In vivo model for microbial invasion of tooth root dentinal tubules

ABSTRACT Objective Bacterial penetration of dentinal tubules via exposed dentine can lead to root caries and promote infections of the pulp and root canal system. The aim of this work was to develop a new experimental model for studying bacterial invasion of dentinal tubules within the human oral cavity. Material and Methods Sections of human root dentine were mounted into lower oral appliances that were worn by four human subjects for 15 d. Roots were then fixed, sectioned, stained and examined microscopically for evidence of bacterial invasion. Levels of invasion were expressed as Tubule Invasion Factor (TIF). DNA was extracted from root samples, subjected to polymerase chain reaction amplification of 16S rRNA genes, and invading bacteria were identified by comparison of sequences with GenBank database. Results All root dentine samples with patent tubules showed evidence of bacterial cell invasion (TIF value range from 5.7 to 9.0) to depths of 200 mm or more. A spectrum of Gram-positive and Gram-negative cell morphotypes were visualized, and molecular typing identified species of Granulicatella, Streptococcus, Klebsiella, Enterobacter, Acinetobacter, and Pseudomonas as dentinal tubule residents. Conclusion A novel in vivo model is described, which provides for human root dentine to be efficiently infected by oral microorganisms. A range of bacteria were able to initially invade dentinal tubules within exposed dentine. The model will be useful for testing the effectiveness of antiseptics, irrigants, and potential tubule occluding agents in preventing bacterial invasion of dentine.


INTRODUCTION
Root caries, pulpitis, and dentine hypersensitivity are becoming increasingly more problematic as the dentate human population ages 17 recession can lead to exposure of dentine and tooth wear can result in opening of dentinal tubules on the exposed surface. When dentine becomes exposed as a result of gingival recession, or through dental caries, cracks, or microleakage around restorations, microorganisms are able to gain access to the tubules 15 . More microorganisms are found in the dentine adjacent to periodontal pockets than in healthy radicular dentine, and more bacteria are dentine 1 . Bacteria can also laterally invade the root surface along the incremental lines of cementum and then infiltrate the dentine 21 . Bacteria can penetrate through hypomineralized enamel into the dentine and contribute to pulpal pain symptoms of teeth with molar incisor hypomineralization 10 .
Bacterial persistence within the dentinal tubules may exacerbate development of root caries, and the formation of complex microbial communities within deeper dentine or root canal space 26 may failure 18 . Evidence suggests that the bacteria that initially invade dentinal tubules are often from the genera Enterococcus and Streptococcus 15 . Enterococci in particular readily penetrate dentinal tubules 13,18 . Root dentinal tubule invasion models in vitro have been utilized widely to study different bacterial penetration capabilities 14 and model pulpal infections 15 , and to test the effects of antimicrobials 3,4 and irrigants 12 . The models have provided valuable information on the mechanisms involved in growth and penetration of dentine 5 , and the potential for various agents, such as photodynamic therapy 29 to help with controlling infection. However, the various laboratory models usually incorporate dentine environment in order to achieve infection with relevant microorganisms. Under natural conditions, dentine would be exposed to salivary components, gingival fluid, immune system molecules, and potentially hundreds of different microorganisms 19 . Most in vitro dentine infection models employ conditions that are quite different from the natural in vivo infection environment.
Dentine studies in vitro have also included testing various compounds for ability to occlude tubules as desensitizing agents 2 . Valuable information has been obtained about the properties and effectiveness of such agents 27 , but these are only just beginning to be tested under suitable in vivo conditions. For example, West, et al. 28 (2011) determined the abilities of desensitizing toothpaste technologies to occlude patent dentinal tubules in a clinical environment. Healthy subjects wore lower intraoral appliances retaining dentine samples, and these were analyzed after 4 d of treatment for degree of occlusion 28 . We have utilized the basis of that study to develop a model for microorganism invasion of dentinal tubules in vivo. This will provide a suitable platform by which to investigate bacterial invasion of dentine within a clinical environment, and to test for effectiveness of tubule-occluding or antimicrobial agents to prevent bacterial invasion of dentine.

