Association between bacteria occurring in the apical canal system and expression of bone-resorbing mediators and matrix metalloproteinases in apical periodontitis.

AIM: To evaluate the association between the presence of selected bacterial species/groups in the apical root canal and expression of mediators of soft and bone tissue destruction in apical periodontitis lesions. Relationships between bacteria and some other features of apical periodontitis were also investigated. METHODOLOGY: Seventeen freshly extracted teeth with pulp necrosis and apical periodontitis were included. The apical root segment was sectioned and cryopulverized; DNA was extracted and evaluated for the presence of 9 bacterial species/groups using real-time polymerase chain reaction. Lesions were processed for histopathological and immunohistochemical analyses, which targeted matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9), receptor activator of NFκB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG). Associations of the target bacteria with expression of these mediators, presence of symptoms, lesion size and histopathological diagnosis were evaluated. Data were analysed using the chi-square, Fisher's exact, Mann-Whitney and Pearson tests. P values lower than 0.05 were considered significant. RESULTS: All pulverized apical root samples were positive for bacteria. The most prevalent taxa were Actinobacteria (53%), Streptococcus species (35%), Fusobacterium species and Parvimonas micra (18%). The target mediators exhibited a high mean expression in the lesions (MMP-2: 82%; MMP-9: 73%; RANK: 78%; RANKL; 81%; OPG; 83%). Mean RANKL:OPG ratio was significantly higher in granulomas than cysts (P < 0.05, Mann-Whitney test). Actinobacteria were associated with granulomas, higher MMP-2 expression, lower OPG expression, and higher RANKL:OPG ratio (P < 0.05 for all, Fisher's exact test or Mann-Whitney test). No other significant associations were found. CONCLUSION: Actinobacteria may play an important role in the active phase of soft and bone tissue destruction in apical periodontitis.

Methyl mercaptan and hydrogen sulfide products stimulate proinflammatory cytokines in patients with necrotic pulp tissue and endodontically treated teeth.

Bacterial infections of the residual dentin or infected pulp tissue are responsible for most cases of endodontic treatment failures. Persisting microorganisms in necrotic pulp tissue produce sulphur components such as methyl mercaptan and hydrogen sulfide as well as thioether derivatives. Although there is emerging evidence that these sulphur compounds stimulate immune cells and induce the inflammatory cascade, the immunological mechanisms of local and systemic inflammation have not been described. In this retrospective study we evaluated the ex-vivo immune response of peripheral blood mononuclear cells to sulphur compounds in 53 patients with clinical or radiologic endodontic treatment failure, 20 patients with clinical discomfort or radiological findings without previous endodontic treatment and a control group of 31 patients who had received successful endodontic treatment at least five years previously. Patients with endodontic abnormalities showed significantly higher ex-vivo sulphur compound-stimulated interferon-gamma (IFN-γ) and interleukin-10 (IL-10) levels as compared to the control group. The association between ex-vivo-stimulated cytokines and endodontically derived sulphur compounds was further substantiated by the fact that the number of IFN-γ and/or IL-10-positive patients decreased significantly 3-8 months after re-treatment of the root canal or tooth extraction. Furthermore, serum tumor necrosis factor-alpha (TNF-α) levels were higher in patients than in controls, and at the same time, the TNFA -308 G/A polymorphism was associated with endodontic treatment failure in our study population. We conclude that a cellular immune response to sulphur compounds contributes to the inflammatory process observed in relation to endodontic treatment failures.

Root canal content from primary endodontic infection and upregulation of gelatinases in fibroblast cells.

AIM: To investigate endotoxin levels from primary endodontic infections before and after chemomechanical preparation (CMP) and to determine their antigenicity against 3T3 fibroblasts through gelatinolytic activity of matrix metalloproteinases (MMPs). METHODOLOGY: Twenty-four root canals with primary endodontic infection and apical periodontitis were selected. Samples were collected using paper points before (S1) and after chemomechanical preparation (CMP) (S2). The limulus amebocyte lysate assay was used for endotoxin measurement. Fibroblasts were stimulated with root canal contents for 24 h. Supernatants of cell cultures stimulated with root canal contents were collected after 24 h to determine the levels of MMP-2 and MMP-9 gelatinolytic activity using the zymography technique. Friedman and Wilcoxon tests were used to compare the amount of endotoxin before (S1) and after CMP (S2) (P < 0.05). Data obtained from gelatinolytic activity were analysed using anova and Tukey's tests (P < 0.05). RESULTS: Endotoxin was recovered in 100% of the samples. There was a significant reduction in endotoxin levels after CMP (P < 0.05). A correlation was found between the levels of endotoxins and MMP-2 expression (P < 0.05). Root canal contents of initial samples (S1) induced significantly greater MMP-2 expression by fibroblasts when compared to S2 and the nonstimulated group (P < 0.05). No gelatinolytic activity of MMP-9 was observed in S1, S2 and control group. CONCLUSIONS: Root canal contents from primary endodontic infections had gelatinolytic activity for MMP-2. Moreover, CMP was effective in reducing endotoxin levels and their antigenicity against fibroblasts on gelatinolytic activity.

