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Introduction: The 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., Methods: Periapical 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., Results: VIP 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., Conclusions: Our 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., (Copyright © 2019 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Understanding protein expression profiles of apical periodontitis may contribute to the discovery of novel diagnostic or therapeutic molecular targets., Methods: Periapical tissue samples (n = 5) of patients with lesions characterized as nonhealing were submitted for proteomic analysis. Two differentially expressed proteins (heat shock protein 27 [HSP27] and serpin family B member 1 [SERPINB1]) were selected for characterization, localization by immunofluorescence, and association with known biomarkers of acute inflammatory response in human apical periodontitis (n = 110) and healthy periodontal ligaments (n = 26). Apical periodontitis samples were categorized as stable/inactive (n = 70) or progressive/active (n = 40) based on the ratio of expression of receptor activator of nuclear factor kappa-B ligand (RANKL)/osteoprotegerin (OPG). Next, the expression of HSP27, SERPINB1, C-X-C motif Chemokine Receptor 1 (CXCR1), matrix metalloproteinase 8 (MMP8), myeloperoxidase (MPO), and cathepsin G (CTSG) messenger RNA was evaluated using real-time polymerase chain reaction. Data analysis was performed using the Shapiro-Wilk test, analysis of variance, and the Pearson test. P values <.05 were considered statistically significant., Results: Proteomic analysis revealed 48 proteins as differentially expressed in apical periodontitis compared with a healthy periodontium, with 30 of these proteins found to be expressed in all 4 lesions. The expression of HSP27 and SERPINB1 was ∼2-fold higher in apical periodontitis. Next, an increased expression of HSP27 was detected in epithelial cells, whereas SERPINB1 expression was noted in neutrophils and epithelial cells. HSP27 and SERPINB1 transcripts were highly expressed in stable/inactive lesions (P < .05). Significant negative correlations were found between the expression of HSP27 and SERPINB1 with biomarkers of acute inflammation including CXCR1, MPO, and CTSG., Conclusions: Our data suggest HSP27 and SERPINB1 as potential regulators of the inflammatory response in apical periodontitis. Additional functional studies should be performed to further characterize the role of these molecules during the development/progression of apical periodontitis., (Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: This study evaluated the physicochemical, antimicrobial, and biological properties of white mineral trioxide aggregate (MTA) mixed with aqueous and propylene glycol extracts of Arctium lappa L. and Casearia sylvestris Sw., Methods: The setting-time test was performed according to American Society for Testing and Materials 266/2008 and flowability by American National Standards Institute/American Dental Association 57/2012 standard specifications. The following parameters were assessed: volumetric change by micro-computed tomography; pH level and calcium ion release measured after 3 hours and 1, 3, 7, and 15 days; antimicrobial effect by the dentin decontamination method with intratubular Enterococcus faecalis viability, verified by using confocal laser scanning microscopy; and biocompatibility by histologic-morphometric analyses of inflammatory infiltrate at subcutaneous implant sites after 15, 30, and 60 days. In addition, rat alveolar tissues with implants were processed to measure tumor necrosis factor-alpha and interleukin-10 cytokines by enzyme-linked immunosorbent assay., Results: The plant extracts associated with MTA significantly increased the final setting time; however, they did not influence volumetric change (P > .05) and maintained medium alkalinity and calcium ion release. Propylene glycol extracts showed higher flowability. Casearia sylvestris increased the cementing effect against E. faecalis after 24-hour and 168-hour periods. Histologic evaluation of inflammatory infiltrate showed no significant differences between plant extracts groups and the distilled water group for all periods. Tumor necrosis factor-alpha and interleukin-10 expression was similar among groups (P > .05)., Conclusions: Casearia sylvestris extracts increased the antimicrobial effect of MTA and did not influence biocompatibility but changed some physicochemical properties., (Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Vascular endothelial growth factor (VEGF) is a signal protein that stimulates angiogenesis and vasculogenesis and has been used in tissue regeneration and pulp regeneration experimental models. The purpose of this study was to develop a delivery system composed of a biodegradable fiber and controlled release of VEGF to promote cell viability and secure an adequate blood supply for the survival of human stem cells of the apical papilla (SCAP) favoring endodontic regenerative procedures., Methods: We developed a polydioxanone fiber, 50 μm in diameter, loaded with VEGF at a linear concentration of 12.2 