Root dentine
Non-carious, unrestored human canine or premolar teeth with single root canals were obtained from orthodontic extractions. Teeth were obtained with informed consent and the study was approved by Central and South Bristol Ethics Committee (REC ref. 04/Q2006/50). Following extraction, teeth were soaked in 2% sodium hypochlorite (NaOCl) for 48 h and any soft tissue remaining was removed. Prior to sectioning, roots were washed in copious amounts of water, and rinsing was repeated following sectioning to ensure no traces of NaOCl remained. Teeth were stored in sterile distilled H 2 O at 4°C until required. Roots were sectioned using a water cooled steel bladed cutting machine (Isomet Saw, Buhler Ltd., Evanston, IL, USA). In brief, the crown and root tip were removed, the remaining root was cut into 0.5 cm lengths, and the cervical segments were longitudinally sectioned in such a way that the root canal was exposed. The root sections were then autoclaved (121°C, 20 min) in distilled H 2 O, which did not visually affect tubule structures 5 , and stored at 4°C.

Preparation of intra-oral appliances
For each subject, a lower alginate impression was recorded in a perforated stock tray. Within dental stone and subsequently two lower-oral appliances were constructed from Forestacryl ® self curing acrylic (Pearson Dental Supply Co., Sylmar, loops were constructed in an anterior and posterior trench region to hold the dentine samples in place ( Figure 1). The cervical region root sections were mounted into the appliances in such a way that below, the level of the surrounding acrylic surface. Before placement in the appliance, the root sections were dipped in sterile distilled water, the face to be in contact with the appliance was dried and the sample mounted onto a small drop of molten sticky wax within the trench of the appliance. Once all four sections were in place they were then further secured in position with the wire loop that was built into the appliance (Figure 1). The appliances were stored overnight at 4°C in a sterile airtight container containing damp tissue to prevent them drying out.

Experimental design
Ethical approval for this work was obtained from the Institutional Ethical Review Board (REC ref. 04/Q2006/50). Inclusion criteria were healthy volunteers aged 18 or over that could accommodate a lower buccal appliance. Exclusion criteria were: pregnancy, lactation, gross caries, unstable periodontal disease, antimicrobial medication within 7 d previously, orthodontic appliances that would interfere with the study evaluations, and tongue or lip piercing. Four subjects, age range 20-26 appliances which were then worn over the course of the next 15 d between 09:00 and 21:00 h. They were removed for 1 h twice a day for mealtimes and also for the period over which any drink other than water was consumed. At 21:00 h, subjects removed the appliances, brushed them with tap water, rinsed them in running water for 20 s, and then stored them overnight in an airtight container. Subjects brushed their teeth morning and evening trial on day 15, root samples were removed from the appliances at 15:00 h. There were no adverse events.

Microscopic analysis of bacterial invasion
Six pieces of dentine from the appliances were being demineralized in 10% formic acid containing 2% formalin for 7 d. Samples were then dehydrated (70% IMS-denatured alcohol x2, 90% IMS x2, being blocked in wax. Fifteen transverse sections, 6 mm thick and 60 mm apart from the next section, were cut from each dentine sample, mounted on using the Brown and Brenn method 6 .
Penetration of bacteria into dentine was were examined for each of the 15 root sections. The extent of invasion was initially expressed as the tubule invasion index (TI) 16 , where 1 to 20 tubules (per invaded scored 2; and >50 tubules invaded scored 3. These scores were then converted to Tubule Invasion Factor (TIF) that took depth of invasion of tubules into account 5 . The TIF was obtained by multiplying the TI by the invasion depth score: x1, tubules per 5 .
Two root pieces from each appliance were rinsed in sterile distilled water and stored at -80°C. One of the specimens in each case was used to optimize Once this had been achieved, the second sample from each subject was transferred into a microfuge tube containing 0.1 mL sterile 10% EDTA (pH 6.5), vigorously vortex-mixed, and incubated at room temperature for 30 min to partially decalcify. Samples were then transferred into 0.1 mL 2 M citric acid (pH 1.6) and incubated for 30 min. Samples were extensively rinsed in sterile distilled H 2 Figure 1-View of dental appliance in place on a dental mould (A) and a close-up view showing positioning of dentine samples (B). This shows the lower right appliance and the dentine pieces with pulpal faces outwards labelled A-D. A similar appliance was placed on the lower left, with four dentine pieces designated E-H. The dentine samples were retained with wax on the base side and with a metal retaining wire on the exposed side (arrowed in panel A). Appliances were custom made for each individual human subject Releaser (Cambio, Cambridge, Cambs, UK) for DNA extraction according to the manufacturer's instructions.
The DNA extracts were used as templates in 25 to amplify a product of 160 bp. The presence of gel electrophoresis. Subsequently, aliquots (6 mL) denaturant gradient) and the separated bands were ethidium bromide-stained and visualized under UV light (344 nm) (Figure 2A). Bands were excised from the gel lanes, transferred to tubes containing 0.3 mL TE buffer (5 mM Tris-HCl, 10 mM EDTA, pH 7.5) and the DNA was allowed to elute from the gel fragments for 16 h at 4°C. Each of the eluted gel bands was then subjected to further PCR amplification using electrophoresis ( Figure 2B). The fragments were Manchester, Lancs, UK), ligated into plasmid pCR2.1 (Invitrogen, Thermo Fischer Scientific Inc., Waltham, MA, USA) and transformed into Escherichia coli XL1-Blue by standard procedures.
Plasmids were extracted from transformant colonies using QIAprep Spin MiniPrep Kit (Qiagen), checked by agarose gel electrophoresis, and the 160-bp Cambridge, Cambs, UK). The partial 16S rRNA gene sequences were then compared with 16S rRNA nucleotide NCBI/BLAST program.

