Human Periapical Lesions Research Articles

Presence and location of bacteria in human periapical pathosis: A histopathological study

Periapical granulomas are sterile or infected have been controversial since long. Various studies mostly used culture methods to evaluate the microbial flora of the periapical lesions, but still landed up with variable and sometimes false positive results. Present study used Modified Brown and Brenn stain to study the presence and location of bacteria in human periapical lesions. Thirty three periapical lesions attached to non-endodontically treated extracted teeth were fixed in 10% buffered formalin, decalcified in 10% HNO3 and were processed for paraffin embedding. Serial sectioning was done and the slides were stained with H&E stain, and Modified Brown and Brenn stain. Of 33 lesions, bacteria were found in 28 lesions (17 granulomas and 11 cysts). Five granulomas were found to be sterile. Majority of the bacteria that were seen were gram-positive cocci, followed by gram-positive bacilli. Gram-negative bacilli were rarely seen. Yeast like structures were also observed in 12 lesions. Knowledge in this field is essential to the clinicians to effectively carryout the endodontic treatment.

Localization and Signaling Patterns of Vascular Endothelial Growth Factors and Receptors in Human Periapical Lesions

IntroductionVascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key players in vasculogenesis and are also involved in pathologic conditions with bone destruction. Vasculogenesis is critical for disease progression, and bone resorption is a hallmark of apical periodontitis. However, the localization of VEGFs and VEGFRs and their gene signaling pathways in human apical periodontitis have not been thoroughly investigated. The aim of this study was to localize VEGFs and VEGFRs and analyze their gene expression as well as signaling pathways in human periapical lesions. MethodsTissue was collected after endodontic surgery from patients diagnosed with chronic apical periodontitis. Periodontal ligament samples from extracted healthy wisdom teeth was also collected and used as control tissue. In lesion cryosections, VEGFs/VEGFRs were identified by immunohistochemistry/double immunofluorescence by using specific antibodies. A human VEGF signaling polymerase chain reaction array system was used for gene expression analysis comparing lesions with periodontal ligament samples. ResultsThe histologic evaluation revealed heterogeneous morphology of the periapical lesions with various degrees of inflammatory infiltrates. In the lesions, all investigated factors and receptors were identified in blood vessels and various immune cells. No lymphatic vessels were detected. Gene expression analysis revealed up-regulation of VEGF-A and VEGFR-3, although not significant. Phosphatidylinositol-3-kinases, protein kinase C, mitogen-activated protein kinases, and phospholipases, all known to be involved in VEGF-mediated angiogenic activity, were significantly up-regulated. ConclusionsThe cellular and vascular expressions of VEGFs and VEGFRs in chronic apical periodontitis, along with significant alterations of genes mediating VEGF-induced angiogenic responses, suggest ongoing vascular remodeling in established chronic periapical lesions.

Antigen recognition and presentation in periapical tissues: a role for TLR expressing cells?

Bacteria are the prime cause of periapical diseases and root canal microbiology is a well-researched area of endodontics. Antigen-presenting cells (APCs) are present in periapical lesions of endodontic origin and play a substantial role in recognizing, processing and presenting pathogenic antigens to the adaptive immune system such as an effective and long-lasting immune response is generated against the specific pathogens. Toll-like receptors (TLRs) are germ-line encoded pathogen recognition receptors (PRR) expressed by various APCs which induce their maturation, lead to gene transcription in the nucleus and the production of several pro- and anti-inflammatory cytokines. Thirteen TLRs have been discovered, 10 of which have been identified in humans so far. Preliminary studies of dental pulp tissue have demonstrated various cell types expressing different TLRs in response to commonly encountered microorganisms. However, there is little information available regarding the expression and function of the various TLRs in human periapical lesions. This review discusses the interactions of various APCs in periapical lesions and the possible roles of different TLRs and APCs in pulp/periapical pathogen recognition and presentation to the adaptive immune system in the initiation and sustaining of periapical diseases.

