Final irrigation protocols can be used to promote stable long-term bond strength of AH Plus to dentin.

Irrigation solutions might affect dentin surface characteristics and, consequently, endodontic sealers adhesion. This study analyzed the effect of different final irrigation protocols on push-out bond strength (BS) of AH Plus to dentin seven days and 20 months after obturation. Scanning electron micrographs were obtained from the dentin surface of one sample/group after final irrigation. Canals of bovine incisors were instrumented and received final irrigation with (n=21): G1 - 2.5% sodium hypochlorite (NaOCl) + distilled water; G2 - 2.5% NaOCl + 17% EDTA; G3 - 2.5% NaOCl + 17% EDTA + 2.5% NaOCl; G4 - 2.5% NaOCl + 17% EDTA + 2% chlorhexidine (CHX); G5 - mixture 5% NaOCl + 18% etidronate (HEDP); and G6 - mixture 5% NaOCl + 10% tetrasodium EDTA (Na4EDTA). After irrigation, one root/group was split and images were obtained by scanning electron microscopy (SEM). The other 20 roots/group were filled with only AH Plus sealer. Three slices/root were used for push-out assessment seven days and 20 months after obturation. One-way analysis of variance and Tukey (α<0.05) were used to compare the results among experimental groups, and unpaired t-test (α<0.05) was used to compare the results of the same group over time. The photomicrographs showed that, excepting G1, all groups completely removed the smear layer from the samples. In G2 and G4, the opening of the dentin tubules enlarged. In G3, erosion was observed in the peritubular and intertubular dentin. Values of the BS in the seven days were G2=G3=G4=G5>G6=G1 and in the 20 months were G3=G5>G6=G4>G1=G2. G3, G5, and G6 presented values of BS in 20 months similar to the values of seven days (P>0.05). The final irrigation protocols tested produced dentin surfaces with different characteristics. Only G3 and G5 presented high BS values that were stable over time.

Final irrigation protocols can be used to promote stable long-term bond strength of AH Plus to dentin

Abstract Irrigation solutions might affect dentin surface characteristics and, consequently, endodontic sealers adhesion. Objective This study analyzed the effect of different final irrigation protocols on push-out bond strength (BS) of AH Plus to dentin seven days and 20 months after obturation. Scanning electron micrographs were obtained from the dentin surface of one sample/group after final irrigation. Methodology Canals of bovine incisors were instrumented and received final irrigation with (n=21): G1 - 2.5% sodium hypochlorite (NaOCl) + distilled water; G2 - 2.5% NaOCl + 17% EDTA; G3 - 2.5% NaOCl + 17% EDTA + 2.5% NaOCl; G4 - 2.5% NaOCl + 17% EDTA + 2% chlorhexidine (CHX); G5 - mixture 5% NaOCl + 18% etidronate (HEDP); and G6 - mixture 5% NaOCl + 10% tetrasodium EDTA (Na4EDTA). After irrigation, one root/group was split and images were obtained by scanning electron microscopy (SEM). The other 20 roots/group were filled with only AH Plus sealer. Three slices/root were used for push-out assessment seven days and 20 months after obturation. One-way analysis of variance and Tukey (α<0.05) were used to compare the results among experimental groups, and unpaired t-test (α<0.05) was used to compare the results of the same group over time. Results The photomicrographs showed that, excepting G1, all groups completely removed the smear layer from the samples. In G2 and G4, the opening of the dentin tubules enlarged. In G3, erosion was observed in the peritubular and intertubular dentin. Values of the BS in the seven days were G2=G3=G4=G5>G6=G1 and in the 20 months were G3=G5>G6=G4>G1=G2. G3, G5, and G6 presented values of BS in 20 months similar to the values of seven days (P>0.05). Conclusions The final irrigation protocols tested produced dentin surfaces with different characteristics. Only G3 and G5 presented high BS values that were stable over time.

MicroRNAs: emerging players in apical periodontitis.