Histologic evaluation and immunohistochemical localization of STRO-1 and BMP-4 in rat immature teeth: a comparison between vital and induced pulp necrosis.

OBJECTIVE: To assess histological features and the expression of STRO-1 and BMP-4 in dental pulp and periapical tissues in vital or necrotic rat immature teeth. DESIGN: The lower left first molars of male Wistar rats ageing four weeks (n=24) had their pulps exposed to the oral environment for 3, 6, 9 and 12 weeks (animals ageing 7, 10, 13 and 16 weeks-old, respectively; n=24). The right lower first molars served as control untouched teeth. After sample harvesting the jaws were dissected and processed for histology and immunodetection of STRO-1 and BMP-4. RESULTS: Necrotic teeth had root development arrested, while control animals showed development of dental tissues. Immunohistochemistry showed that detection of BMP-4 was restricted to vital pulps. For both groups, STRO-1 expression was evident around blood vessels walls. Neither BMP-4 nor STRO-1 was observed in the apical papilla region. CONCLUSION: STRO-1-positive precursor cells were not detected in the apical papilla. BMP-4 expression has not been detected during infection.

Nemotic human dental pulp fibroblasts promote human dental pulp stem cells migration.

Dental pulp inflammation has long been perceived as a negative factor leading to pulp disruption. Previous studies have suggested that the inflammatory reaction might be a prerequisite for the burst of progenitors implicated in pulp repair. To investigate the migration of human dental pulp stem cells (hDPSCs) in response to human dental pulp fibroblasts (HDPFs) nemosis, an in vitro model of nemosis-induced inflammation in three-dimensional culture was used in this study. We observed HDPF spheroid formation and that cell-cell adhesion between HDPFs leads to necrosis. Cell death detection and cell counting kit-8 assays showed reduced live cell numbers and increased levels of cell membrane leakage in HDPF spheroids. HDPFs spheroids expressed cyclooxygenase-2 and released an increasing amount of prostaglandin E2 and interleukin-8, indicating inflammation in response to nemosis. The Transwell assays showed that the conditioned medium from HDPFs spheroids significantly induced hDPSCs migration more than the medium from the monolayer. Taken together, these results indicate that HDPFs spheroids induce nemosis and contribute to the migration of hDPSCs. This model might provide a potential research tool for studying interactions between fibroblasts and stem cells, and studies concerning nemosis-targeted stem cells might help treat pulp inflammation.

Effects of calcium hydroxide on cytokine expression in endodontic infections.

INTRODUCTION: The use of calcium hydroxide is an effective step in killing bacteria that remain after cleaning and shaping procedures. It also induces hard-tissue formation and is effective for stopping inflammatory exudates. METHODS: The aim of this study was to assay and to compare the influence of calcium hydroxide on periapical interstitial fluid from human root canals. The mRNA expression levels of the cytokines interferon (IFN)-γ, tumor necrosis factor-α, interleukin (IL)-1ß, IL-17A, and IL-10 as well as the chemokine MCP-1 were assayed by real-time polymerase chain reaction immediately after root canal cleaning and 15 days later. RESULTS: Levels of IL-1ß, IFN-γ, IL-10, and the chemokine CCL2/MCP-1 were increased in teeth without endodontic dressings. With calcium hydroxide interappointment dressings, no statistically significant changes were observed in cytokine mRNA expression. However, when comparing teeth that received the medication with those that did not, expression levels of IL-1ß, IFN-γ, and IL-10 were statistically lower in those teeth that received calcium hydroxide. CONCLUSIONS: Analyses of cytokines and the chemokine CCL-2/MCP-1 demonstrated the benefits of calcium hydroxide as a root canal dressing because it impedes the increase of all mediators during the experimental time.

Clinical diagnosis of pulp inflammation based on pulp oxygenation rates measured by pulse oximetry.

