Bacterial adhesins--their role in tubule invasion and endodontic disease

Bacterial invasion of dentinal tubules is critical to the progression of dental caries and the development of pulp and periapical disease, and may also influence the progression of periodontal disease. However, little is known about the host or bacterial mechanisms involved in tubule invasion. Recent work has demonstrated that bacterial interactions with dentine, and salivary and tissue molecules influence invasion. Salivary molecules such as mucin and immunoglobulin G (IgG) co-aggregate with bacterial cells, which inhibits dentine invasion, while deposition of dentinal tubule fluid molecules e.g. albumin, IgG, or fibrinogen within dentinal tubules also inhibits invasion. Dentine invasion by streptococci has been shown to be associated with a bacterial growth response and adhesion to unmineralized collagen, which are mediated by streptococcal cell-surface antigen I/II polypeptides. These adhesins possess diverse binding properties including binding to salivary glycoprotein, other bacteria, and to collagen. Additionally, some antigen I/II polypeptides facilitate species-specific co-invasion between streptococci and obligate anaerobes that lack the ability to invade by themselves. An understanding of the mechanisms involved in bacterial invasion of dentine should allow development of new control strategies.     

Dentinal tubule invasion and adherence by Enterococcus faecalis

Aim To investigate dentinal tubule invasion and the predilection of Enterococcus faecalis for dentinal tubule walls. Methodology The invasion of dentinal tubules in extracted human teeth by E. faecalis was measured ex vivo after 8 weeks of incubation. The canal walls of 16 root sections were either intact or instrumented with or without smear layer present. Extent and maximum depth of tubule invasion were assessed histologically and compared between groups. In the adherence study, 44 vertically split root samples were prepared to expose longitudinally aligned dentinal tubules and fractured orthodentine (OD). Surfaces were exposed to E. faecalis (erythromycin resistant strain, JH2‐2 carrying plasmid pGh9:ISS1) and incubated aerobically for 2 h. Samples were processed for analysis using scanning electron microscopy. Bacterial adhesion to tubule walls versus fractured OD was calculated as number of cells per 100 μm². Results The strain of E. faecalis used in this study showed moderate to heavy tubule invasion after 8 weeks. In the adhesion studies, significantly more bacteria adhered to fractured OD than to dentinal tubule walls (anova , P < 0.001). With respect to the tubule wall, adherence was greater in inner versus outer dentine (P = 0.02) and greater when bacterial adhesion was tested in chemically defined medium than in phosphate‐buffered saline (anova , P < 0.001). Conclusions Although E. faecalis readily invaded tubules, it did not adhere preferentially to tubule walls. Initial colonization of dentinal tubules by E. faecalis may depend primarily on other factors.     

Qualitative comparison of sonic or laser energisation of 4% sodium hypochlorite on an Enterococcus faecalis biofilm grown in vitro

The effectiveness of sonic activation, laser activation and syringe irrigation of 4% sodium hypochlorite in removing an Enterococcus faecalis biofilm was compared. Biofilms were grown in extracted human single rooted teeth using a flow cell apparatus. After 4 weeks' growth, teeth were subjected to each treatment using 4% sodium hypochlorite and radicular dentinal surfaces of the root canals were analysed by scanning electron microscopy. Results showed that sonic activation and syringe irrigation with sodium hypochlorite showed reduced numbers of bacterial cells on the radicular dentine but were not effective in eliminating E. faecalis in the dentinal tubules. Laser activation of sodium hypochlorite resulted in clean dentine walls and undetectable levels of bacteria within dentinal tubules. Qualitatively, sonic or laser activation of 4% NaOCl resulted in greater bacterial reduction compared with syringe irrigation, with laser activation producing the greatest overall reduction.     

