Immunohistochemical evaluation of angiogenesis and tryptase-positive mast cell infiltration in periapical lesions

Introduction

Several studies have linked mast cells (MCs) with angiogenesis. The aim of this study was to correlate angiogenesis with MCs in radicular cyst (RC) and periapical granuloma (PG) cases.

Methods

Forty-eight samples of periapical lesions, diagnosed as RC (n = 24) and PG (n = 24), were included. The microvessel density and microvessel area measured through the immunoexpression of CD105 and CD34 and the MC density measured through the immunoexpression of tryptase were performed.

Results

MCs were detected in all RCs and PGs (P = .888), mainly in perivascular location and within fibrous stroma. CD34 stained all vessels present in all RC and PG cases. CD105 revealed differential expression, stained preferentially vessels of greater lumen, and showed variable location inside fibrous stroma in both lesions. There was a significant difference of microvessel density determinate by CD34 and CD105 in RCs (r = 0.634, P = .002) and in PGs (r = 0.5709, P = .0036). The difference was also observed when comparing age of the patient in both lesions. Considering the microscopic association between highest concentration of MCs and vascularization, CD105-positive vessels in 50% of RCs and 70.8% of PGs and CD34-positive vessels in 66.7% of RCs and 87.5% of PGs showed areas of close association with MCs.

Conclusions

These results suggest differential expression of CD105 within RC and PG. There is no difference in angiogenesis and MC density between RC and PG. Moreover, because of the association between MCs with vessels and fibrous stroma, other possible roles of tryptase, in addition to the angiogenic properties, should also be considered.     

Effects of induced periapical abscesses on rat pregnancy outcomes

Objective

The objective of this study was to determine the effects of induced periapical abscesses on pregnant rats.

Design

In 1/2 of the animals (n = 16), the pulps of the maxillary right molars were exposed to the oral environment, which resulted in a periapical abscess. The other 1/2 (n = 16) were sham-operated. 1/2 of the animals of both groups became pregnant 2 weeks later. The pregnancy duration, and weight and number of pups were assessed at delivery. Serum, liver and uterine horn samples were taken from all animals at euthanasia and serum IL-6, endothelin-1, TNF-α, IL-10, cortisol and insulin were determined by ELISA. Liver concentrations of IL-6, CRP and IL-6 and uterine horn concentrations of IL-6, vascular endothelial growth factor (VEGF), TNF-α, IL-10 and IL-1-β were assessed by ELISA. Blood glucose concentrations were determined using a glucometer. Outcome variables were compared by factorial ANOVA, a post hoc Tukey test, and Pearson’s correlation test.

Results

Pregnant rats with periapical abscesses had a significantly longer pregnancy and delivered pups with a significantly higher birthweight (p < 0.05). They had significantly higher concentrations of IL-6, VEGF, IL-1-β, and IL-10 within the uterine horn and IL-6, CRP and TNF-α within the liver (p < 0.01). Blood glucose and serum TNF-α, IL-6, endothelin-1, IL-10, and insulin concentrations were significantly higher in the pregnant animals with pulpal abscesses (p < 0.01).

Conclusion

The significant increase in serum TNF-α, taken together with significant increases in blood glucose and serum insulin concentrations, suggest that animals with induced periapical abscesses developed insulin resistance, which significantly affected their pregnancy outcomes.     

Receptor for advanced glycation end products (RAGE)-expressing endothelial cells co-express AGE and S100 in human periapical granulomas

Objective

The engagement of the receptor for advanced glycation end products (RAGE) by AGE or S100 perturbs homeostatic mechanisms and provides a basis for cellular dysfunction in pathological situations. To assess the mechanism of vascular immune reactions in chronic periapical periodontitis, we analysed co-expression of RAGE and AGE or S100 in periapical granulomas.

Methods

Surgically removed periapical lesions, which had been diagnosed as chronic periodontitis, were inspected histologically using paraffin-embedded sections stained with haematoxylin and eosin. Cryostat sections of the tissues, which were identified histologically as periapical granulomas, were then examined by double immunohistochemistry using polyclonal antibodies raised against human CD34 and monoclonal antibodies specific for human RAGE, AGE or S100. Dual-colour immunofluorescence image analysis was also performed to assess the co-expression of RAGE and AGE or RAGE and S100 by endothelial cells.