Apical periodontitis is an inflammatory disorder of periradicular tissues developed from endodontic infections. Understanding its pathophysiology and the underlying molecular mechanisms is key to the advancement of endodontics. MicroRNAs (miRNAs), a group of evolutionarily conserved small non-coding RNAs, may be phenotypically and functionally associated with the pathogenesis of apical periodontitis. Several studies have focused on the role of miRNAs in the pulp and periradicular biology, and they have demonstrated their essential functions, such as initiating odontogenic differentiation and promoting pro- or anti-inflammatory responses in pulpitis. Up to date, over 2,000 miRNAs have been discovered in humans; however, only few have been reported to associate with apical periodontitis. Therefore, identifying miRNAs involved in diseased apical tissues and conducting functional studies are important in expanding our current knowledge of pulp and periradicular biology and exploring novel therapeutic avenues. In this review, we revisit current models of apical periodontitis and miRNA biogenesis, analyze existing evidence of the involvement of miRNAs in diseased apical tissues, and discuss their diverse functions and potential values. Based on their sheer abundance, prolonged stability in biofluid, and relative ease of sampling, miRNAs may be a useful tool to be developed as diagnostic biomarkers for apical periodontitis. Furthermore, it can be used as therapeutic targets in conjunction with conventional endodontic therapies.

MicroRNAs: emerging players in apical periodontitis

Abstract Apical periodontitis is an inflammatory disorder of periradicular tissues developed from endodontic infections. Understanding its pathophysiology and the underlying molecular mechanisms is key to the advancement of endodontics. MicroRNAs (miRNAs), a group of evolutionarily conserved small non-coding RNAs, may be phenotypically and functionally associated with the pathogenesis of apical periodontitis. Several studies have focused on the role of miRNAs in the pulp and periradicular biology, and they have demonstrated their essential functions, such as initiating odontogenic differentiation and promoting pro- or anti-inflammatory responses in pulpitis. Up to date, over 2,000 miRNAs have been discovered in humans; however, only few have been reported to associate with apical periodontitis. Therefore, identifying miRNAs involved in diseased apical tissues and conducting functional studies are important in expanding our current knowledge of pulp and periradicular biology and exploring novel therapeutic avenues. In this review, we revisit current models of apical periodontitis and miRNA biogenesis, analyze existing evidence of the involvement of miRNAs in diseased apical tissues, and discuss their diverse functions and potential values. Based on their sheer abundance, prolonged stability in biofluid, and relative ease of sampling, miRNAs may be a useful tool to be developed as diagnostic biomarkers for apical periodontitis. Furthermore, it can be used as therapeutic targets in conjunction with conventional endodontic therapies.

Evaluation of Passive Ultrasonic Irrigation and GentleWave System as Adjuvants in Endodontic Retreatment.

INTRODUCTION: Nonsurgical endodontic retreatment continues to be a challenge in endodontics, particularly when dealing with a complex tooth anatomy. This study evaluated the efficacy of passive ultrasonic irrigation (PUI) and the GentleWave system as supplementary techniques to remove remaining filling materials from oval-shaped root canals. METHODS: Twenty distal roots of human mandibular molars with single and oval-shaped canals were shaped with R40 (40.06) instrument and filled with gutta-percha and AH Plus sealer using warm vertical obturation. Initial filling material removal was performed with R50 (50.05) instrument, followed by the use of PUI (n = 10) or GentleWave system (n = 10). Micro-computed tomographic images were obtained after obturation, initial material removal, and after the use of PUI and GentleWave. The volume of remaining filling material was calculated for the entire canal as well as for the coronal, middle, and apical thirds. Statistical analyses were performed by using analysis of variance, Kruskal-Wallis and Mann-Whitney tests. P ≤ .05 was considered significant. RESULTS: The use of PUI and GentleWave as supplementary techniques significantly reduced the volume of remaining filling material after initial instrumentation (P < .05). However, none of these techniques was able to render canals free from filling materials. PUI showed better performance by removing 18% of the remaining filling material, whereas the GentleWave system was able to remove approximately 10% (P = .02). CONCLUSIONS: The use of supplementary techniques optimized filling material removal after initial instrumentation. PUI enhanced the overall cleaning of the root canal system during endodontic retreatment in oval-shaped canals.

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

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.

TBX21-1993T/C polymorphism association with Th1 and Th17 response at periapex and with periapical lesions development risk.