Adherence of Streptococcus gordonii to smeared and nonsmeared dentine

The purpose of this study was to investigate the adherence of Streptococcus gordonii to smeared and non-smeared dentine and to assess the influence of patent dentinal tubules on bacterial retention. In order to examine bacterial adherence, 10 non-smeared (group 1) and 10 smeared samples (group 2) of outer root dentine were prepared from teeth exhibiting dentine sclerosis. Ten non-smeared samples from inner coronal root dentine that did not exhibit sclerosis were prepared in order to study the influence of patent tubules on bacterial retention (group 3). Cells of the bacterium were radioactively labelled and an adhesion assay was performed. The number of bacteria adhering to the dentine surface was determined by scintillation counting. The results show that the number of bacteria adhering to both smeared and non-smeared outer sclerotic dentine was low (0.3% of inoculum), and there was no significant difference between the groups (P > 0.3). A significantly higher number of bacteria was retained on the inner non-smeared coronal root dentine (P < 0.0001) compared to groups 1 or 2. The results suggest that dentinal smear layers do not enhance or impede bacterial adherence to the dentine matrix. Dentinal surfaces with patent dentinal tubules retain more bacteria than a smeared surface.     

Does mechanical pressure force bacteria into the dentinal tubules of carious and non-carious teeth--an exploratory study.

BACKGROUND: Although many researchers have confirmed bacterial migration within dentine, no evidence was found to show whether bacteria can be easily forced into dentine during caries treatment. OBJECTIVES: This exploratory study was to see if bacteria could be easily forced into dentinal tubules of carious and non-carious teeth using hand excavation and air pressure. METHODS: Two (2) carious and ten (10) non-carious teeth were used. Class I (Black's Classification) cavities were created using rotary instruments in the non-carious teeth, while the carious teeth were excavated using hand instruments to remove the caries. The cavity of one carious tooth was etched and in the other tooth was not. Five (5) of the non-carious tooth cavities were etched, of which, one tooth was also disinfected. The remaining five non-carious teeth were not etched and included one tooth that was disinfected. Bacterial cultures of S. mutans, E. coli and Veilonella were placed in the prepared cavities and hand excavators and air pressure were used in an attempt to force bacteria down into the dentinal tubules. Thereafter, the teeth were fixed, decalcified and prepared for electron or light microscopy. RESULTS: One etched, and one etched and disinfected non-carious tooth showed bacteria in the dentinal tubules. One carious tooth (etched) showed bacterial presence in the dentinal tubules. CONCLUSION: This study shows that it is difficult to force bacteria into dentinal tubules using hand excavation (as in the Atraumatic Restorative Treatment technique) so a definitive study is not indicated.     

The effect of tissue molecules on bacterial invasion of dentine

Bacterial invasion of dentinal tubules is a critical step in the pathogenesis of dental caries and pulp and periapical disease. The purpose of this study was to determine the effect some molecules commonly found in saliva and dentinal tubule fluid may have on the bacterial invasion of dentine. The results showed that invasion of Streptococcus gordonii or Enterococcus faecalis cells was inhibited when the bacterial cells were in solution with mucin, immunoglobulin G (IgG) and serum, and this was related to bacterial cell aggregation, as a result of interaction with agglutinins, and/or inhibition of collagen binding. When dentine was soaked in growth media containing fibrinogen, IgG, albumin or serum prior to inoculation, bacterial invasion was inhibited. It is suggested that this may be due to reduced dentine permeability as a consequence of the deposition of the compounds within dentinal tubules.     

Predominant obligate anaerobes invading the deep layers of root canal dentine

This study was carried out to investigate the presence and types of bacteria invading the deep layers (0.5-2.0 mm from the surface of the root canal wall) of infected dentine of human root canals by sampling with an anaerobic glove box system the split surfaces of eight freshly extracted teeth. More bacteria were recovered after incubation in an anaerobic glove box than after aerobic incubation in air with 30 per cent CO2. Out of 256 predominant bacterial isolated, 205 isolates (80 per cent) were obligate anaerobes. These findings suggest that the environment of deep layers of endodontic dentinal lesions is anaerobic and favours the growth of anaerobes. Among the obligate anaerobic isolates, strains belonging to Lactobacillus (30 per cent) and Streptococcus (13 per cent) were predominant, followed by Propionibacterium (9 per cent). No strains of obligate anaerobic Gram-negative rods were isolated. The microflora of deep layers of infected root dentine is somewhat similar to that of the deep layers of carious lesions in coronal dentine.     

