Experimental Periapical Lesions Research Articles

Periapical lesions in two inbred strains of rats differing in immunological reactivity.

To investigate the influence of strain differences in immune responses on the pathogenesis of experimental periapical lesions in Dark Agouti (DA) and Albino Oxford (AO) inbred strains of rats. Periapical lesions were induced in male DA and AO rats by pulp exposure of the first mandibular right molars to the oral environment. Animals were killed 21days after pulp exposure. The mandibular jaws were retrieved and prepared for radiographic, pathohistological, immunohistochemical analysis, real-time PCR and flow cytometry. Blood samples and the supernatant of periapical lesions were collected for measurement of cytokines and oxidative stress marker levels. Statistical analysis was performed using the Kruskal-Wallis H and Mann-Whitney U non-parametric tests or parametric One-Way anova and Independent Samples T-test to determine the differences between groups depending on the normality of the data. A significant difference was considered when p values were <.05. DA rats developed significantly larger (p<.05) periapical lesions compared to AO rats as confirmed by radiographic and pathohistological analysis. The immunohistochemical staining intensity for CD3 was significantly greater in periapical lesions of DA rats compared to AO rats (p<.05). In DA rats, periapical lesions had a significantly higher (p<.05) percentage of CD3+ cells compared to AO rats. Also, the percentage of INF-γ, IL-17 and IL-10 CD3+CD4+ cells was significantly higher in DA rats (p<.05). DA rats had a significantly higher Th17/Th10 ratio. RT-PCR expression of IL-1β, INF-γ and IL-17 genes was significantly higher in periapical lesions of DA compared to AO rats (p<.05). The receptor activator of nuclear factor kappa-Β ligand/osteoprotegerin ratio was higher in DA compared to AO rats with periapical lesions (p<.05). Systemic levels of TNF-α and IL-6 were significantly higher in DA compared to AO rats (p<.05). Levels of lipid peroxidation measured as thiobarbituric acid reactive substances and reduced glutathione were significantly higher (p<.05) in the supernatant in the periapical lesions of DA rats. After pulp exposure, DA rats developed much larger periapical lesions compared to AO rats. Genetically determined differences in immunopathology have been demonstrated to be a significant element defining the severity of periapical lesions.

A Follicle-Stimulating Hormone Exacerbates the Progression of Periapical Inflammation Through Modulating the Cytokine Release in Periodontal Tissue.

Menopause is directly related to a systemically low grade of inflammation, indicating that postmenopausal women might be more prone to the development of inflammation. The high levels of circulating follicle-stimulating hormone (FSH) may cause hypogonadal bone loss during postmenopausal osteoporosis independent of estrogen. Previous research revealed that FSH could aggravate alveolar bone loss during experimental periapical lesions in ovariectomized rats; however, the mechanisms for these effects remain unclear. In this study, we showed that FSH enhanced the expression and secretion of the proinflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha to a significant degree in human periodontal ligament (hPDL) cells. Furthermore, FSH upregulated Porphyromonas gingivalis lipopolysaccharide (pg LPS)-induced proinflammatory cytokine production. Furthermore, in vivo studies demonstrated that FSH increased the levels of the aforementioned cytokines in the serum and enhanced the expression of Toll-like receptor 4 at both the messenger RNA and protein levels in hPDL cells and periodontal tissues. Our research suggests that high FSH levels may regulate the immune status of periodontal tissues during the postmenopausal period and, to a certain extent, suggested that postmenopausal women might be more prone to the development of inflammation of the periapical periodontitis and more obvious bone loss.

