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.

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.

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.

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.

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.

Endotoxic activity in teeth with necrotic pulps.

From root canals of 13 teeth with necrotic pulps, samples were taken for analysis of endotoxin content by means of the Limulus by lysate technique. The results were related to the viable bacteria count of root canal samples, phenol/water-extracted lipopolysaccharides (LPS) from different gram-negative anaerobic bacteria, and to different fractions of inflammation exudate. It was found that the endotoxic activity of the root canal samples was correlated with the presence and the number of gram-negative bacteria in the root canal.

Dental pulp response to bacterial cell wall material.

Lipopolysaccharides (LPS) from Bacteroides oralis and Veillonella parvula and cell wall material from Lactobacillus casei were studied for their capacity to induce leukocyte migration in the dental pulp and in an implanted wound chamber. Three adult monkeys were challenged using lyophilized material sealed into buccal Class V cavities prepared in dentin. Pulp tissue responses were observed histologically eight and 72 hours after initiation of the experiment. Subjacent to cut dentinal tubules, bacterial materials induced polymorphonuclear leukocyte (PMN's) infiltration in the pulp tissue of the majority of test teeth examined. Responses were similar for the three bacterial test materials at both time periods. Topical applications of bovine serum albumin (BSA), used as a control, induced significantly less accumulation of PMN's. Assessments of induced exudate volumes and leukocyte densities in chambers implanted in rats showed comparable rankings with pulpal experiment between test (i.e., bacterial) and control (BSA) materials. Analysis of the data indicates that high-molecular-weight complexes of bacterial cell walls may adversely affect pulpal tissue across freshly exposed dentin.

Immunocompetent cells in the normal dental pulp.

The existence and location of various immunocompetent cells in the human dental pulp were investigated. Pulp tissue for analysis was obtained both from clinically intact pre-molars and from third molars without restorations or caries. Frozen and acetone-fixed pulp tissue sections were subjected to indirect immunohistochemistry with monoclonal antibodies to the following cell types: all peripheral T cells, helper/inducer T cells, cytotoxic/suppressor T cells, macrophages, B cells, and Class II antigen-expressing cells. Dendritic cells expressing Class II antigens (HLA-DR, -DQ), indicating a capacity for presentation of antigen to T helper cells, were seen in the odontoblastic layer as well as in the central portions of the pulp tissue. T lymphocytes, divided into helper/inducer and cytotoxic/suppressor cells, were observed in all pulp specimens. B cells were not seen in any of the pulp samples examined. The data demonstrate that the human dental pulp is equipped with immunocompetent cells essential for the initiation of immunological responses.

Dendritic cells and macrophages expressing class II antigens in the normal rat incisor pulp.

This study has identified and characterized class II (Ia) antigen-expressing cells in the normal rat incisor pulp by immunohistochemistry and flow cytometry. Two types of Ia-expressing cells occurred: one with a pronounced dendritic appearance located primarily in the periphery of the pulp, and one with morphological characteristics similar to those of macrophages. The latter cells were mainly observed in the central portion of the pulp. A numerical ratio of 1:4 was established between the two cell types. The existence of Ia-expressing cells suggests an inherent capacity of the pulp to process and present foreign antigens.

Inhibition of bacterial growth under composite restorations following GLUMA pretreatment.

The purpose of this investigation was to test in five adult monkeys the effects of a glutaraldehyde-containing dentin bonding agent, GLUMA, on bacterial colonization in Class V cavities restored with composite resin. Experimental groups consisted of immediate placement of GLUMA and composite resin as well as placement of GLUMA or Scotchbond (control) in acid-etched cavities that had been left open to the oral environment for 48 hours. Various procedures for pretreatment of the cavities were included. Tissue specimens were prepared for light microscopy for observation of bacterial presence and pulp tissue reactions after eight days and 90 days. Bacteria were not detected in any of the 54 cavities treated with GLUMA regardless of observation period or use of enamel-etching procedure prior to placement of composite resin. When cavities were restored with composite resin without prior GLUMA pretreatment or with Scotchbond, bacteria were present under the majority of restorations at both time intervals. Pulpal inflammation of varying extent and character was seen after eight days in teeth that had been previously infected. At 90 days, pulps showed repair and healing regardless of treatment protocol. Data indicate that GLUMA has a distinct in vivo antibacterial effect that seems to prevent bacterial growth in tooth/restoration interfaces.