Bacterial invasion of non-exposed dental pulp

Anaerobic procedures were adopted to demonstrate the early bacterial invasion of non-exposed dental pulps, and to isolate and identify the bacteria. Of 19 freshly extracted teeth which originally exhibited deep dentinal lesions, clinical examination and electric pulp testing showed that nine of them had no pulpal exposure. Thus the pulps of these teeth were covered by clinically sound dentine beneath the carious lesion. Bacteria were found to have invaded the pulps of six of these nine teeth. The predominant bacteria were obligate anaerobes belonging to the genera Eubacterium, Propionibacterium and Actinomyces. Other obligate anaerobes were Lactobacillus, Peptostreptococcus, Veillonella and Streptococcus. The bacterial composition resembled that of the deep layers of dentinal lesions described previously, suggesting that the bacteria isolated in this study had passed through some individual dentinal tubules, to invade the dental pulp.     

Tricalcium silicate induced mineralization for occlusion of dentinal tubules

Background: The aim of this study was to evaluate the dentinal tubule occluding ability of tricalcium silicate (Ca₃SiO₅ or C₃S) in vitro. Methods: Dentine discs were prepared from extracted caries‐free human third molars and etched with 0.02 M citric acid (pH 4) for 3 minutes in order to produce patent dentinal tubules. Tricalcium silicate pastes of different viscosities were applied to the surface of the dentine specimens for 3 minutes and then removed with a swab followed by deionized water rinse. The dentine specimens were immersed in artificial saliva for 7 days. The tubule occlusion, sealing depth and chemical composition of the dentine specimens and bonding force between the dentine matrix and mineralized layer were evaluated by scanning electron microscopy, energy dispersion X‐ray spectroscopy, X‐ray diffraction analysis and a nanoscratch test. Results: A homogeneous layer of mineral crystals was precipitated onto the dentine surface and caused significant occlusion of the dentinal tubules. The dentine permeability and sealing depth of the C₃S increased with the decrease of viscosity of the paste. With a proper viscosity of C₃S pastes, the interface between the mineralized layer and the dentine matrix bonded well. Conclusions: This study suggests that C₃S may be an effective agent for the treatment of dentine hypersensitivity.     

Dentin tubule invasion by Enterococcus faecalis under stress conditions ex vivo

Enterococcus faecalis is the species most frequently isolated from failed endodontic treatments because it can survive under stress conditions imposed by root canal treatment. The objective of this study was to determine the ability of E. faecalis to invade dentine tubules under alkaline and energy‐starvation stress and to explore the potential mechanisms. Roots from single‐rooted human teeth were infected with E. faecalis under alkaline and energy‐starvation stress conditions. After 4 wk of culture, samples were processed to establish the tubule‐penetration distance. In addition, the hydrophobicity of E. faecalis cells under these conditions was analysed and the expression of genes involved in adhesion was quantified by real‐time quantitative PCR. Culture of E. faecalis under alkaline and energy‐starvation stress conditions resulted in a marked reduction of tubule‐penetration distance, a significant increase in hydrophobicity of the bacterial surface, and marked down‐regulation of most adhesin genes compared with E. faecalis cultured in tryptic soy broth. The results indicate that the dentine tubule invasion ability of E. faecalis was markedly decreased under alkaline and glucose‐starvation stress conditions, possibly because of the increase of hydrophobicity and down‐regulation of some adhesion genes.     

Ultrastructural observations on bacterial invasion in cementum and radicular dentin of periodontally diseased human teeth