Th17/Treg immunoregulatory balance in human periapical lesions (47.5)

Abstract Cysts and granulomas are chronic periapical lesions mediated by a set of inflammatory mediators that develop in order to contain a periapical infection. This study analyzed the inflammatory infiltrate, mast cells and in situ expression of cytokines, chemokines and FoxP3 in human periapical granulomas and cysts compared to controls. Fifty-five lesions (25 cysts, 25 granulomas and 5 controls) were analyzed. The type of inflammatory infiltrate was evaluated by HE staining and the presence of mast cells was analyzed by toluidine blue staining. Indirect immunohistochemistry was used to evaluate the expression of cytokines, chemokines and FoxP3. The inflammatory infiltrate mainly consisted of mononuclear cells. In cysts, mononuclear infiltrates were significantly more frequent than mixed (polymorphonuclear/mononuclear) infiltrates (p=0.04). Mixed inflammatory infiltrates were significantly more frequent in patients presenting fistulae (p=0.0001). The number of mast cells was significantly higher in granulomas than in cystic lesions (p=0.02). A significant difference in the expression of IL-17 (p=0.001) and TGF-β (p=0.003) was observed between cysts and granulomas and the control group. Significantly higher IL-17 levels were also observed in cases of patients with fistulae (p=0.03). Mast cells, TGF-β and IL-17 probably participate in the modulation of the inflammatory process in established periapical lesions, especially in granulomas.

Comparative Study of the Presence of Mast Cells in Periapical Granulomas and Periapical Cysts by Toluidine Blue and Astra Blue: Possible Role of Mast Cells in the Course of Human Periapical Lesions

Comparative Study of the Presence of Mast Cells in Periapical Granulomas and Periapical Cysts by Toluidine Blue and Astra Blue: Possible Role of Mast Cells in the Course of Human Periapical Lesions

Production of proinflammatory and immunoregulatory cytokines by inflammatory cells from periapical lesions in culture

The role of cytokines in pathogenesis of periapical lesions is not well understood. The aim of this study was to study the correlation between proinflammatory and immunoregulatory cytokines in periapical lesions and their relationship with cellular composition and clinical presentation. Inflammatory cells were isolated from 67 human periapical lesions and cultivated for 24 h. The levels of proinflammatory cytokines: interleukin-1 beta (IL-1beta), IL-6, IL-8 and tumour necrosis factor alpha (TNF-alpha) and immunoregulatory cytokines: transforming growth factor-beta (TGF-beta) and IL-10 were determined in culture supernatants using a fluorescent bead immunoassay or ELISA. The phenotype of cells was analysed by immunocytochemistry. Inflammatory cells from symptomatic lesions which contained higher proportion of granulocytes, secreted higher levels of IL-1, IL-6 and IL-8 compared with asymptomatic lesions. Large-size lesions contained lower percentages of mononuclear phagocytes, higher percentages of CD8(+) T cells and produced higher levels of TNF-alpha, IL-6 and IL-10 compared with small-size lesions. There were negative correlations between the concentrations of TGF-beta and proinflammatory cytokines. TGF-beta, added to cultures, downregulated the levels of proinflammatory cytokines more strongly than IL-10, independently of clinical presentation of the lesions. By contrast, exogenous IL-10 was mainly immunosuppressive in cultures of asymptomatic lesions. Symptomatic lesions are characterized by higher production of proinflammatory cytokines. Immunoregulatory cytokines are more important for suppression of inflammation in asymptomatic lesions and in this context the effect of TGF-beta is more potent and different from IL-10.

The Correlation between Serum Immunoglobulin A and Immunoglobulin G Levels and the Presence of Treponema denticola in Human Periapical Lesions

The aim of this study was to compare the serum immunoglobulin A (IgA) and immunoglobulin G (IgG) levels and the presence of Treponema denticola in the root canals in a group of teeth with/without periapical lesion. A total of 66 children aged 8 to 13 years old were involved in this study. Five milliliters of blood samples were taken to detect the serum IgA and IgG levels. Sixty-six endodontic samplings were also obtained to determine the presence of T. denticola by polymerase chain reaction. The presence of T. denticola between the groups with/without periapical lesion was statistically significant (p = 0.026). A significant negative correlation was found between serum IgG and IgA levels and the presence of T. denticola (p = 0.023 and 0.038, respectively). This study may support the hypothesis that the presence of T. denticola in the root canals is mainly related to the periapical lesions, and the higher levels of serum IgG and IgA levels may protect against T. denticola.

Production of interleukin‐8 in vitro by mononuclear cells isolated from human periapical lesions

Interleukin-8 (IL-8) is an important mediator of inflammation. However, little is known about its production in chronic dental periapical lesions and this was the main aim of this work. Inflammatory cells were isolated from clinically different periapical lesions and analyzed by morphological criteria. The mononuclear cells were isolated, phenotypically analyzed by immunocytochemistry and cultivated in vitro. IL-8 was measured in culture supernatants of these periapical lesion mononuclear cells (PL-MNC) using a microbeads fluorescence assay. We found a relatively high production of IL-8 in 19 out of 21 periapical lesions included in the study. The level of IL-8 and the proportion of neutrophil granulocytes were significantly higher in the group of symptomatic lesions, compared to the asymptomatic lesions, but there was no statistically significant correlation between these parameters. According to the predominance of CD3(+) T cells and Ig(+)/CD19(+) B cells and plasma cells, lesions were divided into T-type and B-type lesions, respectively. The levels of IL-8 were significantly higher in the culture supernatants of PL-MNC in the T-type lesions and were positively correlated with the proportion of macrophages/dendritic cells (CD11c(+) cells) and CD4(+) T cells. Such a correlation was not shown in B-type lesions. These results suggest that PL-MNC are a significant source of IL-8, which is probably an important chemokine for the migration and function of different cell types at the site of chronic inflammation.

