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Is it really penetration? Locomotion of devitalized Enterococcus faecalis cells within dentinal tubules of bovine teeth
Archives of Oral Biology 83 (2017) 289–296
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Is it really penetration? Locomotion of devitalized Enterococcus faecalis cells within dentinal tubules of bovine teeth
MARK
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Jasmin Kirscha, , Sabine Baschea, Jörg Neunzehnb, Maria Dedea, Martin Dannemannc, Christian Hanniga, Marie-Theres Webera a
Clinic of Operative Dentistry, Medical Faculty Carl Gustav Carus, TU Dresden, Fetscherstr. 74, D-01307 Dresden, Germany Technische Universität Dresden, Institute of Material Science, Chair for Biomaterials, TU Dresden, Budapester Strasse 27, 01069 Dresden, Germany c Technische Universität Dresden, Institute of Lightweight Engineering and Polymer Technology (ILK), Faculty of Mechanical Engineering, Holbeinstrasse 3, Dresden, 01307, Germany b
A R T I C L E I N F O
A B S T R A C T
Keywords: Dentinal tubule Bacterial penetration E. faecalis Live/Dead staining DAPI
Objective: The aim of the present study was to evaluate the penetration characteristics of devitalized and vital E. faecalis cells into root dentinal tubules. Design: Thirteen root canals were incubated with devitalized (4 days, 7 days, 14 days, 28 days) and vital (28 days) E. faecalis strains (streptomycin-resistant strains) after root canal enlargement (size 80, taper 0.02) with 3 % NaOCl solution. The smear layer was intentionally removed with 20 % EDTA before inoculation. Samples were processed for analysis by scanning electron microscopy (SEM) and DAPI (4′,6-diamidino-2-phenylindole) staining. DAPI was conducted for fluorescence microscopic visualization of the bacterial penetration into dentinal tubules. The penetration depth was calculated with the measurement tool of the Axio Vision program (Zeiss, Jena, Germany). Results: Devitalized E. faecalis strains were able to penetrate into dentinal tubules of the root canal. Apikal penetration depths of the devitalized cells were 100.67 μm ± 26.54 μm after 7 days, 230.67 μm ± 111.5 μm after 14 days and 266.5 μm ± 92.63 μm after 28 days of incubation. The total number and penetration depth of E. faecalis cells was lower compared to a vital suspension of E. faecalis (1002.45 μm) after 28 days. It was noted that bacterial penetration was not common to all of the dentinal tubules in the vital E. faecalis control and especially in the devitalized control. Conclusions: Increased exposure times of devitalized bacteria into root canals lead to an increased number of penetrated dentinal tubules as well as to a deeper penetration.
1. Introduction
Normally, oral bacteria cannot bind directly to hydroxyapatite unless it is modified (Love, 2002). Dentinal tubules contain several tissue molecules that may allow bacterial adhesion, colonization, and invasion of these microorganisms (Love, 2002). The results of the study of Love et al. suggest that reinfection of a treated root canal might be associated with the ability of E. faecalis to remain viable within the tubule system even after the root canal filling, thereby maintaining their ability to migrate into dentinal tubules (Love, 2002). Interestingly, even though they are not motile these microorganisms can still penetrate a significant depth (Perez, Calas, de Falguerolles, & Maurette, 1993). There are different types of models why and how bacteria penetrate dentinal tubules. Perez et al. explained the progression of microorganisms by an active phenomenon, which is based on a regular rate of migration and multiplication instead of a passive slow culture medium penetration through the tubules (Perez, Calas, & Rochd, 1996). Other
To achieve the best possible result of an endodontic treatment, bacterial populations withi;1;n the root canal should be either removed completely or significantly reduced in order to allow periradicular tissue healing (Siqueira & Rocas, 2008). Intraradicular infection is one reason for failure of the endodontic treatment (Nair, Sjogren, Krey, Kahnberg, & Sundqvist, 1990) as bacteria have to resist root canal disinfection methods and are able to adjust to the environmental conditions in the root canal after instrumentation (Siqueira & Rocas, 2008). Enterococcus faecalis (E. faecalis) is thereby the most commonly found species in root canal treated teeth with persistent disease (Engström, 1964; Gomes et al., 2008; Haapasalo, Ranta, & Ranta, 1983; Molander et al., 1998; Pinheiro et al., 2003; Rocas, Jung, Lee, & Siqueira, 2004; Siqueira & Rocas, 2004; Sundqvist, Figdor, Persson, & Sjogren, 1998).
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Corresponding author. E-mail addresses: [email protected], [email protected] (J. Kirsch).
http://dx.doi.org/10.1016/j.archoralbio.2017.08.012 Received 3 May 2017; Received in revised form 10 August 2017; Accepted 21 August 2017 0003-9969/ © 2017 Elsevier Ltd. All rights reserved.
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hypochlorite (3 %, Transcodent GmbH & Co.KG, Kiel, Germany). The smear layer was intentionally removed by a 20 % EDTA-solution (1 min rinsing with 2 ml) and a final rinse with 5 ml sodium chloride (0.9 %) took place. Lastly, the teeth were cleaned in an ultrasonic bath with 20 % EDTA (10 min) and Aqua dest. (1 h). Afterwards, the teeth were placed in an ultrasonic bath for 10 min with tryptic soy broth (TSB), followed by autoclaving (10 min, 121 °C). To check the sterility of the samples, the teeth were incubated in TSB for two days at 37 °C. Afterwards, the teeth were embedded in a 5 ml Eppendorf tube (Eppendorf AG, Hamburg, Germany) with 3 % agarose.
hypothesis are multiplication processes (Morse, 1971). or reinfection of endodontically treated teeth by leakage (Peters, Wesselink, & Moorer, 2000). Thereby, bacterial penetration into the dentinal tubules is going on to a certain extent, not all dentinal tubules are penetrated by bacteria. One reason could be that the cells tend to form colonies and thereby the penetration of dentinal tubules is partially blocked (Perez et al., 1993). In addition, the odontoblastic prolongment inside the dentinal tubules represents a potential barrier. Besides, the presence of a smear layer prevents penetration of bacteria into the tubules too. In order to study the effect of different treatment procedures, it is necessary to know more about these possible ways of infection into the dentinal tubules, including the penetration process and the amount of present bacteria. Why single bacterial cells can be found spread out in the tubule system without obvious nutrient sources needs to be examined. In vitro and in vivo studies have shown that bacteria can penetrate up to 2000 μm into the dentinal tubules (Ando & Hoshino, 1990; Berkiten, Okar, & Berkiten, 2000; Horiba, Maekawa, Matsumoto, & Nakamura, 1990; Love, 1996; Meryon & Brook, 1990; Orstavik & Haapasalo, 1990; Peters et al., 2001; Peters, Wesselink, & Moorer, 1995; Peters et al., 2000; Sen, Piskin, & Demirci, 1995) and that they remain viable for a period of time. Most studies have used various techniques to visualize bacteria in dentinal tubules, including transmission and scanning electron microscopy, light microscopy, and culturing techniques (Haapasalo, 1989; Siqueira, De Uzeda, & Fonseca, 1996; Sundqvist, 1976). On average, these results are 20 to 30 years old. To date, no one has investigated the penetration ability of viable and dead bacteria into dentinal tubules. It is particularly important to verify whether or not the examined bacteria are viable. When investigating the success of endodontic irrigants, microscopic approaches often verify the existence of bacteria in the dentinal tubules. It may then be questioned whether the bacteria in the dentinal tubules will survive after the instrumentation and obturation and if they can multiply and grow to sufficient numbers and produce enough endotoxins to maintain or develop periapical inflammation (Peters et al., 1995). Otherwise, it is inconclusive as to whether or not the irrigant or endodontic treatment procedure was successful. A further goal of the present study was the question why bacteria are detectable at penetration depths above 2000 μm although they are not motile. Therefore, the migration might not be based on a multiplication process of the bacterial cells, but rather on diffusion. Although, devitalized bacteria cannot multiply, virulence factors such as lipoteichoic acid may still be harmful and could induce a defense response in the tissues surrounding the tooth (Hong et al., 2016). As there is no evidence whether or not dead bacteria can also penetrate dentinal tubules, the aim of the present study was to verify if devitalized E. faecalis cells could penetrate dentinal tubules as much as viable bacteria and how the results could affect bacterial findings with microscopic approaches. A new approach was used to detect the bacteria in the dentinal tubules via fluorescence staining with DAPI, a fluorescent dye to visualize bacteria by binding to AT-nucleic acids of double-stranded DNA, forming fluorescent units (Kensche, Basche, Bowen, Hannig, & Hannig, 2013; Morikawa & Yanagida, 1981).
