Periapical Tissue Destruction Research Articles

Platr3/NUDT21/NF-κB Axis Mediates P. gingivalis-Suppressed Cementoblast Mineralization.

Porphyromonas gingivalis (P. gingivalis) is one of the major pathogens causing periodontitis and apical periodontitis (AP). Long noncoding RNA (lncRNA) can regulate cellular mineralization and inflammatory diseases. The aim of this study was to investigate the role and mechanism of lncRNA in P. gingivalis-stimulated cementoblast mineralization. In vivo, C57BL/6 mice were divided into the healthy, the AP, and AP + P. gingivalis groups (n = six mice per group). Micro computed tomography, immunohistochemistry staining, and fluorescence in situ hybridization were used to observe periapical tissue. In vitro, cementoblasts were treated with osteogenic medium or P. gingivalis. Pluripotency associated transcript 3 (Platr3), interleukin 1 beta (IL1B), and osteogenic markers were analyzed by quantitative real-time polymerase chain reaction and western blot. RNA pull-down and RNA immunoprecipitation assays were used to detect proteins that bind to Platr3. RNA sequencing was performed in Platr3-silenced cementoblasts. In vivo, P. gingivalis promoted periapical tissue destruction and IL1B expression, but inhibited Platr3 expression. In vitro, P. gingivalis facilitated IL1B expression (P < 0.001), whereas suppressed the expression of Platr3 (P < 0.001) and osteogenic markers (P < 0.01 or 0.001). In contrast, Platr3 overexpression alleviated the repressive effect of P. gingivalis on cementoblast mineralization (P < 0.01 or 0.001). Furthermore, Platr3 bound to nudix hydrolase 21 (NUDT21) and regulated the nuclear factor-κB (NF-κB) signaling pathway. Knocking down NUDT21 suppressed osteogenic marker expression and activated the above signaling pathway. Collectively, the results elucidated that Platr3 mediated P. gingivalis-suppressed cementoblast mineralization through the NF-κB signaling pathway by binding to NUDT21.

Effects of melatonin against experimentally induced apical periodontitis in rats.

Apical periodontitis is an inflammatory condition resulting from microbial invasion in the root canal system, causing periapical tissue destruction and bone resorption. This study investigated melatonin's effects, known for its antioxidant and anti-inflammatory properties, on experimentally induced apical periodontitis in rats. Three groups of rats were studied: control, apical periodontitis and apical periodontitis with melatonin treatment. Proinflammatory cytokines and enzyme levels in blood serum were measured, and micro-CT analysis assessed bone resorption. Results showed significantly elevated cytokines and enzyme levels in the apical periodontitis group compared to the control. However, in the melatonin-treated group, these levels were significantly reduced (p < 0.01-0.001). Micro-CT analysis indicated decreased periapical resorption cavity volume and surface area with melatonin treatment. This suggests that systemic melatonin administration can mitigate inflammation and reduce bone resorption in experimentally induced apical periodontitis in rats, potentially holding promise for human endodontic disease treatment pending further research.

ОЦЕНКА РЕЗУЛЬТАТОВ ПОВТОРНОГО ЭНДОДОНТИЧЕСКОГО ЛЕЧЕНИЯ ХРОНИЧЕСКОГО АПИКАЛЬНОГО ПЕРИОДОНТИТА С ИСПОЛЬЗОВАНИЕМ БИОМАТЕРИАЛОВ. КЛИНИЧЕСКОЕ НАБЛЮДЕНИЕ