TBX21-1993T/C (rs4794067) polymorphism increases the transcriptional activity of the Tbx21, essential for interferon gamma (IFNg) transcription, but its functional impact on development Th1- response in vivo remains unclear, as well its potential influence over inflammatory osteolytic conditions, such as periapical lesions. Therefore, this study comprises a case-control and functional investigation of Tbx21 genetic variations impact on Th1 response in vivo and in vitro, and its impact on periapical lesions risk and outcome, performed with a population of healthy controls (H; N = 283) and patients presenting periapical lesions (L; N = 188) or deep caries (DC; N = 152). TBX21-1993T/C genotyping demonstrated that the polymorphic allele C, as well TC/TC+CC genotypes, was significantly less frequent in the L patients compared to H and DC groups. Additionally, gene expression analysis demonstrates that T-cell-specific T-box transcription factor (Tbet) and IFNg transcripts levels were downregulated whereas IL-17 levels were upregulated in the TBX21-1993 C carriers (TC/TC+CC) in comparison with the TT group. Also, while TT and TC+CC genotypes are equally prevalent in the lesions presenting low IFN/IL17 ratio, a significant decrease in polymorphic TC+CC genotypes was observed in lesions presenting intermediate and high IFN/IL17 ratio. In vitro experiments confirmed the predisposition to Th1 polarization associated with TBX21-1993, since PBMC CD4 T cells from T allele carriers produce higher IFNg levels upon CD3/CD28 stimulation than the C group, in both standard/neutral and Th1-polarizing culture conditions. In conclusion, the TBX21-1993 T allele and TC/CC genotypes predispose to Th1-type immune response development in vitro, influence immune response polarization in vivo, and consequently account for the risk for apical periodontitis development.

CCR2 Contributes to F4/80+ Cells Migration Along Intramembranous Bone Healing in Maxilla, but Its Deficiency Does Not Critically Affect the Healing Outcome.

Bone healing depends of a transient inflammatory response, involving selective migration of leukocytes under the control of chemokine system. CCR2 has been regarded as an essential receptor for macrophage recruitment to inflammation and healing sites, but its role in the intramembranous bone healing on craniofacial region remains unknown. Therefore, we investigated the role of CCR2 on F4/80+ cells migration and its consequences to the intramembranous healing outcome. C57BL/6 wild-type (WT) and CCR2KO mice were subjected to upper right incisor extraction, followed by micro-computed tomography, histological, immunological, and molecular analysis along experimental periods. CCR2 was associated with F4/80+ cells influx to the intramembranous bone healing in WT mice, and CCR2+ cells presented a kinetics similar to F4/80+ and CCR5+ cells. By contrast, F4/80+ and CCR5+ cells were significantly reduced in CCR2KO mice. The absence of CCR2 did not cause major microscopic changes in healing parameters, while molecular analysis demonstrated differential genes expression of several molecules between CCR2KO and WT mice. The mRNA expression of TGFB1, RUNX2, and mesenchymal stem cells markers (CXCL12, CD106, OCT4, NANOG, and CD146) was decreased in CCR2KO mice, while IL6, CXCR1, RANKL, and ECM markers (MMP1, 2, 9, and Col1a2) were significantly increased in different periods. Finally, immunofluorescence and FACS revealed that F4/80+ cells are positive for both CCR2 and CCR5, suggesting that CCR5 may account for the remaining migration of the F4/80+ cells in CCR2KO mice. In summary, these results indicate that CCR2+ cells play a primary role in F4/80+ cells migration along healing in intramembranous bones, but its deficiency does not critically impact healing outcome.

Saving Natural Teeth: Intentional Replantation-Protocol and Case Series.

INTRODUCTION: Intentional replantation is a reliable and predictable treatment for cases in which nonsurgical endodontic retreatment failed or is impractical and endodontic surgery is hampered because of anatomic limitations. METHODS AND RESULTS: This article presents a protocol for intentional replantation illustrated with some interesting cases. The cases presented here are from patients (average age, 61 years) with no contributing medical history. The cases are molars with previous failed endodontic treatment/retreatment and diagnosed with apical periodontitis. Treatment procedures included atraumatic extractions with minimal manipulations of the periodontal ligament, followed by root-end resection, root-end preparation with ultrasonic tips, root-end fill with bioceramic cement, and rapid tooth replacement into the socket. Granulomatous tissue was gently curetted when applicable. All procedures were performed under the microscope. CONCLUSIONS: Intentional replantation with careful case selection may be considered as a last option for preserving hopeless teeth. Atraumatic extraction by using state-of-the-art equipment, instruments, and materials, minimal extra-alveolar time, and maintaining an aseptic technique are key factors for success.