Results

Marked expression of RAGE, AGE, and S100 by CD34⁺ endothelial cells was noted. Dual-colour immunofluorescence image analysis revealed that the RAGE-expressing endothelial cells co-expressed AGE and S100; however, the number of RAGE-AGE-expressing endothelial cells was significantly higher than that of RAGE-S100-expressing endothelial cells.

Conclusions

Co-expression of RAGE and AGE by endothelial cells in periapical granulomas is more relevant than that of RAGE and S100. The possible engagement of RAGE and AGE may trigger cellular activation and mediate tissue injury.     

Midkine expression in human periapical granulomas

Introduction

The expression of midkine (MK), a heparin-binding growth factor, is increased in various human tumors, making it a promising tumor marker and target for tumor therapy. MK is also related to the regulation of the development and etiology of chronic or autoimmune diseases; however, the involvement of MK in apical periodontitis has never been examined. This study compared the localization of MK-expressing cells and MK messenger RNA expression in periapical granulomas with healthy gingival tissues.

Methods

Periapical lesions were removed surgically from chronic apical periodontitis patients, and serial tissue sections were stained with hematoxylin-eosin. The lesions diagnosed as periapical granulomas pathologically were examined by immunohistochemistry using human MK monoclonal antibodies. MK messenger RNA expression was also detected using real-time polymerase chain reaction analysis. Healthy gingival tissues were analyzed in the same manner.

Results

MK was expressed by inflammatory cells, such as macrophages, lymphocytes, and neutrophils, as well as by endothelial cells in periapical granulomas but not in healthy gingival tissues. The MK-expressing inflammatory cells were seen adjacent to blood vessels, which contained MK-expressing endothelial cells, suggesting the interaction of MK among these cells during the process of inflammatory cell infiltration. Quantitative analysis of MK messenger RNA expression revealed that periapical granulomas expressed significantly more MK than healthy gingival tissues.

Conclusions

These findings suggest that MK is involved in the pathogenesis of periapical granulomas.     

Immunohistochemical detection of factors related to cellular proliferation and apoptosis in radicular and dentigerous cysts

Introduction

This study proposed to investigate aspects of cell proliferation and death in the epithelium of radicular (RCs) and dentigerous (DCs) cysts.

Methods

Serial sections of 17 RCs and 9 DCs were prepared for immunohistochemical detection of caspase-3, Bcl-2, and Ki-67 antigens.

Results

Caspase-3 was detected mainly in the suprabasal and superficial epithelial cells of RCs and DCs, whereas Ki-67 was detected predominantly in the basal layer. Both markers had significant expression in hyperplastic epithelium related to an intense inflammation in the capsule. Immunoreactivity for Bcl-2 was restricted to the basal layer and was significantly higher in atrophic epithelium of DCs than that of RCs.

Conclusions

These results suggest that epithelial proliferation is balanced by apoptosis and that the presence of inflammation inhibits the Bcl-2 expression. DCs and RCs have different formation mechanisms but have similar biological behavior in the presence of intense inflammatory infiltrate.     

Cells isolated from inflamed periapical tissue express mesenchymal stem cell markers and are highly osteogenic

Introduction

We previously reported the presence of mesenchymal stem/progenitor cells (MSCs) in inflamed pulp tissue. Here we asked whether MSCs also exist in inflamed periapical tissues resulting from endodontic infection. The objectives of this study were to detect the expression of MSC markers in periapical inflammatory tissues and to characterize isolated cells from these tissues.

Methods

Human periapical inflammatory tissues were collected and processed to detect MSC marker expression by immunohistochemistry. Cells were isolated and tested for cell surface marker expression by using flow cytometry and examined for multiple differentiation potential into osteogenic and adipogenic pathways. In vivo formation of mineralized tissues was assessed in a mouse model.