Relevance. In the practice of a dentist, there are clinical situations when the chronic apical periodontitis, due to the ineffectiveness of the endodontic treatment, ends with a root apex resection, while the destruction of bone tissue in the periapical tissues remains. This paper presents cases of effective endodontic re-treatment of such teeth using biomaterials. The diagnostic value of the X-ray method was noted with an assessment of the density of the periapical tissue destruction focus using the densitometry method.&#x0D; The aim of the study is to determine the effectiveness of endodontic treatment of chronic apical periodontitis using biomaterials after root apex resection.&#x0D; Material and methods. The study included 42 patients aged 25–35 years with chronic apical periodontitis with root apex resection. All patients underwent clinical examination methods and an X-ray method with an assessment of the density of the periapical tissue destruction focus by densitometry. &#x0D; Results and discussion. The proposed method of endodontic re-treatment included temporary obturation of the root canal system with Bioimplant with Collapan gel in a 1:1 ratio, for a period of 15 to 30 days, followed by permanent filling of the root canal system according to the generally accepted technique. After 6 months, the optical density of the periapical tissues in 27 patients was within the normal range. In 15 patients, these indicators were 82% of the norm. After 12 months, the optical density was within the normal range in 90% of cases, in 10% – the optical density parameters approached normal values and amounted to 115.0-125.0 arbitrary units. &#x0D; Conclusions. When carrying out repeated endodontic treatment, it is recommended to temporarily obturate the root canal system using the Bioimplant biomaterial with Collapan gel, which will increase not only the density of periapical tissues to normal values, but also preserve the function of the tooth, which is important for practical health care.

Clinical influence of calcium hydroxide intracanal medications on matrix metalloproteinases and tissue inhibitors of metalloproteinases in apical periodontitis.

This study investigated the influence of calcium hydroxide intracanal medications on the levels of metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in apical periodontitis (AP). Twenty primarily infected root canals with AP were randomly divided into two groups: Ca(OH)2 + sterile saline solution (SSL) group and Ca(OH)2 + 2% chlorhexidine gel (CHX gel) group. We collected samples from the periradicular tissue fluid (PTF) before (s1) and after 14days of intracanal medication (s2). MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 were measured by ELISA assay. MMP-1, MMP-2, MMP-9, TIMP-1, and TIMP-2 were detected in all PTF samples at s1 and s2 (20/20). At s1, MMP-2 and MMP-9 were detected at higher levels than MMP-1 (p < .05). Higher levels of TIMP-1 than TIMP-2 were found in AP (p < .05). Additionally, we detected higher MMP-1, MMP-2, and MMP-9 over TIMP-1 and TIMP-2 levels in AP (p < .05). At s2, Ca(OH)2 + SSL was as effective as Ca(OH)2 + 2% CHX gel in lowering the levels of MMP-1, MMP-2, and MMP-9 after 14days of intracanal medication, with no significant difference between them (p > .05). Both Ca(OH) 2 intracanal medications had no significant impact on the levels of TIMP-1 and TIMP-2 (both p > .05). At s2, TIMP-1 levels were higher than TIMP-2 (p < .05). Moreover, there were positive correlations between the levels of MMP-1 and TIMP-1 and MMP-1 and TIMP-2 (p < .05). Calcium hydroxide medications effectively lowered the levels of MMP-1, MMP-2, and MMP-9 in periapical tissues after 14days of treatment, with no difference between them. Moreover, the calcium hydroxide intracanal medications tested here had no impact in TIMP-1 and TIMP-2 in periapical tissues. MMPs and TIMPs play an essential role in the degradation of the extracellular matrix. The imbalance MMPs and TIMPs can cause periapical tissue destruction. Therefore, the reestablishment of the balance between activated MMPs and TIMPs with root canal therapy is essential to restore tissue homeostasis.