Proteomic Profiling and Differential Messenger RNA Expression Correlate HSP27 and Serpin Family B Member 1 to Apical Periodontitis Outcomes.

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.

Characterization of a Vascular Endothelial Growth Factor-loaded Bioresorbable Delivery System for Pulp Regeneration.

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.

MMP1-1607 polymorphism increases the risk for periapical lesion development through the upregulation MMP-1 expression in association with pro-inflammatory milieu elements.

OBJECTIVE: In this study, we evaluated the association between the MMP1-1607 polymorphism (rs1799750) and pro-inflammatory milieu elements with MMP-1 mRNA levels in vivo. MATERIAL AND METHODS: MMP1-1607 SNP and the mRNA levels of MMP-1, TNF-a, IFN-g, IL-17A, IL-21, IL-10, IL-4, IL-9, and FOXp3 were determined via RealTimePCR in DNA/RNA samples from patients presenting periapical granulomas (N=111, for both genotyping and expression analysis) and control subjects (N=214 for genotyping and N=26 for expression analysis). The Shapiro-Wilk, Fisher, Pearson, Chi-square ordinal least squares regression tests were used for data analysis (p<0.05 was considered statistically significant). RESULTS: The MMP1-1607 1G/2G and 1G/2G+2G/2G genotypes were significantly more prevalent in the patients than in controls, comprising a risk factor for periapical lesions development. MMP-1 mRNA levels were higher in periapical lesions than in healthy periodontal ligament samples, as well as higher in active than in inactive lesions. The polymorphic allele 2G carriers presented a significantly higher MMP-1 mRNA expression when compared with the 1G/1G genotype group. The ordered logistic regression demonstrated a significant correlation between the genetic polymorphism and the expression levels of MMP-1. Additionally, the pro- and anti-inflammatory cytokines IL-17A, IFN-g, TNF-a, IL-21, IL-10, IL-9, and IL-4 were significant as complementary explanatory variables of MMP-1 expression. CONCLUSION: The MMP1-1607 SNP was identified as a risk factor for periapical lesions development, possibly due to its association with increased MMP-1 mRNA levels in periapical lesions. The MMP-1 expression is also under the control of the inflammatory milieu elements, being the cytokines TNF-a, IL-21, IL-17A, and IFN-g associated with increased MMP-1 levels in periapical lesions, while IL-10, IL-9, or IL-4 presented an inverse association.

MMP1-1607 polymorphism increases the risk for periapical lesion development through the upregulation MMP-1 expression in association with pro-inflammatory milieu elements

ABSTRACT Increased matrix metalloproteinases (MMPs) activity is a hallmark of periapical granulomas. However, the factors underlying the MMPs expression modulation in healthy and diseased periapical tissues remains to be determined. Objective In this study, we evaluated the association between the MMP1-1607 polymorphism (rs1799750) and pro-inflammatory milieu elements with MMP-1 mRNA levels in vivo. Material and Methods MMP1-1607 SNP and the mRNA levels of MMP-1, TNF-a, IFN-g, IL-17A, IL-21, IL-10, IL-4, IL-9, and FOXp3 were determined via RealTimePCR in DNA/RNA samples from patients presenting periapical granulomas (N=111, for both genotyping and expression analysis) and control subjects (N=214 for genotyping and N=26 for expression analysis). The Shapiro-Wilk, Fisher, Pearson, Chi-square ordinal least squares regression tests were used for data analysis (p<0.05 was considered statistically significant). Results The MMP1-1607 1G/2G and 1G/2G+2G/2G genotypes were significantly more prevalent in the patients than in controls, comprising a risk factor for periapical lesions development. MMP-1 mRNA levels were higher in periapical lesions than in healthy periodontal ligament samples, as well as higher in active than in inactive lesions. The polymorphic allele 2G carriers presented a significantly higher MMP-1 mRNA expression when compared with the 1G/1G genotype group. The ordered logistic regression demonstrated a significant correlation between the genetic polymorphism and the expression levels of MMP-1. Additionally, the pro- and anti-inflammatory cytokines IL-17A, IFN-g, TNF-a, IL-21, IL-10, IL-9, and IL-4 were significant as complementary explanatory variables of MMP-1 expression. Conclusion The MMP1-1607 SNP was identified as a risk factor for periapical lesions development, possibly due to its association with increased MMP-1 mRNA levels in periapical lesions. The MMP-1 expression is also under the control of the inflammatory milieu elements, being the cytokines TNF-a, IL-21, IL-17A, and IFN-g associated with increased MMP-1 levels in periapical lesions, while IL-10, IL-9, or IL-4 presented an inverse association.