Results

Immunohistochemistry showed positive staining for MSC markers STRO-1, CD90, and CD146. Isolated cells at passage 0 appeared as typical fibroblastic cells, and a few cells formed colony-forming unit-fibroblasts (CFU-Fs). After passaging, the CFU-F forming ability diminished dramatically, and the population doubling was up to 26. Flow cytometry data showed that these cells at passage 2 expressed low levels of STRO-1 and CD146 and moderate to high levels of CD90, CD73, and CD105. At passage 6, the levels of these markers decreased. When incubated in specific differentiation medium, cells demonstrated a strong osteogenic but weak adipogenic capacity. After in vivo cell transplantation, mineralized tissues formed in immunocompromised mice.

Conclusions

Human periapical inflammatory tissues expressed MSC markers, suggesting the presence of MSCs. Isolated cells exhibited typical mesenchymal cell immunophenotype with a capacity to form mineralized matrix in vitro and in vivo.     

Imbalance of Interleukin-17+ T-cell and Foxp3+ Regulatory T-cell Dynamics in Rat Periapical Lesions

Introduction

Interleukin (IL)-17⁺ T-helper (Th17) cells and Foxp3⁺ regulatory T (Treg) cells are CD4⁺ T-helper cells with reciprocal functions in immunology and bone metabolism. The present study aimed to investigate the expression dynamics of Th17 and Treg cells in rat periapical lesions as well as their correlation with bone resorption.

Methods

Experimental pulp exposures were made in the lower first molars of 28 Wistar rats to induce periapical lesions. Rats were killed on days 0, 7, 21, and 35. Mandibles were prepared for micro–computed tomography scanning, histologic observation, immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis.

Results

Through 3-dimensional and 2-dimensional measurements, the volume and area of periapical lesions visibly increased from day 7 to day 21 and then expanded slowly between days 21 and 35. IL-17–positive cells markedly increased from day 7 to day 35. However, Foxp3-positive cells remained at low levels until day 21 and then dramatically increased by day 35. The IL-17⁺/Foxp3⁺ ratio and number of osteoclasts simultaneously increased from day 7 to day 21 and then decreased on day 35. Finally, the distinct distribution of CD4⁺/IL-17⁺ Th17 and CD4⁺/Foxp3⁺ Treg cells was observed on days 7 and 35.

Conclusions

Our findings imply the imbalance of IL-17⁺ T cell and Foxp3⁺ Treg cell dynamics in induced periapical lesions, which may play an important role in periapical lesion progression.     

Increase of gingival matured dendritic cells number in elderly patients with chronic periodontitis

Objective

The purpose of this study was to evaluate the inflammatory cell subset proportions in the upper gingival connective tissue, including mature dendritic cells (DC) in elderly and younger patients with generalized chronic periodontitis in order to further understand the effect of aging on gingival inflammatory phenomenon.

Methods

Gingival tissue specimens presenting chronic periodontitis from 8 elderly patients aged >75 (test group, group T) and from 8 younger patients aged 50-60 (considered as controls, group C) were analysed by immunohistochemistry using monoclonal antibodies against CD45RB, CD4, CD8, CD19, CD68, DC-SIGN, DC-LAMP molecules. The number of each immunolabelled cells subset was counted using image analysis.

Results

The difference in the number of CD45RB + leucocytes in the upper gingival connective tissue between groups was not significant permitting to use it as reference. As compared to group C, the lymphocyte subsets/CD45RB + leucocytes ratios tended to decrease in group T but the decrease was significant only for CD4 + T lymphocytes/CD45RB + cells ratio (p < 0.03). On the opposite, the ratios of antigen-presenting cells DC-SIGN + cells/CD45RB + cells and DC-LAMP + cells/CD45RB + cells were significantly increased (p < 0.03 and <0.0001, respectively) in group T. Moreover, in group T the DC-LAMP + cells/DC-SIGN + cells ratio was significantly increased (p < 0.05) showing an increased number of matured dendritic cells.

Conclusion

During chronic periodontitis in elderly patients, our results show a decrease in the ratio of gingival CD4 + lymphocyte subset associated with an increase in the ratios of antigen-presenting cells subsets and more particularly maturated DC-LAMP + dendritic cells.     