2.2. Bacteria E. faecalis- isolates from patients with root canal treated teeth exhibiting secondary infection were used in the experiments. An E. faecalis strain with streptomycin resistance ( > 512 μg/ml) was chosen; thereby streptomycin (2 mg/ml) was added to the TSB to inhibit growth of other bacteria. After one hour at 95 °C, E. faecalis was still cultivable with the Colony forming units-method (CFU). The solution was serially diluted afterwards up to 1:106 in physiological sodium chloride solution and plated on TSB agar plates (aerobic and facultative anaerobic bacteria). The TSB plates were then incubated under aerobic conditions with 5 % CO2 for two days at 37 °C (Hannig et al., 2007). After two hours at 95 °C; E. faecalis cells were no longer cultivable with CFU and ready for the incubation in the root canal. In addition Live/Dead staining with the BacLight™ Bacterial Viability Kit (Invitrogen, Molecular probes, Darmstadt, Germany) was performed as described before (Hannig, Follo, Hellwig, & Al-Ahmad, 2010). 2.3. Infection of the root canals The root canals were inoculated E. faecalis culture medium (TSB, Merck, Darmstadt, Germany). Incubation periods for the DAPI-method were 4 days, 7 days, 14 days, and 28 days for devitalized E. faecalis strains (4 teeth, one tooth per incubation period) and 28 days for vital E. faecalis strains (2 teeth). Incubation periods for the SEM were 7 days, 14 days, and 21 days for devitalized E. faecalis strains (3 teeth, one tooth per incubation period) and 28 days for vital E. faecalis strains (4 teeth). The culture medium (TSB, Merck, Darmstadt, Germany) was changed every two days. The purity of the solution was tested regularly. Thereby, it was plated on agar plates and the absence of colony forming units could be monitored. 2.4. Preparation of the specimens for visualization techniques In order to terminate the test series, the teeth were fixated in 4 % formaldehyde (4 °C) for 24 h. Afterwards, the samples were fixated again in 50 % ethanol in PBS for 24 h at 4 °C. Shortly after, the teeth were decalcified with Osteosoft® (Merck, Darmstadt, Germany) for three weeks. The Osteosoft®-solution was changed every three days until the specimens were sliceable with a scalpel. Every root canal was cut into three pieces: a coronal-, medial-, and apical specimen (3 mm before the apex).
2. Materials & methods 2.5. Visualization of dentinal tubule infection and penetration 2.1. Root canal test specimens The specimens were dehydrated in an ascending series of ethanol, degreased by Xylol (Carl Roth GmbH Co. KG, Karlsruhe, Germany) and embedded in paraffin. Afterwards, they were cut with the Microtome (Leica Biosystems Nussloch GmbH, Germany) into 2 μm pieces. The object carrier was silanated, the test species were mounted on top, and the visualization with the DAPI method was performed. The calculation of the bacterial penetration depth took place with the Axio Vision program (Zeiss, Jena, Germany). Thereby, the distance between the entrance of the dentinal tubule and the penetrated bacterial cell was measured.
Thirteen freshly extracted bovine incisors of 3-year-old BSE-free cattle were used in the experiments. The teeth were sectioned transversely below the cemento-enamel junction at a root length of 20 mm using a low speed diamond saw (NTI-Kahla GmbH, Germany). An access cavity was created with a Gates Glidden bur #5 (Kerr, Rastatt, Germany). The root canals’ working length was determined at 19 mm using a K-file #15 (Dentsply Maillefer, Ballaigues, Switzerland). The root canal enlargement was manually prepared to size 80 and taper 0.02. After each instrument, the canal was rinsed with 10 ml sodium 290
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2.6. DAPI DAPI staining (Merck, Darmstadt, Germany) was conducted as described previously (Hannig et al., 2007; Hannig et al., 2013; Jung et al., 2010). DAPI (4′,6-diamidino-2-phenylindole) stains DNA unspecifically by binding to the AT-rich regions of double stranded DNA (Deimling et al., 2007), (Jung et al., 2010). Upon binding to DNA, the DAPI molecule fluoresces intensely at λ = 461 nm. First the samples were rinsed with 0.9 % sodium chloride. For staining, the samples were covered with DAPI stock solution (1.5 μl stock solution in 500 μl PBS (phosphate buffered saline)) in a dark chamber. After 15 min the DAPI solution was removed, and the samples were rinsed several times with PBS before fluorescence microscopic analysis took place (Kensche et al., 2013). Afterwards, the specimens were dried at room temperature and coated with Vectrashield mounting medium (Sigma-Aldrich, Taufkirchen, Germany) and analyzed by epifluorescence microscopy (Axioplan, Zeiss, Oberkochen, Germany). The root canal samples with the dentinal tubules were analyzed at 1000-fold magnification using the light filter for DAPI (BP 365, FT 395, LP 397). The area of ocular grid allowed visualization of the total length of the dentinal tubules. 2.7. Scanning electron microscopy For scanning electron microscopic investigation (SEM), the sectioned root canal specimens of 4 roots inoculated with vital E. faecalis cells and the sectioned root canal specimens of 3 roots inoculated with devitalized E. faecalis cells were fixed with glutaraldehyde, followed by dehydration in an ascending series of isopropanol and chemical drying through the iterative transfer into hexamethyldisilazane (HMDS). The samples were fixed on SEM stubs and sputtered with gold-palladium. Scanning electron microscopy was carried out using a Philips ESEM XL 30 in Hi-Vacuum mode by detecting secondary electrons for imaging.
Fig. 2. Representative scanning electron microscopic pictures (a, b) after 28 days of incubation. E. faecalis colonies adhere to the root canal walls and the entrances of the dentinal tubules. An arrow marks the cells within the state of cell division.
