Bacterial species associated with persistent apical periodontitis exert differential effects on osteogenic differentiation.

AIM: To determine if bacteria associated with persistent apical periodontitis induce species-specific pro-inflammatory cytokine responses in macrophages, and the effects of this species-specific microenvironment on osteogenic differentiation. METHODOLOGY: Macrophages were exposed to Enterococcus faecalis, Streptococcus oralis, Streptococcus mitis, Fusobacterium nucleatum, Treponema denticola or Tannerella forsythia, and levels of TNF-α and IL-1ß elicited were determined by immunoassay. Following treatment of MG-63 pre-osteoblasts with conditioned media from bacteria-exposed macrophages, osteogenic differentiation and viability of osteoblasts were analyzed by Alizarin Red Staining and MTS assay, respectively. Statistical analysis was carried out by one-way anova with the Tukey post-hoc test. Differences were considered to be significant if P < 0.05. RESULTS: Macrophages exposed to Gram-positive bacteria did not produce significant amounts of cytokines. F. nucleatum-challenged macrophages produced up to four-fold more TNF-α and IL-1ß compared to T. denticola or T. forsythia. Only conditioned media from macrophages treated with Gram-negative bacteria decreased mineralization and viability of osteoblasts. CONCLUSIONS: Gram-positive bacteria did not impact osteogenic differentiation and appeared innocuous. Gram-negative bacteria, in particular F. nucleatum elicited an enhanced pro-inflammatory response in macrophages, inhibited osteogenic differentiation and reduced cell viability. The findings suggest that the presence of this organism could potentially increase the severity of persistent apical periodontitis.

Assessment of treatment failure in endodontic therapy.

There is a paucity of guidelines for the dental profession to assess failure of endodontic therapy. While a successful treatment can be well defined by the absence of apical periodontitis and clinical symptoms after a period of observation, failed treatment has escaped a distinct standing over the years. This article highlights aspects of significance and concludes that research ought to better explore the general health properties of persistent apical periodontitis on root-filled teeth and finally confirm the extent there is an association between apical periodontitis and adverse systemic health effects. Clearing this condition will determine whether clinicians should take a serious or relaxed attitude to persistent apical periodontitis subsequent to endodontic treatment.

Strains of Enterococcus faecalis differ in their ability to coexist in biofilms with other root canal bacteria.

AIM: To investigate the relationship between protease production and the ability of Enterococcus faecalis strains to coexist in biofilms with other bacteria commonly recovered from infected root canals. METHODOLOGY: Biofilms with bacteria in mono-, dual- and four-species communities were developed in flow chambers. The organisms used were Lactobacillus salivarius, Streptococcus gordonii and Actinomyces naeslundii and E. faecalis strains, GUL1 and OG1RF. Biovolume and species distribution were examined using 16S rRNA fluorescence in situ hybridization in combination with confocal microscopy and image analysis. The full proteome of the E. faecalis strains was studied using two-dimensional gel electrophoresis. Spots of interest were identified using tandem mass spectroscopy and quantified using Delta 2D software. RESULTS: All bacteria formed biofilms and an anova analysis revealed that the biofilm biomass increased significantly (P ≤ 0.01) between 6 and 24 h. L. salivarius, S. gordonii and A. naeslundii formed mutualistic biofilm communities, and this pattern was unchanged when E. faecalis GUL1 was included in the consortium. However, with OG1RF, L. salivarius and S. gordonii were outcompeted in a 24-h biofilm. Proteomic analysis revealed that OG1RF secreted higher levels of proteases, GelE (P = 0.02) and SprE (P = 0.002) and a previously unidentified serine protease (P = 0.05), than GUL1. CONCLUSIONS: Different strains of E. faecalis can interact synergistically or antagonistically with a consortium of root canal bacteria. A possible mechanism underlying this, as well as potential differences in virulence, is production of different levels of proteases, which can cause detachment of neighbouring bacteria and tissue damage.

Estimates of sensitivity and specificity of electric pulp testing depend on pulp disease spectrum: a modelling study.