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.

Effects of unpolymerized resin components on the function of accessory cells derived from the rat incisor pulp.

Monomeric resin components from dental composites are toxic to fibroblasts in culture and thus may interfere with the local immune system of the pulp, reducing its effective defense potential, either by cytotoxicity or by a more specific immune mechanism. Therefore, the present study was undertaken to observe the cytotoxic effects elicited by certain unpolymerized components of resin composites upon the function of accessory pulp cells in mitogen-induced proliferation of T-lymphocytes. Accessory cells from the rat incisor pulp were released following enzymatic digestion with collagenase. The assay included incubation of these cells with purified T-lymphocytes from cervical lymph nodes for 72 h in the presence of different concentrations of the resin components. The proliferative T-lymphocyte response was monitored by 3H-thymidine incorporation. Initially, we conducted experiments on spleen cells to determine the proper concentration intervals for suitable testing of the resin components. To assess the individual susceptibility of accessory cells and T-lymphocytes, we pre-treated each of these cells with some of the test materials prior to assay. At low concentrations, urethane dimethacrylate (UDMA), bisglycidyl methacrylate (bis-GMA), triethylene glycol dimethacrylate (TEGDMA), and bis-phenol A (BPA) increased spleen cell proliferation to concanavalin A (con A). Purified T-lymphocytes stimulated by pulpal cells did not show enhanced responses to UDMA, bis-GMA, glycidyl mehtacrylate (GMA), or N,N,-dihydroxyethyl-p-toluidine (DHEpT). At higher concentrations, all substances except camphoroquinone (CAMP) showed inhibitory effects in both test systems. The in vitro study shows that resin components can evoke either immunosuppression or immunostimulation on mitogen-driven proliferation of purified T-lyumphocytes and spleen cells.

Difference in capacity between macrophages and dendritic cells from rat incisor pulp to provide accessory signals to concanavalin-A-stimulated T-lymphocytes.

The present study compared the ability of dendritic cells and macrophages derived from the dental pulp to provide accessory signals to Concanavalin A (Con A)-stimulated T-lymphocytes. Pulpal cells from maxillary and mandibular rat incisors were enzymatically released with collagenase. T-lymphocytes were isolated from rat cervical lymph nodes. In initial experiments, suspensions of unseparated pulpal cells were found to provide co-stimulatory help to Con-A-treated T-lymphocytes. The proliferation rate correlated well with the number of cells in the pulp suspension and followed a time course characteristic of a Con-A-driven proliferation of T-lymphocytes. Depletion of class II molecule-expressing cells from the unpurified suspension of pulpal cells resulted in lost ability to provide accessory signals to Con-A-stimulated T-lymphocytes. In contrast, removal of ED2-positive cells, i.e., macrophages, did not affect the ability of the suspension to give this assistance. Partially purified class II molecule-expressing cells enhanced the proliferative response, while addition of enriched macrophages did not. It was concluded that cells in the normal dental pulp with the characteristics of dendritic cells have the capacity to provide help to Con-A-stimulated T-lymphocytes, while cells with the macrophage phenotype lack this ability.

Reduced in vivo cell-mediated immune responses to mumps, tuberculin, and streptokinase/streptodornase but not to Candida albicans in oral lichen planus.

Oral lichen planus is considered to be a T-cell-mediated disease. The purpose of this study was to investigate the capacity of T-lymphocytes in oral lichen planus patients to respond to a number of commonly encountered environmental antigens in vivo. To do this, we assessed dermal delayed-type hypersensitivity responses to mumps, streptokinase/streptodornase, Candida albicans, and purified protein derivative of tuberculin (PPD) in 17 oral lichen planus patients and in matched controls. Reduced induration in response toward mumps, PPD, and streptokinase/streptodornase was demonstrated in oral lichen planus patients compared with controls. In addition, the total sum of induration diameters was decreased in the patients. However, C. albicans stimulation resulted in similar levels of response in both groups. The differences in induration size between matched patients and controls for mumps and PPD were thus significantly greater than the corresponding differences for the C. albicans antigen. This suggests that a selective difference in the response to these antigens exists in oral lichen planus patients. The results may point to a loss of memory T-helper function to infrequently encountered environmental antigens, represented by mumps, PPD, and streptokinase/streptodornase, contrarily to memory function to common antigens (C. albicans), which seem to be unaffected.