3. Results root canal. Representative SEM-images showed that devitalized E. faecalis cells were detectable at the entrance of the dentinal tubules after 7 days (Fig. 3a, arrow). After 14 days the cells had already migrated into the dentinal tubules (Fig. 3b) and after a 21-day period of incubation, no other cells were visible at the entrances of the dentinal tubules (Fig. 3c). Furthermore, the examination with the DAPI method gave additional insight into the penetration depth of the bacterial cells. Devitalized bacterial cells were distributed in small aggregates at the entrances of the dentinal tubules in the apical third of the root canal (Fig. 4a–c) after 4 days of incubation. Individual groups of bacteria penetrated to a depth of 100.67 μm ± 26.54 μm in the apical part of the root canal after 7 days of incubation (n = 3 measuring points per root canal section; Fig. 4f, i). After 14 days of incubation, the penetration depth of the dead bacteria ranged up to 258 μm (n = 1) in the medial part of the root canal and approximately 230.67 μm ± 111.5
Bacterial cells were traceable in all specimens with the DAPIMethod and scanning electron microscopy. The root canals inoculated with vital strains of E. faecalis were heavily infected and microorganisms were observed in all areas of the dentinal tubules (Figs. 2 and 6) with scanning electron microscopy (SEM). The visualization of the vital bacteria under the scanning electron microscope (Fig. 2) showed colonies of E. faecalis especially at the root canal walls and the entrances of the dentinal tubules (Fig. 2). Some bacteria were even in the process of cell division (Fig. 2). E. faecalis was difficult to devitalize. After one hour at 95 °C, E. faecalis was still cultivable with the Colony forming units-method (CFU). After two hours at 95 °C, E. faecalis cells were no longer cultivable with CFU, no longer visualisable under the microscope with the BacLight™ − Live-Dead-staining method (Syto® dye, green fluorescence, Fig. 1) and therefore ready for the incubation in the
Fig. 1. BacLight™ staining, typical example. A) Dead (red) E. faecalis cells are distributed randomly in small aggregates on the root canal surface. B) No vital (green) bacterial cells are detectable. Merely, one cell is situated in a dormant state (orange; Arrow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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4. Discussion During root canal treatment, bacteria spread into ramifications, dentinal tubules, isthmuses, and the apical delta of the root. In vivo, an infection of the root tubules starts from the pulpal side and microorganisms migrate through the tubules from the root canal. Peters et al. suggest that an in vitro model should allow bacteria to penetrate from the pulpal side only (Peters et al., 2000). Other studies show that the penetration from the cemental side is very infrequent (Safavi, Spangberg, & Langeland, 1990) and far less pronounced than from the pulpal side (Orstavik & Haapasalo, 1990). E. faecalis appears to be the best organism for experimental penetration into dentinal tubules leading to gross infections (Orstavik & Haapasalo, 1990; Peters et al., 2000). In addition, other studies have shown that E. faecalis is the most commonly found species in root canal treated teeth exhibiting persistent disease (Gomes et al., 2008; Molander et al., 1998; Pinheiro et al., 2003; Rocas et al., 2004; Siqueira & Rocas, 2004; Sundqvist et al., 1998). It may be concluded that E. faecalis is the most important species in endodontic treatment failures. However, Rocas et al. (2008) show that endodontic treatment failures are frequently associated with persistent mixed bacteria leading to secondary infection. When E. faecalis was present in species-specific PCR (polymerase chain reaction) in teeth with posttreatment apical periodontitis, it was never the dominant species and other taxa were disclosed (Rocas, Hulsmann, & Siqueira, 2008). Furthermore, Rôҫas et al. state that these mixed species (Streptococcus species, Lactobacillus species, Dialister Invisus, Eubacterium infirmum, Prevotella intermedia, Selenomonas sputigena) might be connected with the etiology of secondary infected apical periodontitis (Rocas et al., 2008). Formation of a thick biofilm was not detected in the present study – maybe due to the use of monocultures of E. faecalis instead of mixed culture mediums. Furthermore, the root canal was inoculated with the bacterial solution and afterwards the culture medium was changed every two days. For this reason, it is questionable if another bacterial species or a mixed species solution should have been used for the experiments in the present study or as an alternative, the bacterial solution could have been changed more frequently (Stauffacher, Lussi, Nietzsche, Neuhaus, & Eick, 2017). Nevertheless, this study was not designed to detect the occurrence of specific microbial species. This study was performed to answer the question of whether or not devitalized bacteria are still able to spread into dentinal tubules and if they progress deeper over time, implying a deeper penetration of the bacterial endotoxins. The present study clearly shows that devitalized bacteria are able to penetrate and progress into tubules. This is a vital factor for studies investigating the success of different endodontic treatment regimens and irrigants. The bacteria must be implicated into the dentinal tubules by external factors such as an osmotic process. The cell membrane is damaged during the devitalizing method at 95 °C for two hours as shown with the CFU and BacLight™ method. This procedure should represent devitalized bacteria after chemomechanical treatment of root canals in vivo, in which the cell membrane of E. faecalis becomes penetrable for water. Bacteria are highly proteinaceous cells. They absorb water when the cell membrane is damaged and the bacterial cells follow the concentration gradient to absorb more water. In the in vitro model, this influence is represented by the concentration gradient of the 3 % agarose gel. Since the agarose gel contains more water than the devitalized bacterial suspension in the root canal, the bacterial cells migrate into the dentinal tubules towards the agarose gel in a time dependent manner. The longer the incubation time, the deeper the penetration is into the dentinal tubules. Under in vivo conditions a concentration gradient towards the periodontal ligament also exists. During the root canal treatment, special irrigant solutions such as sodium hypochlorite and chlorhexidine work as antibacterial agents. The bacterial cells mainly die in that process. The present study shows that these devitalized cells are still able to penetrate into the dentinal tubules. When studies
Fig. 3. Representative scanning electron microscopic pictures after 7 days (a), 14 days (b) and 21 days (c) of incubation with devitalized E. faecalis cells. After 7 days, the cells are detectable at the entrance of the dentinal tubules (a, arrow). E. faecalis cells already migrated into the dentinal tubules after 14 days. A solid E. faecalis cell is attached to the root canal surface, no other cells are visible at the entrances of the dentinal tubules after a 21-day period of incubation.
μm in the apical part of the root canal (n = 3 measuring points; Fig. 5a, b). The results of 28 days of incubation with the devitalized cells show that the penetration depth of the bacteria ranged up to 210.75 ± 21.53 μm in the medial part of the root canal and approximately 266.5 μm ± 92.63 μm in the apical part of the root canal (n = 3 measuring points per root canal section; Fig. 5g). While bacterial aggregation at the entrances of the dentinal tubules increased, clusters of bacteria were found after all observed time periods (4 days to 28 days). The total number and penetration depth of E. faecalis cells was lower compared to a vital suspension of E. faecalis (1002.45 μm) after 28 days (Fig. 6a–c). It was noted that bacterial penetration was not common to all of the dentinal tubules in the vital E. faecalis control and especially in the devitalized control. 292
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Fig. 4. DAPI staining, typical examples of bacterial penetration into dentinal tubules after 4 days (a–c) and 7 days (d–i) of incubation with non-vital E. faecalis solution. After 4 days, bacteria (arrow) were distributed in small aggregates at the entrances of the dentinal tubules in the apical third of the root canal (a-c). An enlarged section of d-f is shown in g-i. Bacteria are marked with an arrow. Individual groups of bacteria penetrated to a depth of approximately 128 μm after 7 days (f, i).
