A Horizontal Sequential Cutting Method to Estimate the Effectiveness of Dentin Disinfection by Using Confocal Laser Scanning Microscopy |
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| Institution: | 1. Division of Cariology, Operative Dentistry and Endodontics, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan;2. Division of Microbiology and Infectious Diseases, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan;3. Division of Anatomy and Cell Biology of the Hard Tissue, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan;1. Department of Endodontics, US Army Dental Health Activity, Fort Gordon, Georgia;2. Uniformed Services University of the Health Sciences Postgraduate Dental College, Bethesda, Maryland;3. Department of Endodontics, Dental College of Georgia at Augusta University, Augusta, Georgia;4. Department of Clinical Investigations, Dwight D. Eisenhower Army Medical Center, Fort Gordon, Georgia;1. Department of Conservative Dentistry and Endodontics, University of Cagliari, Cagliari, Italy;2. Department of Endodontology, Academic Centre for Dentistry Amsterdam, Amsterdam, The Netherlands;1. Unit of Endodontology, Division of Restorative Dental Science, University College London Eastman Dental Institute, London, United Kingdom;2. Division of Biomaterials and Tissue Engineering, University College London Eastman Dental Institute, London, United Kingdom;3. The Discoveries Centre for Regenerative and Precision Medicine, University College London, London, United Kingdom;4. Department of Nanobiomedical Science and BK21 Plus NBM, Global Research Center for Regenerative Medicine, Dankook University, Cheonan, Republic of Korea;6. University College London Eastman-Korea Dental Medicine Innovation Centre, Dankook University, Cheonan, Republic of Korea |
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| Abstract: | IntroductionThis study aimed to develop a technique to create sequential slices, allowing the fluorescent visualization of bacterial viability in all parts of an infected dentin.MethodsCylindrical dentin blocks were prepared from freshly extracted human teeth with a single-rooted canal. Each block was immersed in 5% sodium hypochlorite (NaOCl) and 17% EDTA for 5 minutes before being infected with Enterococcus faecalis. The bacteria were allowed to develop inside dentin specimens for 28 days under anaerobic conditions. The specimens were exposed in 2% NaOCl for either 2 minutes or 20 minutes at 20°C, 37°C, and 45°C, respectively. After staining with calcein AM (Thermo Fisher Scientific, Waltham, MA) and propidium iodide, the samples were cryoembedded, mounted on an adhesive film, and sectioned at a thickness of 10 μm along the running of the dentinal tubules. Stacks of fluorescent images were collected in the z dimension using confocal laser scanning microscopy, and the maximum affected distance from a root canal was measured from the 3-dimensional reconstructed image. The reliability of this technique was verified by comparison with a dye bleaching test.ResultsHorizontal sequential sections preserving 3-dimensional bacterial distribution and their viabilities could be made without decalcification. The treatment time contributed to the penetration of NaOCl into dentinal tubules, whereas temperature did not significantly affect the penetration. The judgment by confocal laser scanning microscopic analysis was consistent with that of a dye bleaching test.ConclusionsThe horizontal sectioning method has the advantage of creating sequential sections, allowing information to be imaged at every portion. |
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| Keywords: | Adhesive film method bacterial viability confocal laser scanning microscopy infected root canal |
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