ng/cm. Cytotoxic effects of the VEGF-loaded fiber (VF) on SCAP and mouse fibroblasts were assessed by using a multiparametric assay kit (XTT-NR-CVDE [Xenometrix, Allschwil, Switzerland]). We evaluated VF-induced mRNA expression of downstream growth factors by using a human growth factor Taqman array in real-time polymerase chain reaction. We also assessed the in vivo subcutaneous reaction of C57BL/6 mice to implants of VF alone and human root fragments (10 mm in length) filled with VF after 10, 20, and 45 days. Statistical analyses were performed by using analysis of variance and Student t tests or non-parametric alternatives., Results: Enzyme-linked immunosorbent assay verified detectable concentrations of released VEGF in solution for 25 days. No cytotoxicity was observed on SCAP and mouse fibroblasts treated with VEGF. In addition, VEGF treatment also induced the expression of additional growth factors with roles in tissue and blood vessel formation and neuroprotective function. Implantation of VF and root fragments filled with VF showed biocompatibility in vivo, promoting new blood vessels and connective tissue formation into the root canal space with negligible inflammation., Conclusions: Our results show that the VF used in this study is biocompatible and may be a promising scaffold for additional optimization and use in endodontic regenerative procedures., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Tumor necrosis factor alpha (TNF-α) levels are significantly upregulated in the synovial fluid of patients with temporomandibular joint disorder (TMD). The TNF-α influences pain generation and maintenance. Therefore, the aim of this study was to investigate the influence of single nucleotide polymorphism TNFA-308 (rs1800629) on TMD risk and on the pressure pain threshold., Methods: The genotypic and allelic frequencies of candidate single nucleotide polymorphisms were compared among 152 TMD patients and 91 sex- and age-matched healthy subjects in the control group using the real-time polymerase chain reaction technique. The pressure pain threshold in the temporomandibular joint, anterior fascicle of the temporal muscle, masseter muscle, and Achilles tendon were recorded with an algometer. After the pressure test, all participants received a complete physical examination, including masticatory muscle evaluation, temporomandibular joint palpation, and assessment of mandibular range of motion., Results: The TNFA-308 polymorphism is positively associated with TMD. Subjects with TMD had a 2.87 (95% confidence interval, 1.256-6.569) times greater chance of having the GA genotype than did the control group. Rare A-allele homozygotes demonstrated decreased pain sensitivity for the temporomandibular joint and anterior fascicle of the temporal muscle in the pressure pain threshold test compared with ancestral allele homozygotes., Conclusions: This study presents an unprecedented association between the TNFA-308 (rs1800629) polymorphism and TMD. Future studies are needed to enlighten the association between TNFA-308 G/A single nucleotide polymorphism and mechanical pain sensitivity., (Copyright © 2016 American Association of Orthodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The 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., Methods: Periapical 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)., Results: Treg 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., Conclusions: Because 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., (Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: It has been proposed that individual genetic predisposition may contribute to persistent apical periodontitis. Cytokines are associated with levels of inflammation and are involved in caries, pulpal, and periapical tissue destruction. We hypothesized that polymorphisms in cytokine genes may contribute to an individual's increased susceptibility to apical tissue destruction in response to deep carious lesions., Methods: Subjects with deep carious lesions with or without periapical lesions (≥3 mm) were recruited at the University of Pittsburgh, Pittsburgh, PA, and the University of Texas at Houston, Houston, TX. Genomic DNA samples of 316 patients were sorted into 2 groups: 136 cases with deep carious lesions and periapical lesions (cases) and 180 cases with deep carious lesions but no periapical lesions (controls). Nine single-nucleotide polymorphisms in IL1B, IL6, TNF, RANK, RANKL, and OPG genes were selected for genotyping. Genotypes were generated by end point analysis using TaqMan chemistry (Invitrogen, Carlsbad, CA) in a real-time polymerase chain reaction instrument. Allele and genotype frequencies were compared among cases and controls using the PLINK program (http://pngu.mgh.harvard.edu/purcell/plink/). Ninety-three human periapical granulomas and 24 healthy periodontal ligament tissues collected postoperatively were used for messenger RNA expression analyses of IL1B., Results: A single-nucleotide polymorphism in IL1B (rs1143643) showed allelic (P = .02) and genotypic (P = .004) association with cases of deep caries and periapical lesions. We also observed altered transmission of IL1B marker haplotypes (P = .02) in these individuals. IL1B