strains (Peters et al., 2000). The removal of the smear layer in the present study took place with a 17 % EDTA irrigation solution and subsequently E. faecalis was inoculated into the root canal. With this protocol, vital E. faecalis cells penetrated deep into the root dentinal tubules (Fig. 6). This procedure is in good accordance with other studies (Akpata & Blechman, 1982; Orstavik & Haapasalo, 1990; Peters et al., 2000). The study of Peters et al. also examines the penetration of bacterial cells into dentinal tubules (Peters et al., 2000). They show that in 11 % of the specimens, no bacteria were found in the pulpal or middle grinding sample, whereas bacteria were detected in deeper apical layers. These findings are in good accordance with the present study. The highest number of bacteria penetrating into the tubules was also found in the apical part of the root canal. In addition, they report that light microscopy or SEM (scanning electron microscopy) show the presence of bacteria in tubules but fail to show viability of the organisms. The correlation between viable CFU from grinding samples and histological observations is therefore of interest. Another microscopic technique to detect bacteria is the Confocal Laser Scanning Microscopy (CLSM). This method is a very good approach to detect bacteria on relatively flat surfaces (Jung et al., 2010). However, the curved root canal and the curved dimensions of the dentinal tubules present a major challenge for the examination with the CLSM-method. In the present
investigate the success of new or popular irrigant agents, microscopic approaches such as light-, scanning- or transmission electron microscopy are used to visualize remaining bacteria in the dentinal tubules. The presence of bacteria within the dentinal tubules might be interpreted as a failure of the tested preparation regimen concluding that these bacteria resisted or escaped the intracanal disinfection procedure. However, the present study shows that this conclusion cannot be drawn since possible osmotic processes have an impact on the migration of bacteria into the dentinal tubules. Furthermore, no evidence exists regarding the vitality of the present bacteria since microscopic investigation including light-, scanning- and transmission electron microscopy only detect the presence of bacteria. The bacterial cells could have spread throughout the dentinal tubule system due to a carryover of bacteria during the splitting procedure of the roots. This carryover, however, would lead to an evenly distributed pattern of microorganisms throughout the root. Further, Figs. 4 and 5 show that the penetration of bacteria occured in a time dependent manner. An insufficient devitalization of the E. faecalis cells could certainly also lead to a deeper bacterial penetration over time. The CFU method was therefore performed to proof the devitalization of the bacteria. In an experimental setting, penetration of E. faecalis was found to be greatly enhanced when the smear layer was removed before inoculation with E. faecalis 293
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Fig. 5. DAPI staining, typical examples of bacterial penetration into dentinal tubules after 14 days (a–d) of incubation with nonvital E. faecalis solution. After 14 days, the penetration depth of the dead bacteria ranged between 137 μm and 354 μm (a, b). An enlarged section of a, and c is shown in b, and d. Bacteria are marked with an arrow. After 28 days, the penetration depth of the dead bacteria ranged between 197 μm and 332 μm (g). An enlarged section of e is shown in f. Bacteria are marked with an arrow.
however, it has not yet been carried out to detect bacteria in root canals. In contrast to electron microscopic gold-immunolabelling techniques, this fluorescence microscopic approach does not require timeconsuming embedding procedures and allows the direct visualization of bacterial cells. Another method to examine the bacterial penetration depth is described by the study of Ørstavik and Haapasalo, whom have found Ps. aeruginosa in bur samples of deeper dentine layers, but not in the SEM sections (Orstavik & Haapasalo, 1990). Moreover, with this method it is possible that bacteria were forced deeper into the dentinal tubules during this special grinding procedure. Furthermore, Peters et al. postulated that histological investigations of these bur samples always need to be combined with CFU methods (Peters et al., 2000). In their study, the middle and cemental grinding samples contain low numbers of CFU, whereas bacteria are not seen in histological sections. This is not surprising considering the field of vision under the microscope is limited and low numbers of CFU remain undetected. In contrast to the method described in our study, no bur samples are needed. Therefore, this methodological failure can be neglected. In addition, the
study, the DAPI- method was therefore used to visualize bacteria inside the dentinal tubules for the first time. DAPI stains both living and dead cells. During the devitalization procedure, the DNA of the lysed bacteria leaks out of the cell and is distributed in the surroundings. Therefore, the fluorescent of devitalized cells is less rich in contrast as in vital cells. Nevertheless, a clear differentiation between vital and devitalized cells with the DAPI-method is vague and results in unreliable findings. Hence, with the help of the BacLight™ method (Tawakoli, Al-Ahmad, Hoth-Hannig, Hannig, & Hannig, 2013) it was proven that the utilized cells were effectively devitalized, confirming that DAPI is a proper method to stain devitalized cells in dentinal tubules. Further, no grinding and bur samples of different dentine layers are necessary for examination. Using this fluorescence staining method, the bacterial penetration can be displayed throughout its entire distance under the light microscope (Figs. 4–6) without the use of destructive approaches. The DAPI method is already well known to detect bacteria in in situ biofilm samples (Hannig et al., 2013; Hertel et al., 2016; Kensche et al., 2013); 294
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Fig. 6. DAPI staining, typical examples of bacterial penetration into dentinal tubules after 28 days (a–c) of incubation with vital E. faecalis solution. After 28 days, the penetration depth of the vital bacteria reached up to a penetration depth of 1002.45 μm (c).
teeth are necessary and in preparation to carry out a statistical evaluation for proof of evidence.
combination with the CFU method is not required. Hence, the additional, time consuming and unprecise method is not necessary. Another fact regarding the present study that should be considered is that the diameter of bovine dentinal tubules is 4 to 7 μm and varies only slightly from the diameter of human dentinal tubules (3 to 7 μm) (Berkiten et al., 2000; Jung et al., 2010). The slightly larger diameter might favor an easier bacterial penetration into the tubules. Nevertheless, E. faecalis cells are 0.87 to 1.01 μm in diameter (Kokkinosa, Fasseas, Eliopoulos, & Kalantzopoulos, 1998); therefore it is conceivable that a bacterial penetration occurs to the same extent in human and bovine teeth. In addition, the diameter of bovine dentinal tubules decreases from the outer region towards the inner region of the tooth, which results in a cone-shaped configuration with the larger base at the outer end (Dutra-Correa, Anauate-Netto, & Arana-Chavez, 2007). In contrast, the human dentinal tubules are cone-shaped with their larger base at the pulpal side (Dutra-Correa et al., 2007). Since human and bovine teeth have an opposite relation in their dentinal tubules, the physiology of this complex structure may be different from each other (DutraCorrea et al., 2007). Furthermore, the degree of maturity of the tooth samples should also be taken into account (Perez et al., 1993). Immature teeth have wider entrances of the dentinal tubules than mature teeth. This is an important factor when colonizing these teeth with bacteria. Nevertheless, most studies discussing the topic of dentinal tubule penetration are based on bovine teeth specimens (Berkiten et al., 2000; Haapasalo & Orstavik, 1987; Perez et al., 1993; Siqueira et al., 1996) since they are available in large quantities and all teeth are from the same diet and age group of animals (Jung et al., 2010; Peters et al., 2000). In the present study, only thirteen bovine teeth were used for the experiments. This number is not sufficient to carry out a statistical evaluation. Through the results of the present study, important basic knowledge was gained to understand, interpret, and discuss bacterial findings in microscopic images. Further studies with a larger number of
5. Conclusions Increased exposure times (4 days to 28 days) of devitalized E. faecalis strains into root canals lead to an increased number of penetrated dentinal tubules as well as to a deeper penetration, and the fluorescent labelling technique DAPI is a valuable method to provide information about bacterial penetration into dentinal tubules. Conflict of interests The authors deny any conflicts of interest related to this study. This work was supported by the DFG (German Research Foundation; WE 5838/1-1, DA 1701/1-1). Except as disclosed above, the authors certify that they have no commercial associations that might represent a conflict of interest in connection with the submitted manuscript. Sources of funding This work was supported by the DFG (German Research Foundation; WE 5838/1-1, DA 1701/1-1). Ethical approval No ethical approval was necessary for the present study. Acknowledgements The authors deny any conflicts of interest related to this study. This work was supported by the DFG (German Research Foundation; WE 295