Immunohistochemical Analysis of Cyclooxygenase-2 and Tumor Necrosis Factor Alpha in Periapical Lesions

Introduction

The purpose of this study was to evaluate the expression of cyclooxygenase-2 (COX-2) and tumor necrosis factor alpha (TNF-α) in periapical granuloma (PG) and radicular cyst (RC) samples and to correlate it with the type of lesion, the intensity of the inflammatory infiltrate, and the thickness of the epithelial lining.

Synchrotron radiation microbeam X-ray fluorescence analysis of zinc concentration in remineralized enamel in situ

Objective

Remineralization is an indispensable phenomenon during the natural healing process of enamel decay. The incorporation of zinc (Zn) into enamel crystal could accelerate this remineralization. The present study was designed to investigate the concentration and distribution of Zn in remineralized enamel after gum chewing.

Methods

The experiment was performed at the Photon Factory. Synchrotron radiation was monochromatized and X-rays were focused into a small beam spot. The X-ray fluorescence (XRF) from the sample was detected with a silicon (Si) (lithium (Li)) detector. X-ray beam energy was tuned to detect Zn. The examined samples were small enamel fragments remineralized after chewing calcium phosphate-containing gum in situ. The incorporation of Zn atom into hydroxyapatite (OHAP), the main component of enamel, was measured using Zn K-edge extended X-ray absorption fine structure (EXAFS) with fluorescence mode at the SPring-8.

Results

A high concentration of Zn was detected in a superficial area 10-μm deep of the sectioned enamel after gum chewing. This concentration increased over that in the intact enamel. The atomic distance between Zn and O in the enamel was calculated using the EXAFS data. The analyzed atomic distances between Zn and O in two sections were 0.237 and 0.240 nm.

Conclusion

The present experiments suggest that Zn is effectively incorporated into remineralized enamel through the physiological processes of mineral deposition in the oral cavity through gum-chewing and that Zn substitution probably occurred at the calcium position in enamel hydroxyapatite.     

Comparative evaluation of platelet-rich plasma and guided tissue regeneration membrane in the healing of apicomarginal defects: a clinical study

Introduction

The aim of the study was to compare the healing responses of platelet-rich plasma (PRP), PRP + a collagen sponge, and a collagen membrane used as guided tissue regeneration (GTR) materials for the treatment of apicomarginal defects.

Methods

Thirty patients with suppurative chronic apical periodontitis and apicomarginal communication were selected and allocated randomly into three groups according to the barrier technique to be used during periradicular surgery: the collagen membrane group, the PRP group, and the PRP + collagen sponge group. Clinical and radiographic measurements were determined at baseline and every 3 months after surgery up to 1 year. Cases were defined as healed when no clinical signs or symptoms were present, and radiographs showed complete or incomplete (scar tissue) healing of previous radiolucencies.

Results

The PRP and PRP + collagen sponge groups depicted 83.33% and 88.89% healing, respectively, in terms of combined clinical-radiographic healing as compared with 80% in the collagen membrane group. All the three treatments showed highly significant (P < .05) reductions in the periodontal pocket depth (PD), the clinical attachment level (CAL), the gingival margin position (GMP), the size of the periapical lesion, the percentage reduction of the periapical rarefactions, and periapical healing. No significant differences between the three groups were evident for these parameters (P > .05).

Conclusions

GTR applied to apicomarginal defects using PRP or PRP + collagen sponge lead to similar enhancements of the clinical outcome of periradicular surgery in terms of periapical healing, gain of periodontal support, PD reduction, and PRP may be an alternative treatment for GTR membrane in the treatment of apicomarginal defects.     

Salivary levels of IL-1β, IL-6, IL-8, and TNF-α in patients with burning mouth syndrome

Objective

To compare salivary IL-1β, IL-6, IL-8, and TNF-α levels between patients with burning mouth syndrome (BMS) and controls.

Design

Forty female patients with BMS (mean age: 61.6 ± 10.1 years) and 20 female control subjects (mean age: 65.1 ± 9.0 years) were included in the study. Unstimulated (UWS) and stimulated whole saliva samples (SWS) were collected and their flow rates were determined. Salivary IL-1β, IL-6, IL-8, and TNF-α levels and total protein concentration were also determined. Salivary transferrin level was determined to investigate the level of blood contamination in saliva samples. Gingival index of the subjects was also examined. Student's t-test, Pearson's correlation analysis, and analysis of covariance were used.