Vasoactive Intestinal Peptide Immunoregulatory Role at the Periapex: Associative and Mechanistic Evidences from Human and Experimental Periapical Lesions

IntroductionThe balance between the host proinflammatory immune response and the counteracting anti-inflammatory and reparative responses supposedly determine the outcome of periapical lesions. In this scenario, the vasoactive intestinal peptide (VIP) may exert a protective role because of its prominent immunoregulatory capacity. In this study, we investigated (in a cause-and-effect manner) the potential involvement of VIP in the development of human and experimental periapical lesions. MethodsPeriapical granulomas (n = 124) and control samples (n = 48) were comparatively assessed for VIP and multiple immunologic/activity marker expression through real-time polymerase chain reaction. Experimental periapical lesions (C57Bl/6 wild-type mice) were evaluated regarding endogenous VIP expression correlation with lesion development and the effect of recombinant VIP therapy in lesion outcome. CCR4KO and IL4KO strains and anti-glucocorticoid-induced TNFR-related protein inhibition were used to test the involvement of Treg and Th2 cells in VIP-mediated effects. ResultsVIP expression was more prevalent in periapical granulomas than in controls, presenting a positive association with immunoregulatory factors and an inverse association/correlation with proinflammatory mediators and the receptor activator of nuclear factor kappa B ligand/osteoprotegerin ratio. Endogenous VIP expression up-regulation was temporally associated with lesion immunoregulation and a decline of bone loss. VIP therapy in mice prompted the arrest of lesion development, being associated with an anti-inflammatory and proreparative response that limits the proinflammatory, Th1, Th17, and osteoclastogenic response in the periapex. The VIP protective effect was dependent of Treg migration and activity and independent of interleukin 4. ConclusionsOur results show that VIP overexpression in human and experimental periapical lesions is associated with lesion inactivity and that VIP therapy results in the attenuation of experimental lesion progression associated with the immunosuppressive response involving Treg cells.

Anti-inflammatory and antiresorptive functions of melatonin on experimentally induced periapical lesions.

To investigate the effects of systemically administered melatonin on inflammation and alveolar bone resorption in rats with experimentally induced periapical lesions. Thirty adult Sprague Dawley rats were divided equally into negative, positive control and melatonin groups. The pulp chambers of their mandibular first molars were exposed to the oral environment to induce experimental periapical lesions in the positive control and melatonin groups. The melatonin group received daily intraperitoneal injections of melatonin at a dose of 10mgkg-1 . After 21days, the animals were euthanized; the hemi-mandible parts were prepared for radiological, histopathological, immunohistochemical (IL-1β, RANK, RANKL, OPG and tartrate-resistant acid phosphatase (TRAP) and Brown-Brenn (bacteria) evaluations. Data were analysed by Kruskal-Wallis (for non-parametric data) and one-way anova tests (for parametric data) (P<0.05). The area of radiographic periapical bone loss was significantly smaller in rats that were given daily intraperitoneal injections of melatonin (P<0.01). The histopathological scores of the melatonin group were significantly lower than those of positive control group (P<0.01). Histomorphometrically, the area of periapical bone loss in the melatonin group was significantly smaller than the positive control group (P<0.01). The expression of IL1-β, RANK and RANKL was significantly higher in the positive control group, whereas OPG was significantly higher in the melatonin group (P<0.01). The number of osteoclasts was significantly greater in the positive control group by TRAP staining analyses (P<0.01). The scores for bacteria localization using Brown-Brenn staining in the melatonin group was significantly lower than that of the positive control group (P<0.01). Melatonin demonstrated antiresorptive effects on bone associated with experimentally induced periapical lesions in rats via its anti-inflammatory activity. Further studies are necessary to evaluate its possible effects on the healing of periapical lesions.

Imipramine treatment reverses depressive- and anxiety-like behaviors, normalize adrenocorticotropic hormone, and reduces interleukin-1β in the brain of rats subjected to experimental periapical lesion.