Inhibition of the CXCL9-CXCR3 axis suppresses the progression of experimental apical periodontitis by blocking macrophage migration and activation

Apical periodontitis (AP) is an acute or chronic inflammatory disease caused by complex interactions between infected root canal and host immune system. It results in the induction of inflammatory mediators such as chemokines and cytokines leading to periapical tissue destruction. To understand the molecular pathogenesis of AP, we have investigated inflammatory-related genes that regulate AP development. We found here that macrophage-derived CXCL9, which acts through CXCR3, is recruited by progressed AP. The inhibition of CXCL9 by a CXCR3 antagonist reduced the lesion size in a mouse AP model with decreasing IL-1β, IL-6 and TNFα expression. The treatment of peritoneal macrophages with CXCL9 and LPS induced the transmigration and upregulation of osteoclastogenic cytokines such as IL-1β, IL-6 and matrix metalloprotease 2, a marker of activated macrophages. This suggests that the CXCL9-CXCR3 axis plays a crucial role in the development of AP, mediated by the migration and activation of macrophages for periapical tissue destruction. Our data thus show that CXCL9 regulates the functions of macrophages which contribute to AP pathogenesis, and that blocking CXCL9 suppresses AP progression. Knowledge of the principal factors involved in the progression of AP, and the identification of related inflammatory markers, may help to establish new therapeutic strategies.

Silencing of Ac45 Simultaneously Inhibits Osteoclast-Mediated Bone Resorption and Attenuates Dendritic Cell-Mediated Inflammation through Impairing Acidification and Cathepsin K Secretion.

Endodontic disease is characterized by inflammation and destruction of periapical tissues, leading to severe bone resorption and tooth loss. ATP6AP1 (Ac45) has been implicated in human immune diseases, yet the mechanism underlying how Ac45 regulates immune response and reaction in inflammatory diseases remains unknown. We generated endodontic disease mice through bacterial infection as an inflammatory disease model and used adeno-associated virus (AAV)-mediated Ac45 RNA interference knockdown to study the function of Ac45 in periapical inflammation and bone resorption. We demonstrated that the AAV small hairpin RNA targeting Ac45 (AAV-sh-Ac45) impaired cellular acidification, extracellular acidification, and bone resorption. Our results showed that local delivery of AAV-sh-Ac45 in periapical tissues in bacterium-induced inflammatory lesions largely reduced bone destruction, inhibited inflammation, and dramatically reduced mononuclear immune cells. T-cell, macrophage, and dendritic cell infiltration in the periapical lesion was dramatically reduced, and the periodontal ligament was protected from inflammation-induced destruction. Furthermore, AAV-sh-Ac45 significantly reduced osteoclast formation and the expression of proinflammatory cytokines, such as tumor necrosis factor alpha, interleukin-10 (IL-10), IL-12, IL-1α, IL-6, and IL-17. Interestingly, AAV-sh-Ac45 impaired mature cathepsin K secretion more significantly than that by AAV-sh-C1 and AAV-sh-CtsK Unbiased genome-wide transcriptome sequencing analysis of Ctsk-/- dendritic cells stimulated with lipopolysaccharide demonstrated that the ablation of Ctsk dramatically reduced dendritic cell-mediated inflammatory signaling. Taken together, our results indicated that AAV-sh-Ac45 simultaneously inhibits osteoclast-mediated bone resorption and attenuates dendritic cell-mediated inflammation through impairing acidification and cathepsin K secretion. Thus, Ac45 may be a novel target for therapeutic approaches to attenuate inflammation and bone erosion in endodontic disease and other inflammation-related osteolytic diseases.

Effect of Butyrate on Collagen Expression, Cell Viability, Cell Cycle Progression and Related Proteins Expression of MG-63 Osteoblastic Cells.