was highly expressed in granulomas (P < .001)., Conclusions: Variations in IL1B may be associated with periapical lesion formation in individuals with untreated deep carious lesions. Future studies could help predict host susceptibility to developing periapical lesions., (Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Previous 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., Methods: Periapical 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., Results: MCS 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)., Conclusions: Our 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., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Cells from virtually all organisms respond to a variety of stresses by the rapid synthesis of a highly conserved set of polypeptides termed heat shock proteins (HSPs). HSPs protect cells under adverse conditions such as infection, inflammation, and disease. We hypothesize that endodontic infection might result in an imbalance in the expression of heat shock genes, accounting for different clinical outcomes in periapical lesions., Methods: We analyzed the expression of 44 HSPs genes using a pathway-specific real-time polymerase chain reaction array in 93 human periapical granulomas and 24 healthy periodontal ligament tissues collected postoperatively. Observed variations in the expression of HSP genes were also analyzed based on the classification of periapical granulomas as active or inactive. In addition, U937 cells were differentiated into macrophages, infected with different concentrations of purified Escherichia coli lipopolysaccharide (LPS), and used as templates for the HSP gene array. Protein expression was assessed by immunohistochemistry., Results: The expression of HSP genes was significantly increased in granulomas compared with healthy periodontal ligament (P < .00001). Among the 44 HSP genes, DNAJC3, HSPA4, HSPA6, and HSPB1 showed the highest expression levels in both granulomas and LPS-treated macrophages. DNAJC3, HSPA6, and HSPB1 were highly expressed in active lesions, whereas HSPA4 expression was higher in inactive lesions (P < .005). Higher concentrations of LPS led to increased HSP expression in macrophages (P < .0001). Immunocytochemistry confirmed the expression and colocalization of HSPB1 and HSPA6 proteins in the cytoplasm of LPS-infected macrophages., Conclusions: The observed differential expression patterns of HSPs in periapical granulomas and LPS-infected macrophages suggest that HSP genes and proteins are involved in periapical lesion development and may account for different clinical outcomes. Understanding the role of the heat shock response might provide additional insights into the process of periapical lesion development., (Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Matrix metalloproteinases (MMPs) and the tissue inhibitors of metalloproteinases (TIMPs) are strongly associated with tissue destruction because of inflammation. In this study, we investigated the expression of MMPs and TIMPs messenger RNA and protein levels in apical periodontitis lesions., Methods: Tissue samples from patients presenting clinical signs of chronic apical abscess (CAA) or asymptomatic apical periodontitis (AAP) were collected postoperatively and used for gene expression analysis of MMP-2, -3, -7, -9, -14, -16, and -25; TIMP-1; and TIMP-2 in real-time polymerase chain reaction. Immunohistochemistry was also performed to detect the expression of MMP-7 and TIMP-1 proteins. Lastly, U-937 cells were induced to terminal differentiation into macrophages, infected with purified Escherichia coli lipopolysaccharide, and assessed for the expression of MMP-7 and TIMP-1 using immunocytochemistry and confocal microscopy., Results: Significantly higher messenger RNA levels were found for all genes in AAP and CAA samples when compared with healthy control samples (P < .001). AAP cases exhibited significantly higher TIMP-1 when compared with CAA cases, whereas CAA cases showed higher MMP-2, MMP-7, and MMP-9 messenger RNA levels (P < .05). We also detected positive the expression of MMP-7 and TIMP-1 proteins in the tissue samples. The expression of both MMP-7 and TIMP-1 were increased in lipopolysaccharide-stimulated cells compared with nonstimulated cells and appear to colocalize in the Golgi apparatus., Conclusions: MMPs appear to have an influential role in CAA cases in which ongoing tissue destruction is observed. TIMPs are preferentially associated with AAP, perhaps as a subsequent defense mechanism against excessive destruction. Taken together, our findings implicate MMP and TIMP molecules in the dynamics of inflammatory periapical lesion development., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The 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., Methods: Periapical 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., Results: IL-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., Conclusions: Our results suggest that Th9 and Th22 pathways may contribute to human and experimental periapical lesion stability., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Wound healing process involves the activation of extracellular matrix components, remodeling enzymes, cellular