A periodontal lesion is a consequence of chronic inflammatory processes, itself triggered by a bacterial infection of the pulpal and endodontic microenvironment. Evidence suggests that periodontal lesion induction could alter inflammatory cytokines leading to behavior changes. These effects in the context of anxiety and depressive behavior have been not full investigated. We aimed to observe anxiety- and depressive-like behavioral in rodent subjected to periapical dental lesions. Pro-inflammatory cytokines levels also were investigated in the frontal cortex and hippocampus. Parameters related to hypothalamic-pituitary-adrenal (HPA) axis activation also were evaluated. Wistar rats were divided in groups: control/saline; control/imipramine; periapical lesion/saline; and periapical lesion/imipramine. Three weeks after induction of the periapical dental lesion, they were subjected to behavioral tests. In the periapical lesion group was demonstrated anhedonic behavior and depressive-like behavior. In the elevated plus-maze test the periapical lesion group had an increase in the number of entries and spent more time in the closed arms. Imipramine treatment was able to reverse depressive- and anxiety-like behaviors. In the hippocampus and frontal cortex tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and serum adrenocorticotropic hormone (ACTH) levels were higher in the periapical lesion group. However, rats treated with imipramine had lower IL-1β and ACTH levels. Our results revealed depressive- and anxiety-like behaviors following induction of a specific dental lesion. These effects could be associated to higher levels of brain pro-inflammatory cytokines and HPA axis changes. Antidepressants treatments could be an alternative to treat comorbidities associated to periodontal lesions.

Effects of Rosiglitazone on the Outcome of Experimental Periapical Lesions in Mice

IntroductionThe purpose of this study was to evaluate a protocol for systemic administration of rosiglitazone in mice in order to stimulate apoptosis of osteocytes in the jaws and to evaluate the effect of osteocyte apoptosis induced by rosiglitazone in the progression of periapical lesions in mice at 7, 21, and 42 days. MethodsC57BL/6 mice at 4–5 weeks of age were used. In phase 1, mice (n = 24) were treated with rosiglitazone (gavage, 10 mg/kg dose) or without (phosphate-buffered saline + 10% dimethyl sulfoxide) for 1, 2, or 3 weeks. We used the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and 4′-6-diamidino-2-phenylindole methods for quantification of apoptotic cells. In phase 2, mice (n = 30) received rosiglitazone for 2 weeks or just vehicle for 1 week (n = 30), and periapical lesions were induced for 7, 21, or 42 days. We performed the measurement of periapical lesions, tartrate-resistant acid phosphatase staining, dual-energy x-ray absorptiometry for the evaluation of bone mineral density (BMD) in long bone, and gene evaluation using real time quantitative polymerase chain reaction of osteocyte markers (Sost, Hyou1, and Dmp1) and receptor activator of nuclear factor kappa-B ligand (RANKL) (Tnfsf11). ResultsIt was observed that systemic administration of rosiglitazone for 2 weeks showed apoptosis of osteocytes in a more expressive manner. In phase 2, in the groups that received rosiglitazone, a trend toward larger periapical lesions was observed (P > .05). Rosiglitazone also induced a greater number of osteoclasts and a greater expression of Sost and Hyou1 at 21 days of lesions. Moreover, there were no statistically significant differences in RANKL and Dmp1 expression or in the BMD of femurs. ConclusionsRosiglitazone stimulated apoptosis of osteocytes, interfering in the progression of periapical lesions in mice.

Effects of Zoledronic Acid in Experimental Periapical Lesions in Rats: An Imaging and Histological Analysis.

