u0026nbsp;\u003cem\u003eEnterococcus faecalis\u003c/em\u003e; aPDT: antibacterial photodynamic therapy； ROS: reactive oxygen species；CLSM: confocal laser scanning microscopy；BHI: brain-heart infusion；PI: propidium iodide；qRT-PCT: real-time PCR；EPS extracellular polymeric matrix；VBNC: viable but nonculturable\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eThe study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Science and Ethics Commission of the First Affiliated Hospital of Zhengzhou University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used for the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (No. 81500847 to Tianfeng Du), and the Medical Science and Technology Project of Henan Province (No. 201702042 to Juanfang Zhu).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTianfeng Du and Yiting Wang contributed equally to this study. Conceptualization, Tianfeng Du and Yiting Wang; Funding acquisition, Tianfeng Du and Juanfang Zhu; Methodology, Tianfeng Du, Yiting Wang and Xiaoke Liu; Software, Bowen Yang and Kang Gan; Supervision, Juanfang Zhu; Writing-original draft, Tianfeng Du; Writing-review and editing, Tianfeng Du. All authors have read and agreede to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKakehashi S, Stanley HR, Fitzgerald RJ: \u003cstrong\u003eThe effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats\u003c/strong\u003e.\u0026nbsp;\u003cem\u003eOral Surg Oral Med Oral Pathol\u0026nbsp;\u003c/em\u003e1965, \u003cstrong\u003e20\u003c/strong\u003e:340-349.\u003c/li\u003e\n \u003cli\u003eFulghum RS,\u0026nbsp;Wiggins\u0026nbsp;CB,\u0026nbsp;Mullaney\u0026nbsp;TP:\u0026nbsp;\u003cstrong\u003ePilot study for detecting obligate anaerobic bacteria in necrotic dental pulps\u003c/strong\u003e.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cem\u003eJ\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eDent\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eRes\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e1973, 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\u003cstrong\u003eEffectiveness of endodontic disinfecting solutions against young and old \u003cem\u003eEnterococcus faecalis\u003c/em\u003e biofilms in dentin canals\u003c/strong\u003e. \u003cem\u003eJ Endod\u0026nbsp;\u003c/em\u003e2012, \u003cstrong\u003e38\u003c/strong\u003e(10):1376-1379.\u003c/li\u003e\n \u003cli\u003eAlfredo E, Marchesan MA, Sousa-Neto MD, Brugnera-J\u0026uacute;nior A, Silva-Sousa YT: \u003cstrong\u003eTemperature variation at the external root surface during 980-nm diode laser irradiation in the root canal\u003c/strong\u003e. \u003cem\u003eJ Dent\u003c/em\u003e 2008, \u003cstrong\u003e36\u003c/strong\u003e(7):529-534.\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":"Bacterial biofilms, photodynamic therapy, Enterococcus faecalis, confocal laser scanning microscopy, root canal disinfection","lastPublishedDoi":"10.21203/rs.3.rs-4643951/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4643951/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e Antibacterial photodynamic therapy - aPDT is a medical method that utilizes the activation of a nontoxic photoactive agent or photosensitizer by exposure to visible light of a specific wave-length in the presence of oxygen. The present study aimed to evaluate the killing in vitro effect of aPDT with 0.01% methylene blue (MB) against young and old \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (\u003cem\u003eE. faecalis\u003c/em\u003e) biofilms in bovine dentin with the long-term exposure using confocal laser scanning microscopy (CLSM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Semicylindrical bovine dentin blocks were inoculated with \u003cem\u003eE. faecalis\u003c/em\u003e and incubated in air to form 1- and 3-week-old biofilms. The biofilms in dentin were subjected to aPDT with 0.01% MB, 5% NaOCl and saline with the exposure of 3, 12 and 30 minutes. The dead portions of bacterial cells in \u003cem\u003eE. faecalis\u003c/em\u003e biofilms were analyzed with using LIVE/DEAD bacteria viability staining and CLSM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe visible changes in dentin structure caused by aPDT were verified with scanning electron microscopy. Significantly more bacteria were dead when aPDT with MB and 5% NaOCl were used with the long exposure time (12 and 30 minutes) than with 3 minutes (P \u0026lt; 0.05). The speed of killing was fastest during the first 3 minutes, and few more bacterial cells were killed after 12 minutes in the disinfection groups. Five percent NaOCl exhibited the highest effectiveness of bacterial killing in dentin at each time point than aPDT with MB groups (P \u0026lt; 0.05). The proportion of killed bacteria was higher in young biofilms than in mature biofilms in aPDT with MB and NaOCl groups (P \u0026lt; 0.05). Moreover, there were no clearly visible changes in structure of dentin surfaces subjected to aPDT with MB for 30 minutes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eaPDT with 0.01% MB has the capability to kill bacterial cells in \u003cem\u003eE. faecalis\u003c/em\u003e biofilms on bovine dentin, and does not result in visible changes of dentin structures. The antibacterial effect was time-dependent, but little additional killing was obtained after the first 12 minutes of exposure.\u003c/p\u003e","manuscriptTitle":"Killing effect of antibacterial photodynamic therapy - aPDT with long-term exposure against young and old Enterococcus faecalis biofilms in dentin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-22 10:04:21","doi":"10.21203/rs.3.rs-4643951/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-22T08:11:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-18T09:18:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-15T19:55:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-12T15:34:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-10T08:21:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"164724762326647388036815390142135789426","date":"2024-07-09T08:32:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223977300261292674163944210380022579709","date":"2024-07-07T10:23:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"333926166838009841033536687138052014991","date":"2024-07-05T17:06:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"223322354575504776595620801793460059913","date":"2024-07-05T05:51:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-05T05:19:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-07-04T08:17:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-28T07:20:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-28T07:19:40+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2024-06-26T16:04:15+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":"260b7438-4acc-4081-be1b-035d4351b084","owner":[],"postedDate":"July 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-24T16:05:32+00:00","versionOfRecord":{"articleIdentity":"rs-4643951","link":"https://doi.org/10.1186/s12903-025-05657-4","journal":{"identity":"bmc-oral-health","isVorOnly":false,"title":"BMC Oral Health"},"publishedOn":"2025-02-22 15:57:18","publishedOnDateReadable":"February 22nd, 2025"},"versionCreatedAt":"2024-07-22 10:04:21","video":"","vorDoi":"10.1186/s12903-025-05657-4","vorDoiUrl":"https://doi.org/10.1186/s12903-025-05657-4","workflowStages":[]},"version":"v1","identity":"rs-4643951","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4643951","identity":"rs-4643951","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}