adhesion molecules, growth factors, cytokines and chemokines genes. However, the molecular patterns underlying the healing process at the periapical environment remain unclear. Here we hypothesized that endodontic infection might result in an imbalance in the expression of wound healing genes involved in the pathogenesis of periapical lesions. Furthermore, we suggest that differential expression of wound healing markers in active and latent granulomas could account for different clinical outcomes for such lesions., Methods: Study samples consisted of 93 periapical granulomas collected after endodontic surgeries and 24 healthy periodontal ligament tissues collected from premolars extracted for orthodontic purposes as control samples. Of these, 10 periapical granulomas and 5 healthy periapical tissues were used for expression analysis of 84 wound healing genes by using a pathway-specific real-time polymerase chain reaction array. The remaining 83 granulomas and all 24 control specimens were used to validate the obtained array data by real-time polymerase chain reaction. Observed variations in expression of wound healing genes were analyzed according to the classification of periapical granulomas as active/progressive versus inactive/stable (as determined by receptor activator for nuclear factor kappa B ligand/osteoprotegerin expression ratio)., Results: We observed a marked increase of 5-fold or greater in SERPINE1, TIMP1, COL1A1, COL5A1, VTN, CTGF, FGF7, TGFB1, TNF, CXCL11, ITGA4, and ITGA5 genes in the periapical granulomas when compared with control samples. SERPINE1, TIMP1, COL1A1, TGFB1, and ITGA4 mRNA expression was significantly higher in inactive compared with active periapical granulomas (P < .001), whereas TNF and CXCL11 mRNA expression was higher in active lesions (P < .001)., Conclusions: The identification of novel gene targets that curb the progression status of periapical lesions might contribute to a more accurate diagnosis and lead to treatment modalities more conducive to endodontic success., (Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: Cytokines and chemokines regulate bone remodeling during orthodontic tooth movement. CC chemokine ligand 2 (CCL2) is involved in osteoclast recruitment and activity, and its expression is increased in periodontal tissues under mechanical loading. In this study, we investigated whether the CC chemokine receptor 2 (CCR2)-CCL2 axis influences orthodontic tooth movement., Methods: A coil spring was placed in CCR2-deficient (CCR2(-/-)), wild-type, vehicle-treated, and P8A-treated (CCL2 analog) mice. In a histopathologic analysis, the amounts of orthodontic tooth movement and numbers of osteoclasts were determined. The expression of mediators involved in bone remodeling was evaluated by real-time polymerase chain reaction., Results: Orthodontic tooth movement and the number of TRAP-positive cells were significantly decreased in CCR2(-/-) and P8A-treated mice in relation to wild-type and vehicle-treated mice, respectively. The expressions of RANKL, RANK, and osteoblasts markers (COL-1 and OCN) were lower in CCR2(-/-) than in wild-type mice. No significant difference was found in osteoprotegerin levels between the groups., Conclusions: These data suggested a reduction of osteoclast and osteoblast activities in the absence of CCR2. The CCR2-CCL2 axis is positively associated with osteoclast recruitment, bone resorption, and orthodontic tooth movement. Therefore, blockage of the CCR2-CCL2 axis might be used in the future for modulating the extent of orthodontic tooth movement., (Copyright © 2012 American Association of Orthodontists. Published by Mosby, Inc. All rights reserved.)

Periodontal disease (PD) is a chronic inflammatory and alveolar bone destructive disease triggered by microorganisms from the oral biofilm. Oral inoculation of mice with the periodontopathogen Aggregatibacter actinomycetemcomitans (Aa) induces marked alveolar bone loss and local production of inflammatory mediators, including Macrophage Migration Inhibitory Factor (MIF). The role of MIF for alveolar bone resorption during PD is not known. In the present study, experimental PD was induced in BALB/c wild-type mice (WT) and MIF knockout mice (MIF⁻/⁻) through oral inoculation of Aa. Despite enhanced number of bacteria, MIF⁻/⁻ mice had reduced infiltration of TRAP-positive cells and reduced alveolar bone loss. This was associated with decreased neutrophil accumulation and increased levels of IL-10 in periodontal tissues. TNF-α production was similar in both groups. In vitro, LPS from Aa enhanced osteoclastic activity in a MIF-dependent manner. In conclusion, MIF has role in controlling bacterial growth in the context of PD but contributes more significantly to the progression of bone loss during PD by directly affecting differentiation and activity of osteoclasts., (Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Introduction: Periapical lesions are chronic inflammatory disorders of periradicular tissues caused by etiologic agents of endodontic origin. The inflammatory chemokines are thought to be involved in the latter observed osteolysis. With a murine model of experimental periapical lesion, the objective of this