The aim of this study was to assess the imaging and histological features of experimental periapical lesions, including the adjacent alveolar bone, in rats under zoledronic acid treatment. The study used 40 male Wistar rats distributed into 8 groups of 5 animals each: G1: induction of periapical lesion (PL) and weekly intraperitoneal administration (WIPA) of saline solution (0.9% NaCl) for 4 weeks; G2: PL induction and WIPA of zoledronic acid (0.15 mg/kg/week) for 4 weeks; G3: PL induction and WIPA of saline solution for 8 weeks; G4: PL induction and WIPA of zoledronic acid for 8 weeks; G5:WIPA of saline solution for 4 weeks and subsequent PL induction; G6: WIPA of zoledronic acid for 4 weeks and subsequent PL induction; G7: WIPA of saline solution for 8 weeks and subsequent PL induction; G8: WIPA of zoledronic acid for 8 weeks and subsequent PL induction. The administration of zoledronic acid or saline solution continued after PL induction until the euthanasia. Thus, cone beam computed tomography and histological analysis were performed. Statistical analyzes were performed by ANOVA and Kruskal-Wallis test. Groups treated with zoledronic acid showed significantly smaller size of PL than the groups treated with 0.9% NaCl (p<0.05). PLs were formed by chronic inflammation ranging from mild to moderate, with no difference between groups. In all specimens, no mandibular necrosis was observed. In conclusion, the presence of PLs apparently does not represent an important risk factor for the development of bisphosphonate-related osteonecrosis of the jaws.

High-mobility Group Box 1 Is Associated with the Inflammatory Infiltration and Alveolar Bone Destruction in Rats Experimental Periapical Lesions

IntroductionThis study was conducted to observe the immunohistochemical localization of high-mobility group box 1 (HMGB1) and its receptor, Toll-like receptor 4 (TRL4), in the development of periapical lesions induced in rats. The possible role of these molecules in the pathogenesis of periapical lesions was also explored. MethodsPeriapical lesions developed within 35 days after mandibular first molar pulp exposure in Wistar rats. The animals were randomly killed at 0, 7, 14, 21, 28, and 35 days after pulp exposure. The jaws that contained the first molar were obtained and prepared for histologic analysis, enzyme histochemistry, immunohistochemistry, and double immunofluorescence staining. ResultsFrom day 0 to 35, the areas of periapical bone loss increased and appeared to be stabilized on day 35. A few HMGB1-positive, TLR4-positive cells and osteoclasts could be observed on day 7. From day 7 to 28, the HMGB1 and TLR4 protein expression increased and subsequently remained stable. The number of osteoclasts multiplied from day 0 to 14 and then gradually decreased from day 14 to 35. Double immunofluorescence staining results showed HMGB1-positive, TLR4-positive cells around periapical lesions surrounding the apical foramen. ConclusionsThus, HMGB1 and TLR4 may be associated with the pathogenesis of the periapical lesions.

FSH aggravates bone loss in ovariectomised rats with experimental periapical periodontitis.

Periapical bone loss is one of the prominent pathological and clinical features of periapical periodontitis. Previous studies have demonstrated that follicle-stimulating hormone (FSH) could directly affect skeletal remodelling by stimulating the formation and the function of osteoclasts in vitro and in vivo. However, the effect of FSH on periapical bone loss remained to be fully elucidated. In the current study, a rat model was established in order to verify the effect of FSH in experimental periapical lesions. It was identified that FSH aggravated the bone loss of periapical lesions. In addition, RANKL-, TRAP-, TNF-α- and IL-1β-positive cells were increased significantly in FSH-treated groups, which indicated that the function of FSH in bone loss may be mediated through the increasing activity of osteoclasts and the increased secretion of inflammatory cytokines. The results of the current study suggested that FSH, independent of oestrogen, may aggravate periapical bone loss by FSH receptors, which may serve an important role in the immune and inflammatory response of the host to root canal and periradicular infection during menopause.