Microscopic analysis of bacterial invasion
The tooth root dentine pieces were mounted into appliances as shown in Figure 1. The samples were designated A-D (lower right) ( Figure 1B) and E-H (lower left). After 15 d in vivo, the root pieces were removed and processed as described in Material and Methods for microscopic analyses (A-F). All samples containing patent dentinal tubules showed high levels of bacterial invasion, with TIF values in the range of 5 to 9 (Figure 3). Three dentine samples (Figure 3) could not be assessed for invasion because of disintegration of internal dentine structure.
to type of organisms present within the dentinal tubules, pattern of tubule penetration, depth of enormously across the dentine samples. We invasion, but in the following descriptions we have included only representative micrographs exhibiting distinct features of the invasion processes.
For subject 1, root sample D, there was invasion ( Figure 4A, arrowed b) to depths of >100 m. Root sample F, on the other hand, seemed to be entirely permeated by small cocci bacteria. These stained of deeper (~150 m) invasion ( Figure 4B, arrowed in laboratory studies of dentine invasion by pure cultures of streptococci 16 . In subject 2, sample A carried a dense invasive surface ( Figure 5A) and there was invasion of tubules >150 pattern was seen for root sample D ( Figure 5D), from this subject was one of the samples that could not be properly analyzed, as the internal dentine structure was disintegrated ( Figure 5B). Figure 6 shows sections from blocks C, D, and E from subject 3. Sections through C ( Figure 6A) present a small distance away from the surface of the dentine sample, perhaps having been present on the dentine surface prior to sectioning (Figure negative rods (~5 m length) in well-separated m ( Figure 6B). In sample E, individual tubules contained deep lines of ( Figure 6C).
From subject 4, sample D showed invasion by positive cocci and matrix material ( Figure 7A).

Sample E showed distinct penetration of tubules by
Mean level of microbial cell invasion into dentine samples carried by four subjects (1)(2)(3)(4). Columns A through F correspond to dentine samples (see Figure 1 legend). Invasion is expressed as Tubule Invasion Factor (TIF) (see Material and Methods) which takes into account numbers of tubules containing bacteria and depth of penetration. Samples B, F, and B in subjects 2, 3, and 4 respectively, were not analyzable because of deformed dentine structures. Panel 5, combined dataset mean±standard deviation (8.09±0.87). Error bars are ± standard deviation from microscopic analysis of 75 individual sections (n=21) These results demonstrated that the dentine samples mounted onto the appliances were all readily susceptible to infection by invading microorganisms. The technique therefore was a very effective mean of achieving invasion of dentinal tubules by a variety of different oral bacteria.
Within one of the dentine samples selected at including Klebsiella pneumoniae, Enterobacter species, Enterobacter hormaechei, and sequences similar to those from some uncultivated bacteria from faeces (  (Figures 4-7).