study was to evaluate the role of the chemokine receptor CCR2 in the lesion progression, osteoclast differentiation and activation, and expression of inflammatory osteolysis-related mediators., Methods: For lesion induction, right mandibular first molars were opened surgically with a 1/4 carbine bur, and 4 bacterial strains were inoculated in the exposed dental pulp; left mandibular first molars were used as controls. Animals were killed at 3, 7, 14, and 21 days after surgeries to evaluate the kinetics of lesion development., Results: CCR2 KO mice showed wider lesions than WT mice. CCR2 KO mice also expressed higher levels of the osteoclastogenic and osteolytic factors, receptor activator of nuclear factor kappa B ligand (RANKL) and cathepsin K, of the proinflammatory cytokine tumor necrosis factor-alpha, and of the neutrophil migration related chemokine, KC., Conclusions: These results suggest that CCR2 is important in host protection to periapical osteolysis., (Copyright 2010 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objective: The objective of this study was to determine the expression of matrix metalloproteinase-9 (MMP-9) in apical periodontitis lesions., Study Design: Nineteen epithelialized and 18 nonepithelialized apical periodontitis lesions were collected after periapical surgery. After histological processing, serial sectioning, H&E staining, and microscopic analysis, 10 epithelialized and 10 nonepithelialized lesions were selected for immunohistochemical analysis for MMP-9 and CD 68. At least one third of each specimen collected was frozen at -70 degrees C for further mRNA isolation and reverse transcription into cDNA for real-time-PCR procedures. Geometric averaging of multiple housekeeping genes normalized MMP-9 mRNA expression level., Results: Polymorphonuclear neutrophils, macrophages and lymphocytes presented MMP-9 positive immunostaining in both types of lesions. When present, epithelial cells were also stained. The number and the ratio of MMP-9(+)/total cells were greater in nonepithelialized than epithelialized lesions (P = .0001) presenting a positive correlation to CD68(+)/total cells (P = .045). Both types of lesions presented increased MMP-9 expression (P < .0001) when compared to healthy periapical ligaments. However, no significant differences were observed for MMP-9 mRNA expression between ephithelized and nonephithelized lesions., Conclusion: The present data suggest the participation of several inflammatory cells, mainly CD68(+) cells, in the MMP-9 expression in apical periodontitis lesions. MMP-9 could be actively enrolled in the extracellular matrix degradation in apical periodontitis lesions.

Inflammatory cytokines contribute to periapical tissue destruction. Their activity is potentially regulated by suppressors of cytokine signaling (SOCS), which downregulate signal transduction as part of an inhibitory feedback loop. We investigated the expression of the cytokines tumor necrosis factor alpha (TNF-alpha); interleukin (IL)-10 and RANKL; and SOCS-1, -2, and -3 by real-time polymerase chain reaction in 57 periapical granulomas and 38 healthy periapical tissues. Periapical granulomas exhibited significantly higher SOCS-1, -2, and -3, TNF-alpha, IL-10, and RANKL messenger RNA levels when compared with healthy controls. Significant positive correlations were found between SOCS1 and IL-10 and between SOCS3 and IL-10. Significant inverse correlations were observed between SOCS1 and TNF-alpha, SOCS1 and RANKL, and SOCS3 and TNF-alpha. Increased SOCS-1, -2, and -3 messenger RNA levels in periapical granulomas may be related to the downregulation of inflammatory cytokines in these lesions; therefore, SOCS molecules may play a role in the dynamics of periapical granulomas development.

Receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) are expressed in apical periodontitis, suggesting a role for these molecules during lesion development. However, the profiles of RANKL/OPG expression in periapical lesions remain unknown. In this study we investigated the patterns of RANKL and OPG mRNA expression by real-time polymerase chain reaction in human periapical granulomas (N = 44) and compared them with sites presenting characteristic bone resorbing activity: healthy (n = 14) and orthodontically stretched and compressed periodontal ligament (n = 26), healthy gingiva (n = 24), chronic gingivitis (n = 32), and chronic periodontitis (n = 34) samples. Both RANKL and OPG mRNA expression was higher in periapical granulomas when compared with healthy periodontal ligament. Distinct patterns of RANKL and OPG expression ratio were found in the granulomas and in different physiologic and pathologic conditions, with characteristic bone resorption activity potentially being indicative of the stable or progressive nature of the lesions. Lesions with radiographic image smaller than 5 mm showed higher RANKL/OPG expression than images greater than 5 mm. Periapical granulomas presented heterogeneous patterns of RANKL and OPG expression, ranging from samples with RANKL/OPG ratio similar to that seen in sites with minimal or absent bone resorption to samples with RANKL/OPG expression pattern comparable with active bone resorption sites.