Characterization of the Protective Role of Regulatory T Cells in Experimental Periapical Lesion Development and Their Chemoattraction Manipulation as a Therapeutic Tool

IntroductionThe pathogenesis of periapical lesions is determined by the balance between host proinflammatory immune response and counteracting anti-inflammatory and reparative responses, which include regulatory T cells (Tregs) as potential immunoregulatory agents. In this study, we investigated (in a cause-and-effect manner) the involvement of CCL22-CCR4 axis in Treg migration to the periapical area and the role of Tregs in the determination of outcomes in periapical lesions. MethodsPeriapical lesions were induced in C57Bl/6 (wild-type) and CCR4KO mice (pulp exposure and bacterial inoculation) and treated with anti–glucocorticoid-induced TNF receptor family regulated gene to inhibit Treg function or alternatively with CCL22-releasing, polylactic-glycolic acid particles to induce site-specific migration of Tregs. After treatment, lesions were analyzed for Treg influx and phenotype, overall periapical bone loss, and inflammatory/immunologic and wound healing marker expression (analyzed by real-time polymerase chain reaction array). ResultsTreg inhibition by anti–glucocorticoid-induced TNF receptor family regulated gene or CCR4 depletion results in a significant increase in periapical lesion severity, associated with upregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with decreased Treg and healing marker expression. The local release of CCL22 in the root canal system resulted in the promotion of Treg migration in a CCR4-dependent manner, leading to the arrest of periapical lesion progression, associated with downregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with increased Treg and healing marker expression. ConclusionsBecause the natural and CCL22-induced Treg migration switches active lesion into inactivity phenotype, Treg chemoattractant may be a promising strategy for the clinical management of periapical lesions.

LPS-miR-34a-CCL22 axis contributes to regulatory T cell recruitment in periapical lesions

Regulatory T cells (Tregs) have been shown to regulate the immune response and to control the defense against infection in periapical lesions, but the underlying mechanisms by which Tregs are recruited to these lesions remain unknown. Here we demonstrate that expression of the gene encoding CCL22 (also known as macrophage-derived chemokine), the major chemoattractant that recruits Tregs, is upregulated in periapical tissue during the progression of experimental periapical lesions; this upregulation positively correlated with the number of Tregs that accumulated in the lesions. In terms of mechanism, we determined that lipopolysaccharide (LPS) up-regulates Ccl22 expression in macrophages by suppressing miR-34a. These findings suggest that the LPS-miR-34a-CCL22 axis may contribute to the recruitment of Tregs in periapical lesions, providing a potential therapeutic target for controlling this disease.

ST2 Deletion Increases Inflammatory Bone Destruction in Experimentally Induced Periapical Lesions in Mice

IntroductionST2 is a member of the interleukin (IL)-1 receptor family, and IL-33 is its natural ligand. ST2 signaling promotes Th2 immune response in allergy, autoimmunity, and chronic inflammatory disorders, but its role in the pathogenesis of periapical lesions is unknown. The purpose of this study was to investigate whether ST2 gene deletion affects the development of experimentally induced periapical lesions in mice. MethodsPulps of mandibular molars from wild-type (WT) and ST2 knockout (ST2–/–) BALB/c mice were exposed and left open to the oral environment. After death, hemi-mandibles were isolated and prepared for histologic, immunohistochemical, and flow cytometric analysis. ResultsThe expression of IL-33 and its receptor ST2 was higher in periapical lesions in WT mice compared with normal root apices (both P < .05). The increased periapical bone loss observed in ST2–/– mice was associated with enhanced influx of neutrophils, CD3+ CXCR3+ Th1 cells, and CD3+ CCR6+ Th17 cells and increased number of tartrate-resistant acid phosphatase+ osteoclasts (all P < .05). Furthermore, periapical lesions in ST2–/– mice contained increased percentages of T cells expressing interferon-γ, IL-17, tumor necrosis factor-α, and IL-6 (all P < .05). In comparison with WT mice, CD3+ receptor activator of nuclear factor kappa B ligand+ T cells were increased, whereas CD3+ osteoprotegerin+ T cells were decreased in the lesions of ST2–/– mice (both P < .05). ConclusionsST2 deletion increases inflammatory bone loss in experimental periapical lesions in mice, which is associated with enhanced Th1/Th17 cell mediated periapical immune responses and increased osteoclastogenesis.