Langerhans cells from oral epithelium are more effective in stimulating allogeneic t-cells in vitro than Langerhans cells from skin epithelium.

Dendritic cells, such as Langerhans cells (LC), in different ectodermal compartments may have different functional capabilities. The present study was undertaken to compare oral Langerhans cells (LC) with those of the epidermis in terms of their ability to co-stimulate T-cells in vitro. A Mixed Epithelial Cell Lymphocyte Reaction (MELR) and a mitogen-driven (concanavalin A) T-cell proliferation assay were used. In both assays, LC in a crude cell suspension of freshly isolated oral epithelial cells were found to be five times more effective in mediating T-cell proliferation than freshly isolated epidermal LC. Twenty-four-hour cell culture at 37 degrees C enhanced the T-cell response in the MELR compared with cells cultured at 4 degrees C. This applied to both skin and oral epithelial cells. Oral and skin epithelial cell suspensions depleted of LC lost the capacity to stimulate allogeneic T-cells. Incubation of the epithelial cell suspensions with recombinant Granulocyte/Macrophage-Colony Stimulating Factor (rGM-CSF) did not enhance the co-stimulating capacity of the LC. Titration of different numbers of oral and skin LC to T-cells showed that skin LC were never able to reach more than 44% of the maximal stimulatory capacity of oral LC. Data show that oral LC are more efficient than skin LC in providing co-stimulatory signals to T-cells, suggesting a difference in functional capacity between the two cell populations.

Structural and functional association between substance P- and calcitonin gene-related peptide-immunoreactive nerves and accessory cells in the rat dental pulp.

Defense mechanisms of the dentin/pulp complex involve a variety of biological systems in which immunocompetent cells, the nervous system, and the vascular supply play important roles. In the present study, pulpal accessory cells were examined regarding (i) their structural relationship to nerves and (ii) how the functional capacities of these cells were affected by neuropeptides. Micro-anatomic association was investigated in the normal rat molar pulp with the use of double-immunofluorescence staining and dual-channel confocal laser scanning microscopy. Examinations of confocal laser scanning microscopic images from single focal planes revealed the presence of apparent contacts between thin, varicose nerve fibers and immunocompetent cells, indicating proximity between these two structures. The close associations were most frequently observed in the para-odontoblastic region of the coronal pulp, where more than 70% of class II antigen-expressing (OX6+) cells showed proximity to nerve fibers immunoreactive to calcitonin gene-related peptide. The corresponding figure for substance P was about 50%. ED2+ macrophages closely associated with nerves were less frequently observed. Functional studies conducted in vitro demonstrated that 10(-9) to 10(-7) mol/L of substance P significantly increased (p < 0.05), while 10(-7) to 10(-6) mol/L of calcitonin gene-related peptide suppressed (p < 0.01) proliferation of purified T-lymphocytes stimulated with sub-optimal concentrations of concanavalin A in the presence of rat incisor pulpal cells as accessory cells. These data suggest that pulpal sensory nerve fibers and their products may have an influence upon the immune defense of the dental pulp.

Endodontic complications following periodontal and prosthetic treatment of patients with advanced periodontal disease.

A retrospective analysis was carried out to determine the frequency and onset of endodontic complications occurring in 52 patients treated for advanced periodontal disease. Comparisons were made between teeth which, following periodontal treatment, were used as abutments in fixed prosthetic reconstructions and nonabutment teeth. The study included 672 teeth with initially vital pulps (255 abutment teeth and 417 nonabutment teeth). The observation period varied from 4 to 13 years with a mean of 8.7 years. Pulpal necrosis including periapical lesions developed with a significantly higher frequency in abutment teeth than in nonabutment teeth (15% vs. 3%). The majority of these lesions did not appear until several years following the completion of active treatment. Conceivable reasons for the development of pulpal necrosis in teeth subjected to combined periodontal and prosthetic treatment are discussed.