AIM: To demonstrate how the spectrum of diseased pulps may influence sensitivity and specificity in diagnostic studies on pulp status. METHODOLOGY: An original sample from a previous study consisting of 59 teeth scheduled for root canal treatment was used where the relationship between the response to electric pulp testing and the visual status of the pulp was evaluated. To alter the spectrum of diseased pulps, a hypothetical sample of asymptomatic teeth with deep caries lesions was added to the original sample. Sensitivity and specificity were then compared for the two samples. RESULTS: In the original sample of 59 teeth, sensitivity was 72% and specificity 90%. When the spectrum of diseased pulps was altered, sensitivity decreased to 67% and specificity increased to 97%. The change in disease spectrum also decreased the prevalence of necrotic pulps. CONCLUSIONS: The spectrum of diseased pulps included in a diagnostic study on the accuracy of electric pulp testing, and indirectly also disease prevalence (here pulp necrosis), influences estimates of sensitivity and specificity. This implies that estimates of diagnostic accuracy from one study with a particular tooth population spectrum may not apply to another tooth population with a different disease spectrum.

Fusobacterium nucleatum induces cytokine production through Toll-like-receptor-independent mechanism.

AIM: To determine whether Fusobacterium nucleatum's ability to invade cells allows the bacteria to activate pro-inflammatory response through cytosolic pattern recognition receptors, independent of surface Toll-like receptors (TLRs). METHODOLOGY: HEK293T cells, which lack endogenous TLRs, and overexpressing dominant negative myeloid differentiation primary response gene 88 (MyD88DN) protein, were infected with F. nucleatum and the production of interleukin-8 (IL-8) was determined. The necessity for intracellular invasion of the bacteria for cytokine production was also investigated by blocking bacterial invasion with cytochalasin D. The roles of NFĸB and p38 mitogen-activated protein kinase (MAPK) and nucleotide-binding oligomerization domain-1 (NOD-1) signalling pathways in F. nucleatum-induced IL-8 secretion were determined. RESULTS: Fusobacterium nucleatum-infected HEK293T cells produced IL-8 independent of the MYD88 signalling. This response was inhibited by preventing F. nucleatum invasion into HEK293T cells. p38 MAPK but not the NFĸB signalling pathway was required for F. nucleatum-mediated IL-8 production. HEK293T cells expressed NOD-1 but not NOD-2. Yet, inhibition of NOD-1 signalling did not affect F. nucleatum-induced IL-8 secretion. CONCLUSIONS: Fusobacterium nucleatum invasion led to cytokine production, which is mediated by the p38 MAPK signalling but independent of TLRs, NOD-1, NOD-2 and NFĸB signalling.

Radiological diagnosis of periapical bone tissue lesions in endodontics: a systematic review.

This systematic review evaluates the diagnostic accuracy of radiographic methods employed to indicate presence/absence and changes over time of periapical bone lesions. Also investigated were the leads radiographic images may give about the nature of the process and the condition of the pulp in nonendodontically treated teeth. Electronic literature search included the databases PubMed, Embase and CENTRAL from January 1950 to June 2011. All languages were accepted provided there was an abstract in English. The MeSH terms were 'Cone beam computed tomography (CBCT)', 'Radiography, panoramic', 'Periapical diseases', 'Dental pulp diseases', 'Sensitivity and specificity', 'receiver operating characteristics (ROC) curve', 'Cadaver', 'Endodontics' and 'Radiography dental'. Two reviewers independently assessed abstracts and full text articles. An article was read in full text if at least one of the two reviewers considered an abstract to be potentially relevant. Altogether, 181 articles were read in full text. The GRADE approach was used to assess the quality of evidence of each radiographic method based on studies of high or moderate quality. Twenty-six studies fulfilled criteria set for inclusion. None was of high quality; 11 were of moderate quality. There is insufficient evidence that the digital intraoral radiographic technique is diagnostically as accurate as the conventional film technique. The same applies to CBCT. No conclusions can be drawn regarding the accuracy of radiological examination in identifying various forms of periapical bone tissue changes or about the pulpal condition.

Diagnosis of the condition of the dental pulp: a systematic review.