INTRODUCTION: The objective of this study was to investigate correlations between pulp oxygenation rates (%SpO(2)) and clinical diagnoses of reversible pulpitis (RP), irreversible pulpitis (IP), or pulp necrosis (PN). METHODS: Sixty patients who presented with a tooth with endodontic pathology were grouped according to a clinical diagnosis of either RP (n = 20), IP (n = 20), or PN (n = 20). The clinical diagnosis was based on the patient's dental history, periapical radiographs, clinical inspection, and percussion and thermal sensitivity testing. Pulse oximetry (PO) was used to determine pulp oxygenation rates. For every patient, one additional endodontically treated tooth (negative control [NC], n = 60) and one additional healthy tooth with healthy pulp status (positive control [PC], n = 60) were evaluated. Analysis of variance, the Tukey HSD test, and the Student's t test were used for statistical analysis. RESULTS: The mean %SpO(2) levels were as follows: RP: 87.4% (standard deviation [SD] ±2.46), IP: 83.1% (SD ±2.29), PN: 74.6% (SD ±1.96), PC: 92.2% (SD ±1.84), and NC: 0% (SD ±0.0). There were statistically significant differences between RP, IP, and PN compared with NC and PC and between RP, IP, and PN (all P ≤ .01). CONCLUSIONS: The evaluation of pulp oxygenation rates by PO may be a useful tool to determine the different inflammatory stages of the pulp to aid in endodontic diagnosis.

Protein content in irrigating solutions in contact with pulp tissue.

Endodontic irrigating solutions may have different effects, one of which is dissolving pulp tissue. The capacity of different irrigants to dissolve vital and necrotic pulp tissue was evaluated in vitro by means of a quantitative and qualitative study of total soluble pulp protein. Vital pulps and pulps with induced necrosis from young bovine teeth were used. Pulp was cut into smaller pieces, weighed and placed in 1 ml of 1% and 2.5% sodium hypochlorite, 1% and 5% calcium hydroxide, 0.2% chlorhexidine gluconate, 1% tea and distilled water as a control, and kept at 37 degrees. Samples of 20 microl were taken at 30 and 90 minutes and 20 hours. Total protein was dosed using the Lowry method and soluble protein bands were determined by electrophoresis (12% SDS-Page). The results were analyzed using Anova. Chemical analysis of the electrophoretic runs of bovine pulp protein showed that both concentrations of sodium hypochlorite and calcium hydroxide produce denaturation of proteins. No solvent action was found with chlorhexidine, tea or distilled water.

Quantification of endotoxins in necrotic root canals from symptomatic and asymptomatic teeth.

The purpose of this investigation was to quantify the concentration of endotoxin in necrotic root canals and investigate the possible relationship between the concentration of endotoxin and endodontic signs and symptoms. Samples were collected from root canals of 50 patients requiring endodontic treatment due to necrosis of the pulpal tissue. Anaerobic techniques were used to determine the number of c.f.u. in each sample. A quantitative chromogenic Limulus amoebocyte lysate assay was used to measure the concentration of endotoxin in each sample. The presence of c.f.u. was detected by culture in all samples (range 10(2)-5x10(6)). In samples from cases of patients with spontaneous pain, the mean c.f.u. was 1.43x10(6) while in asymptomatic cases it was 9.1x10(4). Endotoxin was present in all the samples studied [range 2390.0-22100.0 endotoxin units (EU) ml-1]. The mean concentration of endotoxin in samples from patients with spontaneous pain was 18540.0 EU ml-1 while in asymptomatic cases it was 12030.0 EU ml-1. Asymptomatic cases generally had lower levels of endotoxin (i.e. a negative association). A positive association was found between endotoxin and symptomatic cases (e.g. spontaneous pain, tenderness to percussion, pain on palpation, swelling and purulent exudates). This study showed that endotoxin is present in high concentrations in root canals of symptomatic teeth. There was a positive correlation between the concentration of endotoxin in the root canal and the presence of endodontic signs and symptoms.

[Content of fluoride, calcium and magnesium in superficial layer of enamel from teeth with vital and necrotic pulp]. / Porównanie zawartosci fluorków, wapnia i magnezu w powierzchniowej warstwie szkliwa zebów z zywa i martwa miazga.

This work was designed to determine the content of fluorides, calcium, and magnesium in the superficial layer of enamel of adult teeth. The study group consisted of 25 patients, aged 18 to 35 years, appearing for dental treatment. Two microsamples of the superficial layer of enamel were collected from each patient with the acid biopsy technique. Samples were obtained from pulpectomized teeth treated endodontically and from teeth with necrotic pulp qualifying for endodontic treatment. Control samples were obtained from homonymous, caries-free teeth with vital pulp. The results of biochemical tests in the study group were compared with control teeth.

Diffusion of metronidazole through the dentinal tubules of extracted teeth.

Passing of metronidazole from the root canal of extracted gangrenous teeth through the dentinal tubules was proved by agar diffusion and minimum inhibitory concentration assay. The findings explain the excellent clinical experience with metronidazole in root treatment.