Characterization of antigen‐presenting cells in human apical periodontitis lesions by flow cytometry and immunocytochemistry

To analyse phenotypic characteristics of antigen-presenting cells (APC), isolated from human periapical lesions by flow cytometry and immunocytochemistry. Sixteen periapical lesions were digested for 15 min with 0.05% collagenase. Mononuclear cells, separated from other inflammatory cells by density centrifugation, were processed for flow cytometry and/or immunocytochemistry. Single and double immunostainings were performed using monoclonal antibodies specific for human CD45, CD3, CD19, CD14, HLA-DR, CD1a, CD83 and CD123. Antigen-presenting cells (HLA-DR(+) cells) represented 32.9 +/- 17.8% of total mononuclear cells. Amongst them, B cells (HLA-DR(+) CD19(+)) were the predominant APC population, followed by activated macrophages (HLA-DR(+) CD14(+)), dendritic cells (DC) (HLA-DR(+) CD14(-) CD19(-) CD3(-)) and activated T cells (HLA-DR(+) CD3(+)). Based on the predominance of T cells (CD3(+)) or B cells and plasma cells (CD19(+) and CD19(lo), respectively) amongst mononuclear cell infiltrates, lesions were divided into T- and B-types. The percentage of DC in T-type lesions (27.1 +/- 6.8% of total HLA-DR(+) cells) was higher, compared with B-type lesions (10.3 +/- 5.2%) (P < 0.01). Within the DC population, the percentages of CD1a (Langerhans cell type) and CD123 (probably plasmacytoid DC type) did not differ significantly between the groups (P > 0.05). However, the percentage of mature DC (CD83(+)) was significantly higher in T-type periapical lesions (P < 0.05). Flow cytometry and immunocytochemistry are suitable methods for phenotypic analysis of APC after their isolation from human periapical lesions. APC, that were phenotypically heterogeneous, constituted a significant component of infiltrating cells. Lesions with the predominance of T cells were characterized by a higher proportion of mature DC (HLA-DR(+)CD83(+) cells) than lesions with predominance of B cells/plasma cells.

Correlation between phenotypic characteristics of mononuclear cells isolated from human periapical lesions and their in vitro production of Th1 and Th2 cytokines

The development and progression of periapical dental lesions, mediated by the specific immune response, are poorly understood. In these processes, an interplay of different proinflammatory and immunoregulatory cytokines is of crucial importance. To examine the activation of T helper 1 (Th1) and Th2 immune responses in 25 human periapical lesions based on the ex vivo production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) by mononuclear cells (PL-MNC). The levels of IFN-gamma and IL-4 in culture supernatants of PL-MNC, determined by ELISA, were correlated with concentrations of these cytokines in cultures of control MNC from peripheral blood (PB-MNC), cellular composition of inflammatory cells and phenotypic characteristics of PL-MNC. We detected high levels of IFN-gamma in all samples, after cell stimulation with phorbol myristate acetate and Ca(2+) ionophore, that were not statistically different from the levels of IFN-gamma in PB-MNC cultures. IL-4 was detected in 76% samples, but its concentrations were much lower than in PB-MNC samples. The levels of IFN-gamma were higher in cultures of PL-MNC isolated from periapical lesions with predominance of T cells (T-type lesions) and correlated positively with the proportion of antigen-presenting cells (macrophages and dendritic cells), CD4(+) T cells and IgG2(+) B cells/plasma cells. The levels of IL-4 correlated negatively with the proportion of macrophages, but positively with the number of mast cells and IgG4(+) cells. IL-18Ralpha, a stable marker of Th1 cells, was detected on a relatively small proportion of CD3(+) T cells and its expression correlated with the levels of IFN-gamma. However, the expression of ST2L, a stable Th2 cell marker, was not detected. The levels of Th1 and Th2 cytokines did not correlate with clinical characteristics of the lesions, defined by the presence of symptoms. Cumulatively, our results suggest the predominance of Th1 immune response in periapical lesions.