The aim of this systematic review was to appraise the diagnostic accuracy of signs/symptoms and tests used to determine the condition of the pulp in teeth affected by deep caries, trauma or other types of injury. Radiographic methods were not included. The electronic literature search included the databases PubMed, EMBASE, The Cochrane Central Register of Controlled Trials and Cochrane Reviews from January 1950 to June 2011. The complete search strategy is given in an Appendix S1 (available online as Supporting Information). In addition, hand searches were made. Two reviewers independently assessed abstracts and full-text articles. An article was read in full text if at least one of the two reviewers considered an abstract to be potentially relevant. Altogether, 155 articles were read in full text. Of these, 18 studies fulfilled pre-specified inclusion criteria. The quality of included articles was assessed using the QUADAS tool. Based on studies of high or moderate quality, the quality of evidence of each diagnostic method/test was rated in four levels according to GRADE. No study reached high quality; two were of moderate quality. The overall evidence was insufficient to assess the value of toothache or abnormal reaction to heat/cold stimulation for determining the pulp condition. The same applies to methods for establishing pulp status, including electric or thermal pulp testing, or methods for measuring pulpal blood circulation. In general, there are major shortcomings in the design, conduct and reporting of studies in this domain of dental research.

Response to alkaline stress by root canal bacteria in biofilms.

AIM: To determine whether bacteria isolated from infected root canals survive alkaline shifts better in biofilms than in planktonic cultures. METHODOLOGY: Clinical isolates of Enterococcus faecalis, Lactobacillus paracasei, Olsenella uli, Streptococcus anginosus, S. gordonii, S. oralis and Fusobacterium nucleatum in biofilm and planktonic cultures were stressed at pH 10.5 for 4 h, and cell viability determined using the fluorescent staining LIVE/DEAD BacLight bacterial viability kit. In addition, proteins released into extracellular culture fluids were identified by Western blotting. RESULTS: Enterococcus faecalis, L. paracasei, O. uli and S. gordonii survived in high numbers in both planktonic cultures and in biofilms after alkaline challenge. S. anginosus, S. oralis and F. nucleatum showed increased viability in biofilms compared with planktonic cultures. Alkaline exposure caused all planktonic cultures to aggregate into clusters and resulted in a greater extrusion of cellular proteins compared with cells in biofilms. Increased levels of DnaK, HPr and fructose-1,6-bisphosphate aldolase were observed in culture fluids, especially amongst streptococci. CONCLUSIONS: In general, bacteria isolated from infected roots canals resisted alkaline stress better in biofilms than in planktonic cultures, however, planktonic cells appeared to use aggregation and the extracellular transport of specific proteins as survival mechanisms.

The effect of educational intervention on the adoption of nickel-titanium rotary instrumentation in a Public Dental Service.

AIM: To study the influence of two educational programmes on the adoption of nickel-titanium rotary instrumentation (NTRI) amongst general dental practitioners in a short-term as well as a long-term perspective. METHODOLOGY: All dentists employed in the Gothenburg Dental service (n = 148) were enrolled in the study. The clinics in the organization were randomly assigned to one of two educational programmes. In the first programme a 4-h lecture on root canal instrumentation was given. In the second programme the lecture course was supplemented by a 6-h hands-on training session. The short-term effect was measured by a questionnaire distributed 6 months after completed education. The long-term effect was evaluated 4 years later. RESULTS: The overall utilization rate of NTRI increased from 4% to 73%. However, lectures in combination with hands-on training resulted in a better short-term acceptance rate (94%) than if teaching was given only in lecture-format (53%) (P = 0.000). As a consequence, all staff were offered hands-on training. The long-term adoption rate was 88%. Reasons for accepting the new technology usually were found within the 'relative advantage' category. Common reasons for dentists not to adopt NTRI were that they could not get started or that they found no advantage over the old technology. CONCLUSIONS: The short-term adoption of a new technology might be influenced by the design of an introductory educational programme. For clinical procedures, such as root canal instrumentation, the inclusion of hands-on training sessions seems to be important to reach a high acceptance rate.

Improved quality of root fillings provided by general dental practitioners educated in nickel-titanium rotary instrumentation.