AimsButyric acid is one major metabolic product generated by anaerobic Gram-negative bacteria of periodontal and root canal infection. Butyric acid affects the activity of periodontal cells such as osteoblasts. The purposes of this study were to investigate the effects of butyrate on MG-63 osteoblasts.MethodsMG-63 cells were exposed to butyrate and cell viability was estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mRNA and protein expression of type I collagen and cell cycle-related proteins were measured by reverse-transcriptase polymerase chain reaction (RT-PCR), western blotting or immunofluorescent staining. Cellular production of reactive oxygen species (ROS) was analyzed by 2',7'-dichlorofluorescein (DCF) fluorescence flow cytometry.ResultsExposure to butyrate suppressed cell proliferation, and induced G2/M (8 and 16 mM) cell cycle arrest of MG-63 cells. Some cell apoptosis was noted. The mRNA expression of cdc2 and cyclin-B1 decreased after exposure to butyrate. The protein expression of type I collagen, cdc2 and cyclin B1 were decreased, whereas the expression of p21, p27 and p57 was stimulated. Under the treatment of butyrate, ROS production in MG-63 cells markedly increased.ConclusionsThe secretion of butyric acid by periodontal and root canal microorganisms may inhibit bone cell growth and matrix turnover. This is possibly due to induction of cell cycle arrest and ROS generation and inhibition of collagen expression. These results suggest the involvement of butyric acid in the pathogenesis of periodontal and periapical tissue destruction by impairing bone healing responses.

Elevated CD14 (Cluster of Differentiation 14) and Toll-Like Receptor (TLR) 4 Signaling Deteriorate Periapical Inflammation in TLR2 Deficient Mice.

Apical periodontitis (periapical lesions) is an infection-induced chronic inflammation in the jaw, ultimately resulting in the destruction of apical periodontal tissue. Toll-like receptors (TLRs) are prominent in the initial recognition of pathogens. Our previous study showed that TLR4 signaling is proinflammatory in periapical lesions induced by a polymicrobial endodontic infection. In contrast, the functional role of TLR2 in regulation of periapical tissue destruction is still not fully understood. Using TLR2 deficient (KO), TLR2/TLR4 double deficient (dKO), and wild-type (WT) mice, we demonstrate that TLR2 KO mice are highly responsive to polymicrobial infection-induced periapical lesion caused by over activation of TLR4 signal transduction pathway that resulted in elevation of NF-kB (nuclear factor kappa B) and proinflammatory cytokine production. The altered TLR4 signaling is caused by TLR2 deficiency-dependent elevation of CD14 (cluster of differentiation 14), which is a co-receptor of TLR4. Indeed, neutralization of CD14 strikingly suppresses TLR2 deficiency-dependent inflammation and tissue destruction in vitro and in vivo. Our findings suggest that a network of TLR2, TLR4, and CD14 is a key factor in regulation of polymicrobial dentoalveolar infection and subsequent tissue destruction. Anat Rec, 299:1281-1292, 2016. © 2016 Wiley Periodicals, Inc.

Prevalence of Human Cytomegalovirus and Epstein-Barr Virus in Chronic Periapical Lesions

Objective: The aim of this study was to investigate the presence of human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in the tissue of chronic periapical lesions, and to compare the results in relation to the symptoms of patients and the size of the lesion. Methods: Periapical lesions analyzed in the study were collected from the roots of the teeth indicated for extraction. Samples were divided according to the symptoms into groups of symptomatic and asymptomatic, and according the size into groups of small and large lesions. Polymerase chain reaction was used to detect HCMV and EBV. The amplification was performed in a DNA Thermal Cycler (Hybaid). Results: Symptomatic lesions were 7.68 times more likely to be infected with HCMV than asymptomatic lesions (p < 0.001). Large symptomatic lesions were 73.50 times more likely to harbor HCMV than small symptomatic lesions (p < 0.001). Large symptomatic lesions were 7.64 times more likely to be infected with EBV than small symptomatic lesions (p = 0.05). Large symptomatic lesions were 5.38 times more likely to harbor dual HCMV/EBV infection than small symptomatic lesions (p = 0.115). Conclusion: Detection of HCMV and EBV in the samples of periapical lesions suggests an important role of herpesviruses in periapical tissue destruction.

Analysis of Multiple Cytokine Polymorphisms in Individuals with Untreated Deep Carious Lesions Reveals IL1B (rs1143643) as a Susceptibility Factor for Periapical Lesion Development

IntroductionIt 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. MethodsSubjects 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. ResultsA 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). ConclusionsVariations 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.