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Is it really penetration? Locomotion of devitalized Enterococcus faecalis cells within dentinal tubules of bovine teeth
MARK
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Jasmin Kirscha, , Sabine Baschea, Jörg Neunzehnb, Maria Dedea, Martin Dannemannc, Christian Hanniga, Marie-Theres Webera a
Clinic of Operative Dentistry, Medical Faculty Carl Gustav Carus, TU Dresden, Fetscherstr. 74, D-01307 Dresden, Germany Technische Universität Dresden, Institute of Material Science, Chair for Biomaterials, TU Dresden, Budapester Strasse 27, 01069 Dresden, Germany c Technische Universität Dresden, Institute of Lightweight Engineering and Polymer Technology (ILK), Faculty of Mechanical Engineering, Holbeinstrasse 3, Dresden, 01307, Germany b
A R T I C L E I N F O
A B S T R A C T
Keywords: Dentinal tubule Bacterial penetration E. faecalis Live/Dead staining DAPI
Objective: The aim of the present study was to evaluate the penetration characteristics of devitalized and vital E. faecalis cells into root dentinal tubules. Design: Thirteen root canals were incubated with devitalized (4 days, 7 days, 14 days, 28 days) and vital (28 days) E. faecalis strains (streptomycin-resistant strains) after root canal enlargement (size 80, taper 0.02) with 3 % NaOCl solution. The smear layer was intentionally removed with 20 % EDTA before inoculation. Samples were processed for analysis by scanning electron microscopy (SEM) and DAPI (4′,6-diamidino-2-phenylindole) staining. DAPI was conducted for fluorescence microscopic visualization of the bacterial penetration into dentinal tubules. The penetration depth was calculated with the measurement tool of the Axio Vision program (Zeiss, Jena, Germany). Results: Devitalized E. faecalis strains were able to penetrate into dentinal tubules of the root canal. Apikal penetration depths of the devitalized cells were 100.67 μm ± 26.54 μm after 7 days, 230.67 μm ± 111.5 μm after 14 days and 266.5 μm ± 92.63 μm after 28 days of incubation. The total number and penetration depth of E. faecalis cells was lower compared to a vital suspension of E. faecalis (1002.45 μm) after 28 days. It was noted that bacterial penetration was not common to all of the dentinal tubules in the vital E. faecalis control and especially in the devitalized control. Conclusions: Increased exposure times of devitalized bacteria into root canals lead to an increased number of penetrated dentinal tubules as well as to a deeper penetration.
1. Introduction
Normally, oral bacteria cannot bind directly to hydroxyapatite unless it is modified (Love, 2002). Dentinal tubules contain several tissue molecules that may allow bacterial adhesion, colonization, and invasion of these microorganisms (Love, 2002). The results of the study of Love et al. suggest that reinfection of a treated root canal might be associated with the ability of E. faecalis to remain viable within the tubule system even after the root canal filling, thereby maintaining their ability to migrate into dentinal tubules (Love, 2002). Interestingly, even though they are not motile these microorganisms can still penetrate a significant depth (Perez, Calas, de Falguerolles, & Maurette, 1993). There are different types of models why and how bacteria penetrate dentinal tubules. Perez et al. explained the progression of microorganisms by an active phenomenon, which is based on a regular rate of migration and multiplication instead of a passive slow culture medium penetration through the tubules (Perez, Calas, & Rochd, 1996). Other
To achieve the best possible result of an endodontic treatment, bacterial populations withi;1;n the root canal should be either removed completely or significantly reduced in order to allow periradicular tissue healing (Siqueira & Rocas, 2008). Intraradicular infection is one reason for failure of the endodontic treatment (Nair, Sjogren, Krey, Kahnberg, & Sundqvist, 1990) as bacteria have to resist root canal disinfection methods and are able to adjust to the environmental conditions in the root canal after instrumentation (Siqueira & Rocas, 2008). Enterococcus faecalis (E. faecalis) is thereby the most commonly found species in root canal treated teeth with persistent disease (Engström, 1964; Gomes et al., 2008; Haapasalo, Ranta, & Ranta, 1983; Molander et al., 1998; Pinheiro et al., 2003; Rocas, Jung, Lee, & Siqueira, 2004; Siqueira & Rocas, 2004; Sundqvist, Figdor, Persson, & Sjogren, 1998).
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Corresponding author. E-mail addresses: [email protected], [email protected] (J. Kirsch).
http://dx.doi.org/10.1016/j.archoralbio.2017.08.012 Received 3 May 2017; Received in revised form 10 August 2017; Accepted 21 August 2017 0003-9969/ © 2017 Elsevier Ltd. All rights reserved.
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hypochlorite (3 %, Transcodent GmbH & Co.KG, Kiel, Germany). The smear layer was intentionally removed by a 20 % EDTA-solution (1 min rinsing with 2 ml) and a final rinse with 5 ml sodium chloride (0.9 %) took place. Lastly, the teeth were cleaned in an ultrasonic bath with 20 % EDTA (10 min) and Aqua dest. (1 h). Afterwards, the teeth were placed in an ultrasonic bath for 10 min with tryptic soy broth (TSB), followed by autoclaving (10 min, 121 °C). To check the sterility of the samples, the teeth were incubated in TSB for two days at 37 °C. Afterwards, the teeth were embedded in a 5 ml Eppendorf tube (Eppendorf AG, Hamburg, Germany) with 3 % agarose.
hypothesis are multiplication processes (Morse, 1971). or reinfection of endodontically treated teeth by leakage (Peters, Wesselink, & Moorer, 2000). Thereby, bacterial penetration into the dentinal tubules is going on to a certain extent, not all dentinal tubules are penetrated by bacteria. One reason could be that the cells tend to form colonies and thereby the penetration of dentinal tubules is partially blocked (Perez et al., 1993). In addition, the odontoblastic prolongment inside the dentinal tubules represents a potential barrier. Besides, the presence of a smear layer prevents penetration of bacteria into the tubules too. In order to study the effect of different treatment procedures, it is necessary to know more about these possible ways of infection into the dentinal tubules, including the penetration process and the amount of present bacteria. Why single bacterial cells can be found spread out in the tubule system without obvious nutrient sources needs to be examined. In vitro and in vivo studies have shown that bacteria can penetrate up to 2000 μm into the dentinal tubules (Ando & Hoshino, 1990; Berkiten, Okar, & Berkiten, 2000; Horiba, Maekawa, Matsumoto, & Nakamura, 1990; Love, 1996; Meryon & Brook, 1990; Orstavik & Haapasalo, 1990; Peters et al., 2001; Peters, Wesselink, & Moorer, 1995; Peters et al., 2000; Sen, Piskin, & Demirci, 1995) and that they remain viable for a period of time. Most studies have used various techniques to visualize bacteria in dentinal tubules, including transmission and scanning electron microscopy, light microscopy, and culturing techniques (Haapasalo, 1989; Siqueira, De Uzeda, & Fonseca, 1996; Sundqvist, 1976). On average, these results are 20 to 30 years old. To date, no one has investigated the penetration ability of viable and dead bacteria into dentinal tubules. It is particularly important to verify whether or not the examined bacteria are viable. When investigating the success of endodontic irrigants, microscopic approaches often verify the existence of bacteria in the dentinal tubules. It may then be questioned whether the bacteria in the dentinal tubules will survive after the instrumentation and obturation and if they can multiply and grow to sufficient numbers and produce enough endotoxins to maintain or develop periapical inflammation (Peters et al., 1995). Otherwise, it is inconclusive as to whether or not the irrigant or endodontic treatment procedure was successful. A further goal of the present study was the question why bacteria are detectable at penetration depths above 2000 μm although they are not motile. Therefore, the migration might not be based on a multiplication process of the bacterial cells, but rather on diffusion. Although, devitalized bacteria cannot multiply, virulence factors such as lipoteichoic acid may still be harmful and could induce a defense response in the tissues surrounding the tooth (Hong et al., 2016). As there is no evidence whether or not dead bacteria can also penetrate dentinal tubules, the aim of the present study was to verify if devitalized E. faecalis cells could penetrate dentinal tubules as much as viable bacteria and how the results could affect bacterial findings with microscopic approaches. A new approach was used to detect the bacteria in the dentinal tubules via fluorescence staining with DAPI, a fluorescent dye to visualize bacteria by binding to AT-nucleic acids of double-stranded DNA, forming fluorescent units (Kensche, Basche, Bowen, Hannig, & Hannig, 2013; Morikawa & Yanagida, 1981).