DISCUSSION
In this study we have prepared dentine samples in a manner similar to that done for in vitro invasion investigations 16,22 of dentine infection by pure cultures of bacteria such as E. faecalis and  Figure 3 In vivo model for microbial invasion of tooth root dentinal tubules Streptococcus species. These approaches have been undertaken to study the mechanisms involved in dentinal tubule infection, and to investigate the effects of various antiseptics, irrigants, and antimicrobials in preventing dentinal tubule infection. Perhaps one limitation of such in vitro analyses is that they have been undertaken under conditions that are quite different from those that would be encountered in vivo. These include, for example, the presence of whole saliva, salivary Our studies here show that it is possible to readily achieve dentine infection in vivo to the levels and extent that can be obtained in vitro 16 . This model therefore would be useful for testing the effects of new dentinal tubule occluding compounds 27 or agents for preventing root caries 30 in order to complement the in vitro experiments that have been previously employed.
readily penetrate dentinal tubules. Historically, E. faecalis has been considered as a major invader of dentine 13,15 , but more recent molecular studies that do not employ cultivation methods suggest that E. faecalis may not be so prevalent as generally believed 23 . Invasions of dentine have been shown 8 . Penetration in vitro has not been investigated in such detail. Interestingly, periodontal bacteria Porphyromonas gingivalis were found to be unable to invade dentine unless co-cultured with Streptococcus 16 . In this present article we have demonstrated microscopically, and by molecular means, that dentine in vivo can be

Enterobacter, Klebsiella, seemed on
Enterobacter and Klebsiella species have been 8 . More recently, Klebsiella lesions underneath restorations 20 and E. hormaechei was cultivated from human atherosclerotic tissue 24 . Members of the Enterobacteriaceae and Pseudomonadaceae are also found on the human tongue 9 . Our work thus provides further evidence the oral cavity and have the ability to penetrate dentine.
A range of bacterial species were present within a small number of dentine samples analyzed. Three samples showed disintegration of tubule structure, most likely arising from the lengthy dehydration, sectioning). Only a limited number of specimens were employed here because we were results suggest that the model can be applied to future studies of dentine hypersensitivity agents, to occlude tubules and block bacterial invasion 27 . It is acknowledged that the molecular methods used here do not differentiate between live or dead bacteria. However, it might be possible to utilize dentine discs, fracture them, and stain the intratubular bacteria with LIVE/DEAD stain. This method has recently been described in studies evaluating in vitro the antimicrobial effect of a commercial product on residual bacteria in dentinal tubules 11 . One of the samples in the study described here cocci, which corroborates the notion that these dentine 15 . However, E. faecalis was not found in our Granulicatella, S. oralis, S. mitis, and S. gordonii which, with the exception of Granulicatella, have been previously implicated in tubule invasion 15 . In addition, all of these bacteria including Granulicatella are organisms that have been linked with infective endocarditis. Therefore, there could potentially be an association between ability to invade dentine and ability to cause   present within dentinal tubules that have been exposed to many hundreds of different bacteria in vivo 19 . In this study we only utilized four dentine samples to identify bacteria types that could invade the specimens under the condition of compounds or products in occluding tubules employ many more subjects to provide suitable power. However, the molecular studies cannot be directly related to the morphological studies at this stage. We have established though that it is dentine. Our methodology would tend to identify the most prevalent microorganisms that were present within the dentine samples analyzed. We would like to develop these studies further in such a way that we could visualize and identify, by molecular techniques, the bacteria that have invaded the same dentine sample. This could be achieved by extracting bacterial DNA, or by detecting DNA adjacent sections to those histochemically stained.

CONCLUSION
In summary, this study has established a novel in vivo model for studying the infection of dentine by oral microorganisms. Dentine specimens exposed to the human oral environment become infected with microorganisms to similar extent and depth to dentine infected in vitro under laboratory conditions. In addition to streptococci, bacteria from the genera Enterobacter, Klebsiella and Pseudomonas were in vivo in vitro experimental results on the effects of antiseptics, irrigants, or tubule occluding agents on dentine invasion by oral bacteria.