Mesenchymal Stem Cells as Active Prohealing and Immunosuppressive Agents in Periapical Environment: Evidence from Human and Experimental Periapical Lesions

IntroductionPrevious studies describe contrasting molecular profiles of active and inactive periapical granulomas characterized by distinct expression of cytokines, osteoclastogenic factors, and wound healing markers. Although the molecular mechanisms underlying such a dichotomy remain unknown, in this study we investigated the potential involvement of mesenchymal stem cells (MSCs) in determining human and murine periapical lesion activity and outcomes. MethodsPeriapical granulomas (n = 83) and control samples (n = 24) were comparatively assessed for the expression levels of 11 mesenchymal stem cell (MSC) markers using real-time polymerase chain reaction. Experimental periapical lesions induced in mice were evaluated for MSC marker expression and the effects of AMD3100 treatment on lesion outcomes. ResultsMCS marker expression was prevalent in periapical granulomas compared with that in controls, whereas CD29, CD73, CD90, CD146, CD166, NANOG, Stro-1, and CXCR4 expressions were higher in inactive than in active lesions. Experimental periapical lesion inactivity was also associated with an increased expression of MSC markers. The inhibition of MSC mobilization to the periapex by AMD3100 resulted in increased lesion sizes; decreased expression of MSCs and wound healing markers; and increased expression of interleukin 1 beta (IL-17β), interleukin 17 (IL-17), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and nuclear factor kappa-B ligand (RANKL). ConclusionsOur results show that MSC markers are overexpressed in inactive human and experimental periapical lesions and that MSC mobilization results in the attenuation of experimental lesion progression associated with immunosuppressive and prohealing mechanisms.

Immunohistochemical localization of NALP3 inflammasome in experimental periapical lesions

To explore the role of NALP3 inflammasome [NALP3, its effector molecule apoptosis associated speck-like protein (ASC), caspase-1, interleukin (IL)-1β and IL-18] in the development of periapical lesions in rats. Periapical lesions were developed within 21days after mandibular first molar pulp exposure in Sprague-Dawley rats. The animals were randomly sacrificed at 0, 1, 3, 7, 10, 14 and 21days after pulpal exposure. The bilateral mandibles were extracted for histological processing, then they were haematoxylin-eosin (HE) stained to examine inflammation infiltration in the apical region and immunohistochemically examined for the NALP3 inflammasome signalling pathway. Data were analysed by one-way analysis of variance and the Pearson(') s correlation and linear tendency test. NALP3 was detected in the cytoplasm of fibroblasts, monocytes, neutrophils, macrophages and vascular endothelial cells in the periapical region. From day 1 to day 21, the number of NALP3-positive cells ascended and was significantly correlated with the intensity of inflammatory infiltration (r=0.776, P<0.01). ASC, caspase-1, IL-1β and IL-18 were all expressed in the inflammatory periapical tissues. The positive cell counts of IL-1β and IL-18 were significantly correlated with that of NALP3, and r=0.718, P<0.01; r=0.688, P<0.01, respectively. NALP3 inflammasome is expressed in the inflammatory periapical tissues. This cytokine-signalling pathway may therefore be crucial in the regulatory control of inflammatory responses in periapical tissues and the development of periapical lesions.

Expression of a cytokine, interleukin-23, in experimental periapical lesions

To investigate the expression of interleukin (IL)-23, a new member of the IL-12 family, in experimental periapical lesions. Periapical lesions were induced in Wistar rats by occlusal surface pulp exposure in mandibular first molars. The rats were randomly sacrificed 0, 7, 14, 21, 28, 35, 42 and 56 days after pulp exposure. The jaws that contained the first molars were obtained and routinely prepared for radiographic, histological, enzyme histochemical, immunohistochemical and double immunofluorescence analyses. Data were analysed by one-way analysis of variance and the Pearson correlation test. The number of IL-23-positive cells increased from day 7 to day 35 and then gradually decreased. The number of osteoclasts increased and peaked on day 14 and then gradually decreased from day 21 to day 56. A significant positive correlation existed between the number of IL-23 positive cells and the size of bone resorption in periapical lesions from day 7 to day 56 (r = 0.875, P < 0.01). Interleukin-23 can be observed and may be associated with inflammatory response and bone resorption in periapical lesions.