2.2. Bacteria E. faecalis- isolates from patients with root canal treated teeth exhibiting secondary infection were used in the experiments. An E. faecalis strain with streptomycin resistance ( > 512 μg/ml) was chosen; thereby streptomycin (2 mg/ml) was added to the TSB to inhibit growth of other bacteria. After one hour at 95 °C, E. faecalis was still cultivable with the Colony forming units-method (CFU). The solution was serially diluted afterwards up to 1:106 in physiological sodium chloride solution and plated on TSB agar plates (aerobic and facultative anaerobic bacteria). The TSB plates were then incubated under aerobic conditions with 5 % CO2 for two days at 37 °C (Hannig et al., 2007). After two hours at 95 °C; E. faecalis cells were no longer cultivable with CFU and ready for the incubation in the root canal. In addition Live/Dead staining with the BacLight™ Bacterial Viability Kit (Invitrogen, Molecular probes, Darmstadt, Germany) was performed as described before (Hannig, Follo, Hellwig, & Al-Ahmad, 2010). 2.3. Infection of the root canals The root canals were inoculated E. faecalis culture medium (TSB, Merck, Darmstadt, Germany). Incubation periods for the DAPI-method were 4 days, 7 days, 14 days, and 28 days for devitalized E. faecalis strains (4 teeth, one tooth per incubation period) and 28 days for vital E. faecalis strains (2 teeth). Incubation periods for the SEM were 7 days, 14 days, and 21 days for devitalized E. faecalis strains (3 teeth, one tooth per incubation period) and 28 days for vital E. faecalis strains (4 teeth). The culture medium (TSB, Merck, Darmstadt, Germany) was changed every two days. The purity of the solution was tested regularly. Thereby, it was plated on agar plates and the absence of colony forming units could be monitored. 2.4. Preparation of the specimens for visualization techniques In order to terminate the test series, the teeth were fixated in 4 % formaldehyde (4 °C) for 24 h. Afterwards, the samples were fixated again in 50 % ethanol in PBS for 24 h at 4 °C. Shortly after, the teeth were decalcified with Osteosoft® (Merck, Darmstadt, Germany) for three weeks. The Osteosoft®-solution was changed every three days until the specimens were sliceable with a scalpel. Every root canal was cut into three pieces: a coronal-, medial-, and apical specimen (3 mm before the apex).
2. Materials & methods 2.5. Visualization of dentinal tubule infection and penetration 2.1. Root canal test specimens The specimens were dehydrated in an ascending series of ethanol, degreased by Xylol (Carl Roth GmbH Co. KG, Karlsruhe, Germany) and embedded in paraffin. Afterwards, they were cut with the Microtome (Leica Biosystems Nussloch GmbH, Germany) into 2 μm pieces. The object carrier was silanated, the test species were mounted on top, and the visualization with the DAPI method was performed. The calculation of the bacterial penetration depth took place with the Axio Vision program (Zeiss, Jena, Germany). Thereby, the distance between the entrance of the dentinal tubule and the penetrated bacterial cell was measured.
Thirteen freshly extracted bovine incisors of 3-year-old BSE-free cattle were used in the experiments. The teeth were sectioned transversely below the cemento-enamel junction at a root length of 20 mm using a low speed diamond saw (NTI-Kahla GmbH, Germany). An access cavity was created with a Gates Glidden bur #5 (Kerr, Rastatt, Germany). The root canals’ working length was determined at 19 mm using a K-file #15 (Dentsply Maillefer, Ballaigues, Switzerland). The root canal enlargement was manually prepared to size 80 and taper 0.02. After each instrument, the canal was rinsed with 10 ml sodium 290
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2.6. DAPI DAPI staining (Merck, Darmstadt, Germany) was conducted as described previously (Hannig et al., 2007; Hannig et al., 2013; Jung et al., 2010). DAPI (4′,6-diamidino-2-phenylindole) stains DNA unspecifically by binding to the AT-rich regions of double stranded DNA (Deimling et al., 2007), (Jung et al., 2010). Upon binding to DNA, the DAPI molecule fluoresces intensely at λ = 461 nm. First the samples were rinsed with 0.9 % sodium chloride. For staining, the samples were covered with DAPI stock solution (1.5 μl stock solution in 500 μl PBS (phosphate buffered saline)) in a dark chamber. After 15 min the DAPI solution was removed, and the samples were rinsed several times with PBS before fluorescence microscopic analysis took place (Kensche et al., 2013). Afterwards, the specimens were dried at room temperature and coated with Vectrashield mounting medium (Sigma-Aldrich, Taufkirchen, Germany) and analyzed by epifluorescence microscopy (Axioplan, Zeiss, Oberkochen, Germany). The root canal samples with the dentinal tubules were analyzed at 1000-fold magnification using the light filter for DAPI (BP 365, FT 395, LP 397). The area of ocular grid allowed visualization of the total length of the dentinal tubules. 2.7. Scanning electron microscopy For scanning electron microscopic investigation (SEM), the sectioned root canal specimens of 4 roots inoculated with vital E. faecalis cells and the sectioned root canal specimens of 3 roots inoculated with devitalized E. faecalis cells were fixed with glutaraldehyde, followed by dehydration in an ascending series of isopropanol and chemical drying through the iterative transfer into hexamethyldisilazane (HMDS). The samples were fixed on SEM stubs and sputtered with gold-palladium. Scanning electron microscopy was carried out using a Philips ESEM XL 30 in Hi-Vacuum mode by detecting secondary electrons for imaging.
Fig. 2. Representative scanning electron microscopic pictures (a, b) after 28 days of incubation. E. faecalis colonies adhere to the root canal walls and the entrances of the dentinal tubules. An arrow marks the cells within the state of cell division.