DNA Methylation of MMP9 Is Associated with High Levels of MMP-9 Messenger RNA in Periapical Inflammatory Lesions.

INTRODUCTION: Matrix metalloproteinases (MMPs) are the major class of enzymes responsible for degradation of extracellular matrix components and participate in the pathogenesis of periapical inflammatory lesions. MMP expression may be regulated by DNA methylation. The purpose of the present investigation was to analyze the expression of MMP2 and MMP9 in periapical granulomas and radicular cysts and to test the hypothesis that, in these lesions, their transcription may be modulated by DNA methylation. METHODS: Methylation-specific polymerase chain reaction was used to evaluate the DNA methylation pattern of the MMP2 gene in 13 fresh periapical granuloma samples and 10 fresh radicular cyst samples. Restriction enzyme digestion was used to assess methylation of the MMP9 gene in 12 fresh periapical granuloma samples and 10 fresh radicular cyst samples. MMP2 and MMP9 messenger RNA transcript levels were measured by quantitative real-time polymerase chain reaction. RESULTS: All periapical lesions and healthy mucosa samples showed partial methylation of the MMP2 gene; however, periapical granulomas showed higher MMP2 mRNA expression levels than healthy mucosa (P = .014). A higher unmethylated profile of the MMP9 gene was found in periapical granulomas and radicular cysts compared with healthy mucosa. In addition, higher MMP9 mRNA expression was observed in the periapical lesions compared with healthy tissues. CONCLUSIONS: The present study suggests that the unmethylated status of the MMP9 gene in periapical lesions may explain the observed up-regulation of messenger RNA transcription in these lesions.

TBX21-1993T/C (rs4794067) polymorphism is associated with increased risk of chronic periodontitis and increased T-bet expression in periodontal lesions, but does not significantly impact the IFN-g transcriptional level or the pattern of periodontophatic bacterial infection.

Th1-polarized host response, mediated by IFN-γ, has been associated with increased severity of periodontal disease as well as control of periodontal infection. The functional polymorphism TBX21-1993T/C (rs4794067) increases the transcriptional activity of the TBX21 gene (essential for Th1 polarization) resulting in a predisposition to a Th-1 biased immune response. Thus, we conducted a case-control study, including a population of healthy controls (H, n = 218), chronic periodontitis (CP, n = 197), and chronic gingivitis patients (CG, n = 193), to investigate if genetic variations in TBX21 could impact the development of Th1 responses, and consequently influence the pattern of bacterial infection and periodontitis outcome. We observed that the polymorphic allele T was significantly enriched in the CP patients compared to CG subjects, while the H controls demonstrated and intermediate genotype. Also, investigating the putative functionality TBX21-1993T/C in the modulation of local response, we observed that the transcripts levels of T-bet, but not of IFN-γ, were upregulated in homozygote and heterozygote polymorphic subjects. In addition, TBX21-1993T/C did not influence the pattern of bacterial infection or the clinical parameters of disease severity, being the presence/absence of red complex bacteria the main factor associated with the disease status and the subrogate variable probing depth (PD) in the logistic regression analysis.

Analysis of multiple cytokine polymorphisms in individuals with untreated deep carious lesions reveals IL1B (rs1143643) as a susceptibility factor for periapical lesion development.

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.

Mesenchymal stem cells as active prohealing and immunosuppressive agents in periapical environment: evidence from human and experimental periapical lesions.

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-17ß), 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.

Simultaneous analysis of T helper subsets (Th1, Th2, Th9, Th17, Th22, Tfh, Tr1 and Tregs) markers expression in periapical lesions reveals multiple cytokine clusters accountable for lesions activity and inactivity status.