Vitapex can promote the expression of BMP-2 during the bone regeneration of periapical lesions in rats

To investigate the effect of Vitapex on the healing of periapical lesions and the expression of bone morphogenetic protein (BMP-2) during the periapical bone regeneration. Periapical lesions were induced in Sprague-Dawley (S-D) rats by an occlusal pulp exposure in the mandibular first molars and were verified by X-ray. Total of 36 rats were randomly divided into three groups, and they were obturated with Zinc Oxide Eugenol (ZOE), or with Vitapex, or non-treated as negative control group. The rats of three groups were randomly killed at week 0, 2, 4, and 8 after root canal therapy, and then the mandibles were processed for histological examination and immunohistochemistry analysis. At week 0, only a few BMP-2 positive cells could be observed in all rats. While the expression of BMP-2 was dramatically increased in case of Vitapex group at week 2 and week 4, and then climaxed at week 8. However, no apparent changes were observed in ZOE group and negative group at week 2, 4, and 8. These observations suggested that Vitapex has a greater ability in inducing bone regeneration than ZOE by the expression of BMP-2 induction in the treatment of rats experimental periapical lesions.

Gradation criteria for experimentally induced periapical lesions in mice

A periapical lesion occurs as periapical tissue reacts to a dental pulp anaerobic infection. This phenomenon may result from carious lesions, tooth fractures, or iatrogenic and other circumstances that allow for bacteria to penetrate into the pulpal tissues. Objectives: This study histologically evaluated experimentally induced periapical lesions using the Autodesk AutoCAD 2010 software. Additionally, based on the amounts of alveolar bone destruction sizes in the mouse model regions, a proposed experimental periapical lesion gradation criterion was created. Methods: Twelve BALB/c mice were utilizised in a periapical model, whereby their mandibular right first molars were coronally opened, allowing for pulp exposure to host oral bacterial flora. Th ese mice were sacrificed two and four weeks following pulp exposure. Following each sacrifice, hemimandibles were fixed in 4% paraformaldehyde, decalcified in 3% formic acid, embedded in paraffin and cut into 4-μm-thick sections. The sections were stained with haematoxylin- eosin and examined with light microscopy (40x). Section images that included the mandibular first molar distal roots and passed through the apical foramens were selected for analysis. The periodontal ligament sizes were measured using the Autodesk AutoCAD 2010 software. Results: There was a highly significant lesgion size difference at the two different time points following the lesion induction (p=0.002). The periapical lesions were classified according to periapical bone resorption sizes. We determined the values of quartiles (25% and 75%) and median areas (50%) of the mean lesion values at both experimental periods. These data enabled for scoring of the lesions with grades from 1 to 4. The area of the normal periodontal ligament space was assigned with a grade of 0. Conclusion: This newly designed gradation criteria represent a significant advantage compared with the previous descriptive methods used for determining periapical tissue bone destruction levels. It The criteria achieves this advantage by excluding subjectivity, facilitating a numerical presentation of the data and reducing the possibility of making errors by using the highly availably Autodesk AutoCAD 2010 software.