In this study the bacterial invasion in root cementum and radicular dentin of periodontally diseased, caries-free human teeth was examined. In addition, structural changes in these tissues, which could be related to the bacterial invasion, were reported. Twenty-one caries-free human teeth with extensive periodontal attachment loss were studied by light and scanning electron microscopy. At the base of the gingival pocket, bacteria were found in the spaces between remnants of Sharpey's fibers and their point of insertion in the cementum. In teeth that had been scaled and root planed, most of the root cementum had been removed. Bacterial invasion was found in the remaining root cementum. The invasion seemed to start as a localized process, often involving only one bacterium. In other areas bacteria were present in lacunar defects in the cementum. These lacunae extended into the radicular dentin. In 11 teeth bacteria had invaded the dentinal tubules. Most bacteria were located in the outer 300 microns of the dentinal tubules, although occasionally they were found in deeper parts. In two of the nontreated teeth, bacteria were detected on the pulpal wall. No correlation was found between the presence of bacterial invasion and the absence of radicular cementum. No bacteria were found in the portion of the root located apically to the epithelial attachment. These data are in agreement with our results from cultural studies of the bacterial flora in these structures. It was also demonstrated that in spite of meticulous scaling and root planning and personal oral hygiene, bacterial plaque remained present on radicular surfaces. Both the invaded dentinal tubules and the lacunae could act as bacterial reservoirs from which recolonization of treated root surfaces occurs. From these reservoirs bacteria could also induce pulpal pathoses. Since these bacterial reservoirs are not eliminated by conventional mechanical periodontal treatment, it seems appropriate to combine mechanical periodontal therapy with the use of chemotherapeutic agents.     

Microscopy of the dentine of enamel-free areas of rat molar teeth

Dentine of the enamel-free areas (EFA) of first mandibular molar teeth of rats aged between 20-150 days was examined by scanning electron microscopy. Demineralized sections were stained with Gram-Twort's stain and examined for dental caries. In newly erupted teeth, a few dentinal tubules opened on the EFA surface. As teeth became worn down, extensive areas of EFA showed numerous patent tubules. In primary dentine, no occlusion or partial occlusion of tubules was apparent at any age except at the occlusal surface. It is postulated that this surface narrowing was caused either by dentine debris or salivary deposits. The only other covering of tubule openings appeared to be a thin salivary pellicle which in older animals was plaque-like. Patent tubules in primary dentine contained odontoblast processes which even in worn teeth extended to EFA surfaces. Processes in adjacent enamel-covered dentine also extended to the enamel-dentine junction. Only a few examples of bacterial invasion of EFA dentine were seen: in younger animals bacteria occupied cracks in the EFA surface, but in older animals they also occupied tubules. The EFA dentine does not appear to respond to attrition by infilling of the tubules; the persistence of vital odontoblast processes in worn teeth suggests that dead tract formation is not extensive. Continuous occlusal wear may restrict the progress of caries in EFA but odontoblasts in the rat may be also physiologically and immunologically involved in restricting the ingress of bacteria into otherwise poorly protected EFA.     

In vivo study of bacterial invasion in root planed and citric acid treated radicular surfaces of periodontally involved human teeth

The purpose of this study was to investigate whether plaque bacteria invade the exposed radicular dentin after root planing or chemical root treatment in vivo. Eighteen caries-free human periodontally involved teeth with hopeless prognoses were studied. Fourteen teeth were scaled and root planed with hand curette type scalers. The proximal surface of each treated tooth was designated as the RP surface. The remaining half of the proximal surface was treated with citric acid (pH 1.0) for 3 minutes and was designated as the CA surface. Four untreated teeth served as controls. After 4 weeks, the teeth were extracted, and were processed for light microscopy and for scanning electron microscopy concerning bacterial invasion into the supragingival radicular dentin. The following results were obtained. 1. Radicular cementum was present on most untreated tooth surfaces. However, bacteria were never seen in the dentinal tubules. 2. Bacterial invasion into the dentinal tubules was observed in five of the 10 proximal surfaces (50% of the RP surfaces) and in nine of the 10 proximal surfaces (90% of the CA surfaces). 3. The depth (9.5 +/- 24.1 microns vs 84.6 +/- 136.3 microns) and percentage (0.8 +/- 2.1% vs 20.3 +/- 17.3%) of bacterial invasion in the dentinal tubules of the RP surfaces was lower than that of the CA surfaces. 4. Cocci and short rods were present in the supragingival dentinal tubules. 5. Since CA surfaces may accelerate bacterial invasion the citric acid treatment might be harmful in patients with inadequate plaque control.     