A study of periapical lesions correlating the presence of a radiopaque lamina with histological findings

To compare the presence or absence of a radiopaque lamina of 60 human periapical lesions with the histological findings from a case study in dental practice. Paralleling radiographs were taken of 60 teeth with periapical radiolucencies. The periapical radiographs were scanned, and standardized images were evaluated on a computer screen for the presence or absence of a radiopaque lamina by 2 calibrated observers according to agreed criteria. The serially sectioned histological specimens were obtained from the extraction of 13 endodontically treated teeth and 27 nontreated teeth, as well as 20 post-treatment teeth with apical periodontitis removed during periapical surgical procedure; the specimens were classified according to agreed criteria. The findings of the 2 evaluations were compared. Out of 57 electronic images that could be interpreted, 10 lesions had a radiopaque lamina, but of these only 3 were histologically diagnosed as cysts, while 7 were granulomas or abscesses. Out of 47 lesions without a radiopaque lamina, 40 were histologically diagnosed as granulomas or abscesses, while 7 were cysts. The diagnosis of periapical lesions cannot be made on the basis of the presence or absence of a radiopaque lamina, but requires histological examination of serial sections.

Upregulation of Basic Fibroblast Growth Factor in Human Periapical Lesions

Basic fibroblast growth factor is one of a class of heparin-binding growth factors that stimulates endothelial cell proliferation and migration in vitro and angiogenesis in vivo. The purpose of this study was to investigate the expression of basic fibroblast growth factor in chronic periapical lesions of endodontic origin. Ten chronic inflammatory periapical lesions were examined using immunohistochemical staining. The experimental control group consisted of four specimens of uninflamed periodontal ligament tissue. Two independent observers graded the staining intensity for basic fibroblast growth factor. An immunopositive, cytoplasmic, and nuclear reaction for basic fibroblast growth factor, with varying degrees of upregulation was observed in all 10 chronic periapical lesions. We speculate that the formation of granulation tissue and the activation of epithelial cell rests in chronical apical lesions might be associated with a local rise in the tissue level of basic fibroblast growth factor. Therefore, this growth factor could play an important role in the pathogenesis of chronic apical periodontitis and periapical cysts.

Epithelium and bacteria in periapical lesions

The purpose of this study was to evaluate 50 human periapical lesions for bacteria and epithelium in a case study in dental practice. Specimens were obtained from the extraction of 50 untreated teeth that had lesions attached to their apices. The specimens were histologically evaluated using serial sections. Bacteria were found in all teeth, colonizing necrotic tissue in the main canal, dentinal tubules, or apical ramifications, and in the body of the periapical lesion in 18 abscesses or cysts. Twenty-one lesions were epithelialized; 14 abscesses, 20 granulomas, and 16 cysts were distinguished. In 18 root canals inflamed tissue was found in the apical part of the canal. A single foramen was present in 13 cases while apical ramifications were found in 37 cases. Granulomas were most common, and most epithelialized lesions were cysts. Bacteria were only detected periapically in abscesses or cysts. Inflamed tissue was present in the apical root canal in one third of cases.

Importance of Mast Cells in Human Periapical Inflammatory Lesions

The role of mast cells (MCs) in periapical inflammatory lesions is not well understood. The objective of this work was to quantify MC numbers in human periapical lesions with the aim to clarify their role in the pathogenesis of these lesions. We analyzed the slides of 64 human periapical inflammatory lesions stained with pH 8.0 toluidine blue technique, quantified the number of MCs, and evaluated any correlation with age, gender, size, and location. The results of this study suggest that MCs were more numerous in females (p < 0.01); MC numbers were higher in biopsies from granulomas with proliferating epithelium and lower in biopsies from chronic apical abscesses; MC counts did not correlate with patients' age or size. MCs were observed more commonly in areas containing inflammatory infiltrate and degranulation was a frequent finding in these zones. Our results suggest that MCs play an active role in the pathogenesis of the periapical inflammatory lesions. The potential role of MCs related with the initiation, development, and persistence of the periapical inflammatory process are discussed.