Evidence Supporting a Protective Role for Th9 and Th22 Cytokines in Human and Experimental Periapical Lesions

IntroductionThe development of periapical granulomas is dependent on the host response and involves Th1, Th2, Th17, and Treg-related cytokines. The discovery of new Th9 and Th22 subsets, with important immunomodulatory roles mediated by interleukin (IL)-9 and IL-22, respectively, emphasizes the need for reevaluation of current cytokine paradigms in context of periapical lesions. We investigated the expression of IL-9 and IL-22 in active and stable human granulomas and throughout experimental lesion development in mice. MethodsPeriapical granulomas (N = 83) and control specimens (N = 24) were evaluated regarding the expression of IL-9 and IL-22 via real-time polymerase chain reaction. Experimental periapical lesions were induced in mice (pulp exposure and bacterial inoculation) and the lesions evolution correlation with IL-9 and IL-22 expression kinetics was evaluated. ResultsIL-9 and IL-22 mRNA expression was higher in periapical lesions than in control samples; higher levels of IL-9 and IL-22 were observed in inactive than in active lesions. In the experimental lesions model, increasing levels of IL-9 and IL-22 mRNA were detected in the lesions, and inverse correlations were found between IL-9 and IL-22 and the increase of lesion area in the different time point intervals. ConclusionsOur results suggest that Th9 and Th22 pathways may contribute to human and experimental periapical lesion stability.

Protective Effect of Metformin on Periapical Lesions in Rats by Decreasing the Ratio of Receptor Activator of Nuclear Factor Kappa B Ligand/Osteoprotegerin

IntroductionMetformin, one of the antihyperglycemic agents commonly used for the treatment of type 2 diabetes, was shown to inhibit osteoclast formation. The current study aimed to investigate the effects of systemically administered metformin on alveolar bone resorption and on the ratio of receptor activator of nuclear factor kappa B ligand/osteoprotegerin (RANKL/OPG) in rats subjected to experimental periapical lesions. MethodsForty adult male Wistar rats were divided equally into control and experimental groups, and the pulp chambers of their mandibular first molars were exposed to the oral environment to induce periapical lesions. The experimental group received daily intramuscular injections of metformin at 40 mg/kg doses, whereas the control group received only the saline vehicle. The injections were initiated 1 day before the periapical lesion induction and then were continued daily throughout the entire experimental period. Two or 4 weeks after pulp exposure, the rats were killed, and the mandibles were prepared for histologic analysis, enzyme histochemistry, immunohistochemistry, and immunofluorescence. ResultsThe number of RANKL-positive and tartrate-resistant acid phosphatase (TRAP)–positive cells in the metformin-treated groups decreased on day 14, whereas the number of OPG-positive cells increased on day 28. The periapical bone loss area in the metformin-treated group significantly decreased on day 28 compared with the control group. ConclusionsMetformin inhibits the periapical lesions possibly by lowering the RANKL/OPG ratio, subsequently reducing the number of osteoclasts and bone resorption areas.

Effect of alendronate on alveolar bone resorption and angiogenesis in rats with experimental periapical lesions

To investigate the effects of systemically administered alendronate, one of the most potent bisphosphonates (BPs), on alveolar bone resorption and angiogenesis in rats subjected to experimental periapical lesions over two time periods. Forty adult Sprague-Dawley (SD) rats were divided equally into control and experimental groups, and the pulp chambers of mandibular first molars of all rats were exposed to the oral environment to induce periapical lesions. The experimental group received daily subcutaneous injections of alendronate at a dose of 0.25 mg kg(-1), whereas the control group received only the saline vehicle. These injections were initiated 1 week before the periapical lesion induction and then continued daily throughout the entire experimental period. After 2 or 4 weeks following pulp exposure, the rats were killed, and the mandibles were examined histologically for periapical bone loss area, number of microvascular vessels (NMV) and tartrate-resistant acid phosphatase (TRAP) activity. Overall, periapical bone loss area and the number of TRAP-positive cells (osteoclasts) were significantly decreased at 2 and 4 weeks, respectively, after daily subcutaneous injection of alendronate compared with the control group (P < 0.05). There was no significant decrease change in NMV (P > 0.05). Administration of alendronate to rats might inhibit alveolar bone resorption associated with periapical disease, which might not lead to impairment of angiogenesis. However, because of the differences between rats and humans, one has to consider the possible consequences of this treatment in the clinic.