Results

No significant differences were found in the salivary levels of IL-1β, IL-6, IL-8, and TNF-α in BMS patients compared with controls. Salivary flow rates and their total protein concentrations did not differ significantly between the groups. The levels of salivary cytokines and total protein concentration correlated significantly with the level of blood contamination in both UWS and SWS.

Conclusion

There were no differences in the salivary levels of IL-1β, IL-6, IL-8, and TNF-α in BMS patients compared with controls. Cytokine levels in whole saliva were affected mainly by the amount of blood contamination.     

The association of MMP-9 and IL-18 gene promoter polymorphisms with gingivitis in adolescents

Objective

Interleukin 18 (IL-18) is shown to be a proinflammatory cytokine that regulates the expression of matrix metalloproteinase 9 (MMP-9). The aim of this study was to test for differences between Czech adolescents with and without gingivitis in relation to MMP-9 and IL-18 polymorphisms.

Design

A total of 298 Caucasian children, aged 11-13 years, were examined to assess gingival health. DNA for genetic analysis was obtained from buccal epithelial cells, and the MMP-9 −1562C/T and IL-18 −607A/C variants were identified with PCR-RFLP.

Results

Gingivitis was present in 49.3% of the adolescents examined, the rest of the group was considered healthy. The IL-18 −607C and MMP-9 −1562T alleles were found in 58.9% and 8.3% of the healthy subjects, and in 62.2% and 15.0% of the patients with gingivitis, respectively. Although differences in allele frequencies were not significant for IL-18 variant, they were significant for MMP-9 polymorphism (p = 0.01, p_corr < 0.05). Furthermore, a highly significant association of the composite genotype (formed by the variants of the both genes) with gingivitis was found (p = 0.004, p_corr < 0.05).

Conclusions

The −1562 T allele of MMP-9 gene could have a role in gingivitis in adolescents. In addition, interaction of the MMP-9 and IL-18 genes could be considered a risk factor for the development of gingivitis in children.     

Biomimetic Approach to Perforation Repair Using Dental Pulp Stem Cells and Dentin Matrix Protein 1

Introduction

Dentin regeneration could be an ideal treatment option to restore tissue function. This study was conducted to evaluate the ability of dental pulp stem cells (DPSCs) and dentin matrix protein 1 (DMP1) impregnated within a collagen scaffold to regenerate dentin.

Methods

Simulated perforations were created in 18 dentin wafers made from freshly extracted human molars. Six groups were established. They were (1) empty wafers, (2) mineral trioxide aggregate, (3) collagen scaffold, (4) scaffold with DMP1, (5) scaffold with DPSCs, and (6) scaffold with DPSCs and DMP1. One sample was placed subcutaneously in each mouse with three mice in each group. After 12 weeks, the samples were subjected to radiographic, histological, and immunohistochemical evaluations.

Results

DPSCs impregnated within a collagen scaffold differentiated into odontoblast-like cells forming a highly cellular, vascular, and mineralized matrix in the presence of DMP1.

Conclusions

A triad consisting of DPSCs, DMP1, and a collagen scaffold promotes dentin regeneration in a simulated perforation repair model.     

Maxillary sinus floor elevation using a tissue engineered bone complex with BMP-2 gene modified bMSCs and a novel porous ceramic scaffold in rabbits

Objectives

To study the effects of maxillary sinus floor elevation by a tissue engineered bone complex with bone morphogenetic protein-2 (BMP-2) gene modified bone marrow stromal cells (bMSCs) and a novel porous ceramic scaffold (OsteoBone™) in rabbits.

Materials and methods

bMSCs derived from New Zealand rabbit bone marrow were cultured and transduced with adenovirus AdBMP-2 and with AdEGFP gene (without BMP-2 gene sequence) as a control, respectively, in vitro. These bMSCs were then combined with OsteoBone™ scaffold at a concentration of 2 × 10⁷ cells/ml and used to elevate the maxillary sinus floor in rabbits. Eight rabbits were randomly allocated into groups and sacrificed at weeks 2 and 4. For each time point, 8 maxillary sinus floor elevation surgeries were made bilaterally in 4 rabbits for the two groups (n = 4 per group): group A (AdBMP-2-bMSCs/material) and group B (AdEGFP-bMSCs/material). All samples were evaluated by histologic and histomorphometric analysis.