Dentine tubule infection and endodontic therapy implications

A critical review of the literature suggests that the microenvironment of dentinal tubules appears to favour the selection of relatively few bacterial types irrespective of the aetiology of the infection process; coronal dental caries or pulpar necrosis. These bacteria may constitute an important reservoir from which root canal infection and reinfection may occur following pulp necrosis or during and after endodontic treatment. Previous studies of this microflora have utilized microbiological culture techniques which need to be supplemented by those that allow In situ demonstration as well as identification of the bacteria. Newer treatment strategies that are designed to eliminate this microflora must include agents that can penetrate the dentinal tubules and destroy these microorganisms, since they are located in an area beyond the host defence mechanisms where they cannot be reached by systemically administered antimicrobial agents.     

Hemin nutritional stress inhibits bacterial invasion of radicular dentine by two endodontic anaerobes

AIM: To determine if anaerobic bacteria routinely found in infected dentine and root canals require the presence of heme in the environment in order for them to invade dentinal tubules. METHODOLOGY: Noncarious, unrestored human teeth with single root canals were prepared for invasion experiments and soaked in either TSB-M supplemented with hemin (5 microg mL(-1)) (n = 12 roots), TSB-M media (n = 12 roots) or TSB-M media followed by hemin soak (n = 12 roots) for 2 days, then inoculated with either Prevotella intermedia ATCC 25611 or Peptostreptococcus micros ATCC 33270 and incubated anaerobically for 14 days. Roots were prepared for light microscopy, stained with Brown and Brenn or antisera raised to the bacteria, and invasion within tubules assessed using a tubule invasion index (TI). Data were analysed using Student's t-test and Mann-Whitney U-test. RESULTS: Prevotella intermedia (TI = 0.7 +/- 0.04) and P. micros (TI = 0.96 +/- 0.08) showed low invasion when grown in the presence of hemin with cells generally restricted to the superficial 20 microm of the tubules, whilst neither bacteria invaded tubules (TI = 0) when hemin was absent from the growth media (P < 0.01). CONCLUSIONS: Hemin was required in the growth medium for P. intermedia and P. micros to invade dentinal tubules.     

伢典化学机械去龋法的扫描电镜研究

目的：利用扫描电镜技术观察伢典和传统车针去龋后窝洞表面的微观形态。方法：选取累及牙本质深层龋坏的离体牙9颗，分为对照组，机械组，伢典组3组，每组3颗。用扫描电镜观察窝洞表面形态。结果：对照组表面有很多碎屑、残渣等，无牙本质小管完整形态。传统车针组的牙本质表面有不规则的颗粒和碎片，有玷污层形成。牙本质小管口堵塞。伢典组的牙本质表面没有玷污层，牙本质小管口清晰可见。结论：伢典能有效去除玷污层。     

SEM investigations on the human sclerosed dentinal tubules

Sclerotic changes of dentinal tubules appeared in the dentin beneath the carious, attrition and abrasion lesions and in the transparent root dentin were investigated using the scanning electron microscopy. 1) Shape of crystal-like substances appeared in the dentinal tubules could not be closely related to kinds of dentin destruction. 2) In the dentinal tubules beneath the carious lesions, cuboidal or rhomboid-shaped, short rod-like, long rod-like, plate-like and droplet-like crystals were mainly observed. Combination of cuboidal or rhomboid-shaped and short rod-like crystals or that of long rod-like and plate-like crystals were most frequently observed. 3) In the dentinal tubules beneath attrition and abrasion lesions, cuboidal or rhomboid-shaped, short rod-like and droplet-like crystals were mainly observed, however, the size of cuboidal or rhomboid-shaped crystal was usually smaller than that observed beneath carious lesion and they deposited compactly within the tubules. 4) In the sclerosed dentinal tubules in the transparent root dentin, compact deposition of rhomboid-shaped and irregular sand-like crystals were usually observed. Size of rhomboid-shaped crystal is smaller than that observed beneath carious lesion. The deposit of sand-like crystal in the tubules was easily discriminated from the matrix of peritubular dentin. 5) In any case, the kinds of crystals and pattern of deposition were not necessarily same between the neighboring tubules. However, in a tubule, they were almost same in shape and mode of deposition throughout the entire length of tubule from the outer side to the pulpal side. These facts seem to indicate that mineral deposition within the tubule due to sclerosis is performed under individual condition. 6) Finding showing biological control of mineral deposition within the dentinal tubules by the odontoblasts could not be observed.     