AIM: To test the hypothesis that an increased utilization of nickel-titanium rotary instrumentation (NTRI) by general dental practitioners will lead to an increased frequency of good quality root fillings. A second aim was to determine whether the educational format would exert influence on the quality. METHODOLOGY: Dentists were assigned at random to three intervention groups: a 4-h lecture (L-group, n = 40); a 4-h lecture plus a full day hands-on course (LH-group, n = 40); or a control group receiving no instruction (n = 68). The control group received education later on in the study. Radiographs of two root filled molar teeth per dentist were selected at random before the start of the education program and after a 6-month clinical learning period. Using length, seal and shape of root-fillings a 5-level variable was created. RESULTS: The rate of good quality root fillings increased after the introduction of NTRI. Calculated over all types of roots the frequency of excellent (score 1) root fillings increased from 31% to 51% (P = 0.006) in the L-group and from 27% to 47% (P = 0.016) in the LH-group. The frequency of low quality root-fillings (score 5) dropped in the L-group from 22% to 16% (P = 0.29) and in the LH-group from 13% to 9% (P = 0.48). No statistically significant difference was seen among the controls. CONCLUSIONS: When NTRI technology replaced manual stainless steel techniques the rate of good quality root fillings increased. A significant drop in the rate of low quality root fillings was not found.

Low HEMA conjugation induces high autoantibody titer in mice.

2-hydroxyethylmethacrylate (HEMA) is a known causal agent of hypersensitivity to resin composites. We have reported that immunization with HEMA conjugated to mouse serum albumin (MSA) induces an autoantibody response in mice. In this study, we investigated both the activity and the avidity of autoantibodies induced by immunization with various HEMA conjugations to MSA. Female Balb/c mice were given MSA carrying 3, 7, 15, or 22 HEMA molecules. Antigen-specific IgG and IgE antibodies were determined by ELISA, and average antibody avidity by thiocyanate dissociation. Immunization with MSA carrying the lowest number of HEMA molecules induced a significantly higher IgG and IgE anti-MSA autoantibody response, with significantly higher IgG antibody avidity, than did the more heavily conjugated preparations. The results suggest that the lower the degree of HEMA conjugation to self-protein, the higher the risk for autoantibody production to the carrier protein. These findings suggest a mechanism of potential relevance in humans.

Langerhans cells from human oral epithelium are more effective at stimulating allogeneic T cells in vitro than Langerhans cells from skin.

This report is focused on the functional capacity of Langerhans cells (LC) in the epithelium of skin and oral mucosa, which both meet different antigenic challenges. The capacity of LC from human oral and skin epithelium to provide co-stimulatory signals to T cells in vitro was compared. LC in a crude suspension of oral epithelial cells had a significantly enhanced T cell co-stimulatory capacity compared to skin epithelial cells. This applied both to cultures with concanavalin A (con-A)-stimulated syngeneic T cells and to a mixed epithelial cell lymphocyte reaction involving allogeneic T cells. The co-stimulatory capacity of oral and skin epithelial cells was reduced by >70% if monoclonal antibodies against HLA-DR, -DP and -DQ were added to the cultures with allogeneic T cells, indicating the involvement of HLA class II expressing LC. Immunohistochemistry revealed that 6% of the epithelial cells were CD1a + LC in sections from both oral and skin epithelium. Interleukin (IL)-8 production was higher in cultures of oral epithelial cells and con-A stimulated T cells than in corresponding cultures with skin epithelial cells as accessory cells. The results suggest that LC in human oral epithelium are more efficient at stimulating T cells than those of skin.

CONTROVERSIES IN ENDODONTICS.

Diseases of the dental pulp often have an infectious origin, and treatments are aimed to control infections of the root canal system. Endodontic treatment principles originally evolved on the basis of trial and error, and only in recent decades have scientific methods been adopted to support clinical strategies. Yet, relevant research on the disease processes, their diagnoses, and efficient treatment are rare in the endodontic literature. Hence, the advancement of biologically based knowledge significant to clinical endodontics has been slow. Therefore, many differences of opinion still prevail in this field of dentistry. This review highlights and analyzes the background of some of the more heavily debated issues in recent years. Specifically, it deals with disagreements regarding the clinical management of pulpal exposures by caries in the adult dentition, definitions of success and failure of endodontic therapy, and causes of and measures to control infections of the root canal system. Clearly, a most apparent gap in the published endodontic literature is the lack of randomized clinical trials that address the more significant controversial matters relating to the management of pulpal wounds, medication, and the number of appointments required for the treatment of infected root canals. However, trials in endodontics require extremely long follow-up periods if valid conclusions are to be generated. Therefore, it is not to be expected that there will be rapid solutions to these issues in the foreseeable future.

T-cell costimulatory capacity of oral and skin epithelial cells in vitro: presence of suppressive activity in supernatants from skin epithelial cell cultures.