Results

The augmented maxillary sinus height was maintained for both groups over the entire experimental period, while new bone area increased over time for group A. At week 4 after operation, bone area in group A was significantly more than that in group B (P < 0.05), and was more obviously detected in the center of the elevated space. Under a confocal microscope, green fluorescence in newly formed bone was observed in the EGFP group, which suggests that those implanted bMSCs had contributed to the new bone formation.

Conclusion

bMSCs modified with AdBMP-2 gene can promote new bone formation in elevating the rabbit maxillary sinus. OsteoBone™ scaffold could be an ideal carrier for gene enhanced bone tissue engineering.     

Bacteriological findings in radicular cyst and keratocystic odontogenic tumour fluids from asymptomatic patients

Objective

In this study the potential presence of bacteria in radicular cyst (RC) and keratocystic odontogenic tumour(KCOT) fluids from clinically asymptomatic patients was investigated.

Materials and methods

Cyst fluids were collected by needle aspiration from 16 patients with asymptomatic osteolytic lesions (10 RCs and 6 KCOTs) undergoing surgery. All samples were transferred into tubes containing pre-reduced transport medium, delivered to the microbiology laboratory and processed within 1 h. The cysts, surgically enucleated, were sent for standard histopathological examination. Cyst fluid samples were cultured on selective and differential media in anaerobic (for about 2 weeks) and aerobic (for 24–48 h) conditions to detect viable microorganisms. After incubation, the colonies were counted, Gram-stained and identified by biochemical tests.

Results

Cultures were positive for the presence of bacteria in 15 (9 RCs, 6 KCOTs) out of 16 cases. RCs and KCOTs generally yielded low bacterial counts (10²–10⁴ CFU/ml) and were predominantly colonized by obligate anaerobes (64%), whereas less commonly by facultative anaerobes (36%). No significant differences in the detection frequencies of obligate and facultative anaerobes were evidenced between RCs and KCOTs. Propionibacterium acnes was the most common obligate anaerobe recovered both in RC and KCOT fluids. Among facultative anaerobes, Gemella morbillorum was more frequently isolated in KCOTs, whereas Staphylococcus spp. in RCs.

Conclusions

Bacteria may be present and persist within fluids of clinically asymptomatic jaw cystic lesions. The influence of bacteria and latent bacterial infection within cystic jaw lesions should be reconsidered in odontogenic cyst progression.     

Distribution of Mast Cells and Macrophages and Expression of Interleukin-6 in Periapical Cysts

Introduction

Mast cells and macrophages are important components of the inflammatory infiltrate found in inflammatory periapical diseases. Several cytokines participate in the mechanisms of inflammation, tissue repair, and bone resorption associated with periapical cysts. The aim of the present study was to evaluate the distribution of mast cells and macrophages and the expression of interleukin-6 (IL-6) in periapical cysts.

Methods

Thirty periapical cysts were selected for the study, and clinical, demographic, and gross information from the cases was obtained from the laboratory records. Five-micrometer sections stained with hematoxylin-eosin were reviewed for analysis of the microscopic features of the cysts, and 3-μm sections on silanized slides were used for immunohistochemical reactions with anti-tryptase, anti-CD68, and anti–IL-6.

Results

There was no statistically significant difference in the mean number of mast cells and macrophages when comparing superficial and deep regions of the fibrous capsule of the cysts. Mean number of mast cells on the superficial region of the fibrous capsule was higher in cysts showing intense superficial inflammation and exocytosis. Macrophages were more commonly found in areas showing IL-6 expression, and IL-6 was less expressed in deep regions of the fibrous capsule in cysts showing greater gross volume.

Conclusions

The results reinforced the participation of mast cells and macrophages in the pathogenesis of periapical cysts and suggested that IL-6 is not the major bone resorption mediator in larger periapical cysts.