Identification of γδT lymphocytes in human periapical lesions

Endodontic (root canal) therapy is required when the pulp of a tooth becomes necrotic due to a bacterial infection or trauma. A proportion of patients who receive endodontic therapy subsequently have periapical (around the tooth root) lesions detected by radiolucency. Currently, there are no means to identify susceptible patients. Although tissue from periapical lesions has been described as inflammatory, inflammatory cell types and their functions have been poorly characterized. For example, T lymphocytes were identified using pan specific anti‐CD3 mAb, which recognizes both αβ and γδT cells. Using the current model of γδT cells as immunoregulatory cells; γδT cells can mediate protective or destructive milieus. We postulated that patients who have a periapical lesion, as identified by radiographic bone loss, mount a γδT cell response. We collected specimens removed by surgery from both periapical lesions and other oral tissues, generated total RNA and performed reverse‐transcriptase polymerase chain reaction to identify rearranged δ genes. Results were confirmed with semi‐nested polymerase chain reaction. In addition, we demonstrate that these lesions contain a population of CD3+ cells that are αβT cell receptor negative, implying that these cells are γδT cells. Here we show that 36/37 of periapical lesions and only 2/11 of other lesions contain γδT cells (P &lt; 0.0001). Vδ2+ T cells were the most common subtype identified (30/36) in these samples. This is the first report in the literature of the presence of γδT cells in human periapical lesions.

Production of Interleukin–8 and Nitric Oxide in Human Periapical Lesions

Bacterial infection of the pulp and root canal system leads to the recruitment of immunocompetent cells in the periapex and stimulates inflammatory cell responses to produce a variety of inflammatory mediators. Cytokines, reactive oxygen intermediates, and reactive nitrogen intermediates are frequently found at sites of acute inflammation. In this study, we measured the levels of interleukin (IL)-8 and nitric oxide (NO) in the periapical exudate (PE) from human periapical lesions and investigated the association of these mediators with the clinical symptoms of periapical periodontitis. PE samples were collected from root canals during routine endodontic treatments. The IL-8 concentration was measured by the enzyme-linked immunosorbent assay, and the NO level was measured as nitrite/nitrate concentration assayed by the Griess reaction. Detectable levels of IL-8 and nitrite/nitrate were found in 24 and 19 of 27 PE-samples, respectively. Although PE-IL-8 and nitrite/nitrate concentration showed a broad range, a significantly positive correlation was found between both mediators. Also, significantly higher IL-8 levels were found in PE from lesions that had painful symptoms at the sampling visit. However, there was no relationship between elevated NO levels and clinical symptoms. These results suggest that the up-regulation of IL-8 may have a critical role in the development of the symptoms of periapical disease.

CD45/Isotypes Expression in the Immune Cells of Human Periapical Lesions

Expression of some leukocyte antigens (including CD45) and its isoforms (CD2, CD4, CD5, CD6, CD7, and CD8) was examined in the human periapical granulation tissue samples in the present study. The majority of the infiltrating cells expressed heavy molecular-weight isoforms of the CD45 antigen. Expression of CD2, CD5, CD6, and CD7 antigens was also detected, implying significant roles for these antigens in the immune reaction taking place in periapical lesions. This suggests that the immune response taking place at the periapical region is predominantly cellular and the humoral responses to antigenic challenge are conducted mainly by regional lymph nodes.

OR 59 Quantitative analysis of immunoreactive calcitonin gene-related peptide in human periapical lesions

OR 59 Quantitative analysis of immunoreactive calcitonin gene-related peptide in human periapical lesions

Detection of interleukin‐6 in human dental pulp and periapical lesions

Pulpal and periapical diseases are characterized by inflammation. The cytokine IL-6 is a major mediator of the host response to tissue injury and infection. This study examined the level of interleukin-6 (IL-6) in six inflamed human pulps and six human periapical lesions of endodontic origin using ELISA. Pulp samples from eight clinically impacted teeth were used as controls. The periapical samples exhibited significant levels of IL-6 (mean = 78.1 +/- 9 pg/mg protein) as did inflamed pulpal tissues mean = 36 +/- 3.9 pg/mg protein) compared to healthy pulp mean = 0.01 +/- 0.02 pg/mg protein). These data indicated that IL-6 was produced and released locally in the inflamed pulpal and periapical lesions.

Distribution of interleukin-2 receptor alpha-chain and cells expressing major histocompatibility complex class II antigen in chronic human periapical lesions.

In situ distribution of CD2+ T-lymphocytes, CD4+ and CD8+ T-cell subsets, CD14+ macrophages, interleukin-2 receptor alpha-chain (IL-2R alpha) and class II major histocompatibility complex antigen (major histocompatibility complex class II, HLA-DR) expressing cells were determined in 14 chronic human periapical granulomas by immunohistochemical method using monoclonal antibodies. CD2+ lymphocytes were rather evenly distributed within the classical granulation tissue and comprised 55% of the mononuclear cells. Macrophages were distributed all over the periapical area, but their proportion was much less than that of T lymphocytes. Both small, lymphocyte-like mononuclear cells and larger mononuclear cells resembling macrophages displayed mild to strong circumferential staining with the anti-HLA-DR antibody. The majority of lymphocytes expressed IL-2R alpha indicating the activated state of T cells within the lesion.