Bacterial penetration and proliferation in root canal dentinal tubules after applying dentin adhesives in vitro

Endodontic treatment is aimed at eliminating infection and preventing bacterial regrowth in the root canal and dentinal tubules. In the present study the ability of two dentin adhesives to prevent bacterial penetration and subsequent proliferation in dentinal tubules was evaluated. Cylindrical root specimens prepared from freshly extracted bovine teeth were used in an in vitro model of dentinal tubule infection. After removal of the smear layer the intracanal dentinal tubules of the specimens were acid-etched and treated with either Gluma or EBS. Untreated specimens served as controls. Specimens were infected with Enterococcus faecalis and incubated in Brain Heart Infusion for 21 days. Powder dentin samples obtained from within the canal lumina, using ISO 025 to 033 burs, were examined for the presence of vital bacteria by inoculating on agar plates and counting colony-forming units. A significant difference was found between the experimental groups and the untreated group. After application with Gluma specimens showed the least viable bacteria in dentinal tubules. Data suggested that dentin adhesives reduced bacterial invasion into dentin and therefore have a potential role in endodontic treatment.     

Dentine hypersensitivity: the effects of brushing desensitizing toothpastes, their solid and liquid phases, and detergents on dentine and acrylic: studies in vitro

Dentine exhibiting symptoms of dentine hypersensitivity has tubules open at the dentine surface and patent to the pulp. The mechanisms whereby dentinal tubules are exposed is ill understood but probably involves a variety of abrasive and/or erosive agents. This study in vitro examined the quantitative and qualitative effects of toothpastes, their solid and liquid phases and detergents on dentine and acrylic. Abrasion of dentine and acrylic were measured by surfometry. Morphological changes to dentine were assessed by scanning electron microscopy. Abrasion of dentine and acrylic by toothpastes increased with increasing brushstrokes with marked differences in the extent of abrasion between different pastes. Brushing dentine with water or detergents produced progressive abrasion but which appeared to plateau around 2 μm loss. Water and detergents produced minimal effects on acrylic. At 5000 strokes dentine abrasion by solid phases was less than the parent toothpastes but the ranking order of abrasivity was the same as the parent toothpastes. Loss of dentine produced by liquid phases was minimal and in the order of 1–2 μm. Observationally, all toothpastes removed at least the dentine smear layer to expose many tubules; with one desensitizing product leaving a particulate deposit occluding most tubules. The solid phases of the toothpaste produced identical morphological changes to the parent paste. The liquid phases and detergents all exposed dentinal tubules by 5000 strokes. Water had little or no effect on the dentine smear layer. It is concluded that toothpastes, solid phase, liquid phase and detergents have the potential to abrade or erode dentine to a variable degree and result in tubule exposure. The effects of the liquid phases and detergents appear limited to the removal of the smear layer. Such detrimental effects seen in vitro could have relevance to the aetiology and management of dentine hypersensitivity. Toothpaste formulations which despite exposing tubules have ingredients capable of occluding tubules may be an area of development for such products.     

Ultrastructural localization of immunoglobulins in carious human dentine

The ultrastructural localization of IgG, IgM, IgA and the secretory component in carious dentine was studied in vital human molars and premolars using the direct peroxidase labelled antibody method and the indirect peroxidase-antiperoxidase method. These labelled proteins had similar localization and were found exclusively in the carious part of dentine. Positive reactions were noted (1) in bacterial cell walls and tissue remnants located in the superficial carious layer, (2) along the walls of the dentinal tubules and (3) in large and dense vacuoles located in the odontoblasts and their processes. The presence of secretory component in the deep layers of carious dentine is consistent with an exogenous salivary origin of this protein but the pulpal origin for IgA and especially for IgG and IgM cannot be excluded.