3. Results root canal. Representative SEM-images showed that devitalized E. faecalis cells were detectable at the entrance of the dentinal tubules after 7 days (Fig. 3a, arrow). After 14 days the cells had already migrated into the dentinal tubules (Fig. 3b) and after a 21-day period of incubation, no other cells were visible at the entrances of the dentinal tubules (Fig. 3c). Furthermore, the examination with the DAPI method gave additional insight into the penetration depth of the bacterial cells. Devitalized bacterial cells were distributed in small aggregates at the entrances of the dentinal tubules in the apical third of the root canal (Fig. 4a–c) after 4 days of incubation. Individual groups of bacteria penetrated to a depth of 100.67 μm ± 26.54 μm in the apical part of the root canal after 7 days of incubation (n = 3 measuring points per root canal section; Fig. 4f, i). After 14 days of incubation, the penetration depth of the dead bacteria ranged up to 258 μm (n = 1) in the medial part of the root canal and approximately 230.67 μm ± 111.5
Bacterial cells were traceable in all specimens with the DAPIMethod and scanning electron microscopy. The root canals inoculated with vital strains of E. faecalis were heavily infected and microorganisms were observed in all areas of the dentinal tubules (Figs. 2 and 6) with scanning electron microscopy (SEM). The visualization of the vital bacteria under the scanning electron microscope (Fig. 2) showed colonies of E. faecalis especially at the root canal walls and the entrances of the dentinal tubules (Fig. 2). Some bacteria were even in the process of cell division (Fig. 2). E. faecalis was difficult to devitalize. After one hour at 95 °C, E. faecalis was still cultivable with the Colony forming units-method (CFU). After two hours at 95 °C, E. faecalis cells were no longer cultivable with CFU, no longer visualisable under the microscope with the BacLight™ − Live-Dead-staining method (Syto® dye, green fluorescence, Fig. 1) and therefore ready for the incubation in the
Fig. 1. BacLight™ staining, typical example. A) Dead (red) E. faecalis cells are distributed randomly in small aggregates on the root canal surface. B) No vital (green) bacterial cells are detectable. Merely, one cell is situated in a dormant state (orange; Arrow). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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4. Discussion During root canal treatment, bacteria spread into ramifications, dentinal tubules, isthmuses, and the apical delta of the root. In vivo, an infection of the root tubules starts from the pulpal side and microorganisms migrate through the tubules from the root canal. Peters et al. suggest that an in vitro model should allow bacteria to penetrate from the pulpal side only (Peters et al., 2000). Other studies show that the penetration from the cemental side is very infrequent (Safavi, Spangberg, & Langeland, 1990) and far less pronounced than from the pulpal side (Orstavik & Haapasalo, 1990). E. faecalis appears to be the best organism for experimental penetration into dentinal tubules leading to gross infections (Orstavik & Haapasalo, 1990; Peters et al., 2000). In addition, other studies have shown that E. faecalis is the most commonly found species in root canal treated teeth exhibiting persistent disease (Gomes et al., 2008; Molander et al., 1998; Pinheiro et al., 2003; Rocas et al., 2004; Siqueira & Rocas, 2004; Sundqvist et al., 1998). It may be concluded that E. faecalis is the most important species in endodontic treatment failures. However, Rocas et al. (2008) show that endodontic treatment failures are frequently associated with persistent mixed bacteria leading to secondary infection. When E. faecalis was present in species-specific PCR (polymerase chain reaction) in teeth with posttreatment apical periodontitis, it was never the dominant species and other taxa were disclosed (Rocas, Hulsmann, & Siqueira, 2008). Furthermore, Rôҫas et al. state that these mixed species (Streptococcus species, Lactobacillus species, Dialister Invisus, Eubacterium infirmum, Prevotella intermedia, Selenomonas sputigena) might be connected with the etiology of secondary infected apical periodontitis (Rocas et al., 2008). Formation of a thick biofilm was not detected in the present study – maybe due to the use of monocultures of E. faecalis instead of mixed culture mediums. Furthermore, the root canal was inoculated with the bacterial solution and afterwards the culture medium was changed every two days. For this reason, it is questionable if another bacterial species or a mixed species solution should have been used for the experiments in the present study or as an alternative, the bacterial solution could have been changed more frequently (Stauffacher, Lussi, Nietzsche, Neuhaus, & Eick, 2017). Nevertheless, this study was not designed to detect the occurrence of specific microbial species. This study was performed to answer the question of whether or not devitalized bacteria are still able to spread into dentinal tubules and if they progress deeper over time, implying a deeper penetration of the bacterial endotoxins. The present study clearly shows that devitalized bacteria are able to penetrate and progress into tubules. This is a vital factor for studies investigating the success of different endodontic treatment regimens and irrigants. The bacteria must be implicated into the dentinal tubules by external factors such as an osmotic process. The cell membrane is damaged during the devitalizing method at 95 °C for two hours as shown with the CFU and BacLight™ method. This procedure should represent devitalized bacteria after chemomechanical treatment of root canals in vivo, in which the cell membrane of E. faecalis becomes penetrable for water. Bacteria are highly proteinaceous cells. They absorb water when the cell membrane is damaged and the bacterial cells follow the concentration gradient to absorb more water. In the in vitro model, this influence is represented by the concentration gradient of the 3 % agarose gel. Since the agarose gel contains more water than the devitalized bacterial suspension in the root canal, the bacterial cells migrate into the dentinal tubules towards the agarose gel in a time dependent manner. The longer the incubation time, the deeper the penetration is into the dentinal tubules. Under in vivo conditions a concentration gradient towards the periodontal ligament also exists. During the root canal treatment, special irrigant solutions such as sodium hypochlorite and chlorhexidine work as antibacterial agents. The bacterial cells mainly die in that process. The present study shows that these devitalized cells are still able to penetrate into the dentinal tubules. When studies
Fig. 3. Representative scanning electron microscopic pictures after 7 days (a), 14 days (b) and 21 days (c) of incubation with devitalized E. faecalis cells. After 7 days, the cells are detectable at the entrance of the dentinal tubules (a, arrow). E. faecalis cells already migrated into the dentinal tubules after 14 days. A solid E. faecalis cell is attached to the root canal surface, no other cells are visible at the entrances of the dentinal tubules after a 21-day period of incubation.
μm in the apical part of the root canal (n = 3 measuring points; Fig. 5a, b). The results of 28 days of incubation with the devitalized cells show that the penetration depth of the bacteria ranged up to 210.75 ± 21.53 μm in the medial part of the root canal and approximately 266.5 μm ± 92.63 μm in the apical part of the root canal (n = 3 measuring points per root canal section; Fig. 5g). While bacterial aggregation at the entrances of the dentinal tubules increased, clusters of bacteria were found after all observed time periods (4 days to 28 days). The total number and penetration depth of E. faecalis cells was lower compared to a vital suspension of E. faecalis (1002.45 μm) after 28 days (Fig. 6a–c). It was noted that bacterial penetration was not common to all of the dentinal tubules in the vital E. faecalis control and especially in the devitalized control. 292
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Fig. 4. DAPI staining, typical examples of bacterial penetration into dentinal tubules after 4 days (a–c) and 7 days (d–i) of incubation with non-vital E. faecalis solution. After 4 days, bacteria (arrow) were distributed in small aggregates at the entrances of the dentinal tubules in the apical third of the root canal (a-c). An enlarged section of d-f is shown in g-i. Bacteria are marked with an arrow. Individual groups of bacteria penetrated to a depth of approximately 128 μm after 7 days (f, i).