UNLABELLED: Previous studies demonstrate that the balance between pro- and anti-inflammatory mediators determines the stable or progressive nature of periapical granulomas by modulating the balance of the osteoclastogenic factor RANKL and its antagonist OPG. However, the cytokine networks operating in the development of periapical lesions are quite more complex than what the simple pro- versus anti-inflammatory mediators' paradigm suggests. Here we simultaneously investigated the patterns of Th1, Th2, Th9, Th17, Th22, Thf, Tr1 and Tregs cytokines/markers expression in human periapical granulomas. METHODS: The expression of TNF-α, IFN-γ, IL-17A, IL23, IL21, IL-33, IL-10, IL-4, IL-9, IL-22, FOXp3 markers (via RealTimePCR array) was accessed in active/progressive (N=40) versus inactive/stable (N=70) periapical granulomas (as determined by RANKL/OPG expression ratio), and also to compare these samples with a panel of control specimens (N=26). A cluster analysis of 13 cytokine levels was performed to examine possible clustering between the cytokines in a total of 110 granulomas. RESULTS: The expression of all target cytokines was higher in the granulomas than in control samples. TNF-α, IFN-γ, IL-17A and IL-21 mRNA levels were significantly higher in active granulomas, while in inactive lesions the expression levels of IL-4, IL-9, IL-10, IL-22 and FOXp3 were higher than in active granulomas. Five clusters were identified in inactive lesion groups, being the variance in the expression levels of IL-17, IL-10, FOXp3, IFN-γ, IL-9, IL-33 and IL-4 statistically significant (KW p<0.05). Three clusters were identified in active lesions, being the variance in the expression levels of IL-22, IL-10, IFN-γ, IL-17, IL-33, FOXp3, IL-21 and RANKL statistically significant (KW p<0.05). CONCLUSION: There is a clear dichotomy in the profile of cytokine expression in inactive and active periapical lesions. While the widespread cytokine expression seems to be a feature of chronic lesions, hierarchical cluster analysis demonstrates the association of TNF-α, IL-21, IL-17 and IFN-γ with lesions activity, and the association of FOXP3, IL-10, IL-9, IL-4 and IL-22 with lesions inactivity.

Simultaneous analysis of T helper subsets (Th1, Th2, Th9, Th17, Th22, Tfh, Tr1 and Tregs) markers expression in periapical lesions reveals multiple cytokine clusters accountable for lesions activity and inactivity status

Previous studies demonstrate that the balance between pro- and anti-inflammatory mediators determines the stable or progressive nature of periapical granulomas by modulating the balance of the osteoclastogenic factor RANKL and its antagonist OPG. However, the cytokine networks operating in the development of periapical lesions are quite more complex than what the simple pro- versus anti-inflammatory mediators' paradigm suggests. Here we simultaneously investigated the patterns of Th1, Th2, Th9, Th17, Th22, Thf, Tr1 and Tregs cytokines/markers expression in human periapical granulomas. Methods: The expression of TNF-α, IFN-γ, IL-17A, IL23, IL21, IL-33, IL-10, IL-4, IL-9, IL-22, FOXp3 markers (via RealTimePCR array) was accessed in active/progressive (N=40) versus inactive/stable (N=70) periapical granulomas (as determined by RANKL/OPG expression ratio), and also to compare these samples with a panel of control specimens (N=26). A cluster analysis of 13 cytokine levels was performed to examine possible clustering between the cytokines in a total of 110 granulomas. Results: The expression of all target cytokines was higher in the granulomas than in control samples. TNF-α, IFN-γ, IL-17A and IL-21 mRNA levels were significantly higher in active granulomas, while in inactive lesions the expression levels of IL-4, IL-9, IL-10, IL-22 and FOXp3 were higher than in active granulomas. Five clusters were identified in inactive lesion groups, being the variance in the expression levels of IL-17, IL-10, FOXp3, IFN-γ, IL-9, IL-33 and IL-4 statistically significant (KW p<0.05). Three clusters were identified in active lesions, being the variance in the expression levels of IL-22, IL-10, IFN-γ, IL-17, IL-33, FOXp3, IL-21 and RANKL statistically significant (KW p<0.05). Conclusion: There is a clear dichotomy in the profile of cytokine expression in inactive and active periapical lesions. While the widespread ...