Oral Langerhans cells (LC) have better T-cell costimulatory capacity than skin LC. In this study factors affecting this capacity have been assessed in a mixed epithelial cell lymphocyte reaction (MELR) assay. Flow cytometry analysis of freshly recovered cells revealed major histocompatibility complex (MHC) class II molecule expression on 7.5% of the oral epithelial cells and 9.7% of the skin epithelial cells. Monoclonal anti class II antibodies significantly reduced the T-cell proliferation in the MELR. Pretreatment of skin epithelial cells with interleukin-1beta, tumour necrosis factor-alpha or interferon (IFN)-gamma did not affect the MELR proliferation, but incubation with IFNgamma significantly suppressed the T-cell response. Transfer of supernatants from cultures of skin epithelial cells and allogeneic T cells to cultures of oral epithelial cells and T cells resulted in a reduced T-cell proliferation while supernatants from oral epithelial cells and T cells did not reduce proliferation. The higher proliferation in cultures of T cells and oral epithelial cells than in cultures containing skin epithelial cells may be due to the presence of a suppressive factor in the skin epithelial cell suspensions.

Bacterial status in root-filled teeth exposed to the oral environment by loss of restoration and fracture or caries--a histobacteriological study of treated cases.

AIM: To describe histological and microbiological findings in teeth where root fillings had been exposed to caries and the oral environment for a prolonged period. METHODOLOGY: For inclusion in the study, only teeth with a follow-up period of 3 years or more and those that had been without proper restoration for at least a period of 3 months were considered. Some root fillings had been without restoration for several years. In all, 39 roots representing 32 teeth were examined by histology. RESULTS: The majority of the specimens were without a discernible periapical bone lesion as assessed by radiography. Osteolytic lesions were seen with five roots. Longitudinal tissue sections stained with a modified Brown/Brenn staining technique revealed presence of stainable bacteria in abundance at the canal entrance and in dentinal tubules but were absent mid-root and apically in all but two specimens. Soft tissue attached to the root tip and in apical ramifications displayed distinct inflammatory cell infiltrates, suggesting microbial exposure in 7 of the 39 roots examined. In all other specimens, inflammatory cell infiltrates were either nonexistent or sparse and then associated with extruded sealer material. CONCLUSIONS: Well-prepared and filled root canals resist bacterial penetration even upon frank and long-standing oral exposure by caries, fracture or loss of restoration.

Bacteria recovered from teeth with apical periodontitis after antimicrobial endodontic treatment.

UNLABELLED: Chávez de Paz LE, Dahlén G, Molander A, Möller A, Bergenholtz G. Bacteria recovered from teeth with apical periodontitis after antimicrobial endodontic treatment. International Endodontic Journal, 36, 500-508, 2003. AIM: To determine whether there is a pattern for certain bacteria to remain after chemo-mechanical treatment of root canals in teeth with apical periodontitis. METHODOLOGY: Consecutive root-canal samples of 200 teeth receiving root-canal treatment, referred from general practitioners and endodontic specialists for analyses of cultivable microbes, were studied prospectively. To be included, samples had to be taken at a treatment session subsequent to the one at which endodontic therapy was initiated. All samples were from teeth that either presented with clinical or radiographic evidence of apical periodontitis or both. Bacteriological findings were linked to clinical and radiographic parameters including status of the root canal prior to treatment, namely, vital pulp, necrotic pulp or root filled. RESULTS: A total of 248 strains were isolated from 107 teeth giving bacterial growth. Gram-positives predominated (85%). Lactobacillus spp. (22%), nonmutans streptococci (18%), and Enterococcus spp. (12%) were the most common isolates. Gram-negative anaerobes were relatively sporadic. Large radiographic bone lesions, persistent pain and use of intracanal calcium hydroxide dressing correlated with bacterial presence (P < 0.05). CONCLUSIONS: Once established, nonmutans streptococci, enterococci and lactobacilli appear to survive commonly following root-canal treatment of teeth with clinical and radiographical signs of apical periodontitis.

HEMA bound to self-protein promotes auto-antibody production in mice.