strains (Peters et al., 2000). The removal of the smear layer in the present study took place with a 17 % EDTA irrigation solution and subsequently E. faecalis was inoculated into the root canal. With this protocol, vital E. faecalis cells penetrated deep into the root dentinal tubules (Fig. 6). This procedure is in good accordance with other studies (Akpata & Blechman, 1982; Orstavik & Haapasalo, 1990; Peters et al., 2000). The study of Peters et al. also examines the penetration of bacterial cells into dentinal tubules (Peters et al., 2000). They show that in 11 % of the specimens, no bacteria were found in the pulpal or middle grinding sample, whereas bacteria were detected in deeper apical layers. These findings are in good accordance with the present study. The highest number of bacteria penetrating into the tubules was also found in the apical part of the root canal. In addition, they report that light microscopy or SEM (scanning electron microscopy) show the presence of bacteria in tubules but fail to show viability of the organisms. The correlation between viable CFU from grinding samples and histological observations is therefore of interest. Another microscopic technique to detect bacteria is the Confocal Laser Scanning Microscopy (CLSM). This method is a very good approach to detect bacteria on relatively flat surfaces (Jung et al., 2010). However, the curved root canal and the curved dimensions of the dentinal tubules present a major challenge for the examination with the CLSM-method. In the present
investigate the success of new or popular irrigant agents, microscopic approaches such as light-, scanning- or transmission electron microscopy are used to visualize remaining bacteria in the dentinal tubules. The presence of bacteria within the dentinal tubules might be interpreted as a failure of the tested preparation regimen concluding that these bacteria resisted or escaped the intracanal disinfection procedure. However, the present study shows that this conclusion cannot be drawn since possible osmotic processes have an impact on the migration of bacteria into the dentinal tubules. Furthermore, no evidence exists regarding the vitality of the present bacteria since microscopic investigation including light-, scanning- and transmission electron microscopy only detect the presence of bacteria. The bacterial cells could have spread throughout the dentinal tubule system due to a carryover of bacteria during the splitting procedure of the roots. This carryover, however, would lead to an evenly distributed pattern of microorganisms throughout the root. Further, Figs. 4 and 5 show that the penetration of bacteria occured in a time dependent manner. An insufficient devitalization of the E. faecalis cells could certainly also lead to a deeper bacterial penetration over time. The CFU method was therefore performed to proof the devitalization of the bacteria. In an experimental setting, penetration of E. faecalis was found to be greatly enhanced when the smear layer was removed before inoculation with E. faecalis 293
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Fig. 5. DAPI staining, typical examples of bacterial penetration into dentinal tubules after 14 days (a–d) of incubation with nonvital E. faecalis solution. After 14 days, the penetration depth of the dead bacteria ranged between 137 μm and 354 μm (a, b). An enlarged section of a, and c is shown in b, and d. Bacteria are marked with an arrow. After 28 days, the penetration depth of the dead bacteria ranged between 197 μm and 332 μm (g). An enlarged section of e is shown in f. Bacteria are marked with an arrow.
however, it has not yet been carried out to detect bacteria in root canals. In contrast to electron microscopic gold-immunolabelling techniques, this fluorescence microscopic approach does not require timeconsuming embedding procedures and allows the direct visualization of bacterial cells. Another method to examine the bacterial penetration depth is described by the study of Ørstavik and Haapasalo, whom have found Ps. aeruginosa in bur samples of deeper dentine layers, but not in the SEM sections (Orstavik & Haapasalo, 1990). Moreover, with this method it is possible that bacteria were forced deeper into the dentinal tubules during this special grinding procedure. Furthermore, Peters et al. postulated that histological investigations of these bur samples always need to be combined with CFU methods (Peters et al., 2000). In their study, the middle and cemental grinding samples contain low numbers of CFU, whereas bacteria are not seen in histological sections. This is not surprising considering the field of vision under the microscope is limited and low numbers of CFU remain undetected. In contrast to the method described in our study, no bur samples are needed. Therefore, this methodological failure can be neglected. In addition, the
study, the DAPI- method was therefore used to visualize bacteria inside the dentinal tubules for the first time. DAPI stains both living and dead cells. During the devitalization procedure, the DNA of the lysed bacteria leaks out of the cell and is distributed in the surroundings. Therefore, the fluorescent of devitalized cells is less rich in contrast as in vital cells. Nevertheless, a clear differentiation between vital and devitalized cells with the DAPI-method is vague and results in unreliable findings. Hence, with the help of the BacLight™ method (Tawakoli, Al-Ahmad, Hoth-Hannig, Hannig, & Hannig, 2013) it was proven that the utilized cells were effectively devitalized, confirming that DAPI is a proper method to stain devitalized cells in dentinal tubules. Further, no grinding and bur samples of different dentine layers are necessary for examination. Using this fluorescence staining method, the bacterial penetration can be displayed throughout its entire distance under the light microscope (Figs. 4–6) without the use of destructive approaches. The DAPI method is already well known to detect bacteria in in situ biofilm samples (Hannig et al., 2013; Hertel et al., 2016; Kensche et al., 2013); 294
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Fig. 6. DAPI staining, typical examples of bacterial penetration into dentinal tubules after 28 days (a–c) of incubation with vital E. faecalis solution. After 28 days, the penetration depth of the vital bacteria reached up to a penetration depth of 1002.45 μm (c).
teeth are necessary and in preparation to carry out a statistical evaluation for proof of evidence.
combination with the CFU method is not required. Hence, the additional, time consuming and unprecise method is not necessary. Another fact regarding the present study that should be considered is that the diameter of bovine dentinal tubules is 4 to 7 μm and varies only slightly from the diameter of human dentinal tubules (3 to 7 μm) (Berkiten et al., 2000; Jung et al., 2010). The slightly larger diameter might favor an easier bacterial penetration into the tubules. Nevertheless, E. faecalis cells are 0.87 to 1.01 μm in diameter (Kokkinosa, Fasseas, Eliopoulos, & Kalantzopoulos, 1998); therefore it is conceivable that a bacterial penetration occurs to the same extent in human and bovine teeth. In addition, the diameter of bovine dentinal tubules decreases from the outer region towards the inner region of the tooth, which results in a cone-shaped configuration with the larger base at the outer end (Dutra-Correa, Anauate-Netto, & Arana-Chavez, 2007). In contrast, the human dentinal tubules are cone-shaped with their larger base at the pulpal side (Dutra-Correa et al., 2007). Since human and bovine teeth have an opposite relation in their dentinal tubules, the physiology of this complex structure may be different from each other (DutraCorrea et al., 2007). Furthermore, the degree of maturity of the tooth samples should also be taken into account (Perez et al., 1993). Immature teeth have wider entrances of the dentinal tubules than mature teeth. This is an important factor when colonizing these teeth with bacteria. Nevertheless, most studies discussing the topic of dentinal tubule penetration are based on bovine teeth specimens (Berkiten et al., 2000; Haapasalo & Orstavik, 1987; Perez et al., 1993; Siqueira et al., 1996) since they are available in large quantities and all teeth are from the same diet and age group of animals (Jung et al., 2010; Peters et al., 2000). In the present study, only thirteen bovine teeth were used for the experiments. This number is not sufficient to carry out a statistical evaluation. Through the results of the present study, important basic knowledge was gained to understand, interpret, and discuss bacterial findings in microscopic images. Further studies with a larger number of
5. Conclusions Increased exposure times (4 days to 28 days) of devitalized E. faecalis strains into root canals lead to an increased number of penetrated dentinal tubules as well as to a deeper penetration, and the fluorescent labelling technique DAPI is a valuable method to provide information about bacterial penetration into dentinal tubules. Conflict of interests The authors deny any conflicts of interest related to this study. This work was supported by the DFG (German Research Foundation; WE 5838/1-1, DA 1701/1-1). Except as disclosed above, the authors certify that they have no commercial associations that might represent a conflict of interest in connection with the submitted manuscript. Sources of funding This work was supported by the DFG (German Research Foundation; WE 5838/1-1, DA 1701/1-1). Ethical approval No ethical approval was necessary for the present study. Acknowledgements The authors deny any conflicts of interest related to this study. This work was supported by the DFG (German Research Foundation; WE 295
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