While several studies report that acrylic monomers contained in dental materials may cause hypersensitivity reactions, little is known of the associated immune response. Here we address the potential of 2-hydroxyethyl-methacrylate (HEMA) to bind to endogenous protein and elicit auto-antibody production in vivo. Albumin was incubated with HEMA at various times and pH. Following confirmation of the conjugation by inhibition of trinitrophenyl (TNP) binding, female Balb/c mice received HEMA conjugated to mouse serum albumin (MSA) in Freund's incomplete adjuvant or saline subcutaneously. ELISA was used to determine the serum antibody responses to native and modified MSA. IL-2 production in spleen cell cultures stimulated with HEMA-conjugated MSA was measured. HEMA reacted with serum albumin at physiological conditions. HEMA-conjugated MSA induced IL-2 secretion and production of IgG antibodies to native MSA. The results suggest that modification of an endogenous protein like serum albumin with HEMA may defeat the control of immune responses to this self-protein.

Root-dentin sensitivity following non-surgical periodontal treatment.

BACKGROUND, AIMS: Little clinical data exist on the incidence and severity by which root dentin sensitivity (RDS) results from periodontal therapy. The aim of the present clinical trial was to study the degree to which a sample of patients requiring non-surgical periodontal treatment develops RDS. MATERIAL AND METHODS: Alterations in RDS was followed in 35 patients (29-65 years of age) requiring non-surgical treatment for moderate to advanced periodontal disease. Inclusion criteria for participation were need for periodontal treatment in at least 2 quadrants comprising a minimum of 4 teeth with vital pulps, no open caries lesions, no dental treatment in the last 3 months and no ongoing treatment for RDS. Baseline and follow-up recordings included responses of teeth to pain stimuli (directed compressed air) at buccal surfaces as graded by the patient on a 10-cm visual analogue scale (VAS). Periodontal therapy consisted of oral hygiene instruction (OH) followed by supra- and subgingival scaling/root planing by hand and ultrasonic instrumentation of one quadrant per each of the subsequent weeks. Thus, follow-up data included pain assessment after 1-3 weeks of OH alone, and 1-4 weeks post-instrumentation. RESULTS: There was a statistically significant reduction in mean VAS scoring over time in quadrants where only meticulous plaque control had been maintained, while VAS mean values increased significantly after instrumentation (p<0.001). Also the % of subjects reporting higher mean VAS values increased after instrumentation. Changes in mean VAS scores were generally moderate and only 9 patients gave an increase on VAS of >2 cm for 3 or more teeth. A statistically significantly higher increase of RDS was observed for initially sensitive teeth (VAS>0) than for teeth not responding at baseline (p<0.001). Although a reduction in the intensity of RDS could be noticed during the later phase of the 4-week follow-up period after scaling and root planing, the percentage of sensitive teeth remained unchanged. CONCLUSION: The data confirm that meticulous plaque control will diminish RDS problems and that scaling and root planing procedures in periodontal therapy result in an increase of teeth that respond to painful stimuli. However, pain experiences in general appeared minor and only a few teeth in a few patients developed highly sensitive root surfaces following instrumentation.

Periapical status of root-filled teeth exposed to the oral environment by loss of restoration or caries.

OBJECTIVE: Studies in vitro carried out on extracted teeth have demonstrated that bacterial elements may penetrate root fillings from the coronal to the apical end after a period of exposure to artificial saliva or bacterial culture. To address the clinical significance of this so-called issue of coronal leakage, a retrospective cohort analysis was conducted of 55 patients with root fillings that had been exposed to the oral environment because of caries or absent restorations. STUDY DESIGN: Cases were matched 1-to-1 with regard to initial pulpal and periapical diagnosis, period after completion of endodontic therapy, tooth type, age of the patient, and the technical quality of the root filling. Only cases with a follow-up period of 3 years or more were included. Radiographs taken at the last follow-up examination were subjected to a masked evaluation. RESULTS: A total of 14 osteolytic lesions were recorded. In 43 of the 55 matched pairs (78%), there were identical periapical conditions. In 9 pairs, a periapical lesion was present in the "open" tooth category, whereas in 3 pairs, a periapical lesion was seen exclusively in the "intact" tooth. Though the odds-ratio for a lesion to be present in the "open group" was 3. 0, this was not a statistically significant result (P >.10). CONCLUSIONS: Data suggest that the problem of coronal leakage may not be of such a great clinical importance as implicated by numerous studies in vitro, provided instrumentation and root fillings are carefully performed.