MMP9 Deficiency Increased the Size of Experimentally Induced Apical Periodontitis

IntroductionApical periodontitis is an inflammation and destruction of periapical tissues. Matrix metalloproteinase-9 (MMP-9) is thought to be involved in periapical lesion formation and progression. The aim of this study was to evaluate the lesion progression in MMP-9 knockout (KO) mice compared with that in control mice (wild type [WT]). MethodsThe pulps of mouse mandibular first molars were exposed; animals were killed at 0, 7, 14, 21, and 28 days after surgery. Hematoxylin-eosin–stained sections were observed for the description of pulpal, apical, periapical features, and the periapical lesion size. The periapical lesion size was further measured with micro–computed tomographic imaging. The number of osteoclasts was also counted by tartrate-resistant acid phosphatase histoenzymology. Real-time polymerase chain reaction and immunohistochemistry were used to analyze the expression levels of receptor activator of NF-κB (RANK), receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG), interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), MMP-2, and MMP-8. ResultsThere was a significant difference (P < .05) between the 2 types of animals regarding the periapical lesion size, which was larger in MMP-9 KO animals. No significant differences (P > .05) were found between WT and MMP-9 KO mice related to the osteoclast number as well as the pulpal, apical, and periapical features. More neutrophil cells were observed in MMP-9 KO animals than WT mice (P < .05). The expression levels of RANK, RANKL, OPG, IL-1β, TNF-α, MMP-2, and MMP-8 were found up-regulated in MMP-9 KO mice (P < .05). ConclusionsMMP-9 KO animals developed larger periapical lesions with greater inflammatory response, indicating an important role of MMP-9 in the host's immune and inflammatory response to root canal and periradicular infection.

Regulation of Matrix Metalloproteinases, Tissue Inhibitor of Matrix Metalloproteinase-1, and Extracellular Metalloproteinase Inducer by Interleukin-17 in Human Periodontal Ligament Fibroblasts

IntroductionUnder physiological conditions, matrix metalloproteinases (MMPs) are involved in the turnover of periapical tissue, and their activity is tightly regulated by tissue inhibitors of metalloproteinases (TIMPs). Disturbances in the balance between MMPs and TIMPs may result in excessive tissue destruction. In addition, the extracellular metalloproteinase inducer (EMMPRIN) capable of inducing MMPs may also play a role in the pathologic processes. This study aimed to investigate the effects of interleukin (IL)-17 on the mRNA expression and protein production of MMP-1, MMP-2, MMP-9, MMP-13, TIMP-1, and EMMPRIN through human periodontal ligament cells. MethodsThe cells were stimulated with IL-17 (1, 10, and 50 ng/mL) for different time periods. The mRNA levels of MMP-1, MMP-2, MMP-9, MMP-13, TIMP-1, and EMMPRIN were evaluated via quantitative real-time polymerase chain reaction analysis, whereas the protein secretion into the culture medium was assessed via enzyme-linked immunosorbent assay and zymography analysis. ResultsIL-17 significantly up-regulated MMP-1 and MMP-13 mRNA expression but down-regulated MMP-2, MMP-9, and TIMP-1 mRNA expression. Furthermore, IL-17 (50 ng/mL) increased the secreted protein level of MMP-1 and MMP-13 and conversely reduced the level of MMP-2, MMP-9, and TIMP-1. However, IL-17 exerted no effect on EMMPRIN mRNA or protein secretion. ConclusionsThis study first reported the ability of IL-17 to regulate MMP and TIMP-1 production through human periodontal ligament cells, a phenomenon that may contribute to periapical tissue destruction.

Genetic Susceptibility to Periapical Disease: Conditional Contribution of MMP2 and MMP3 Genes to the Development of Periapical Lesions and Healing Response

IntroductionIt has been proposed that individual genetic predisposition may contribute to a persistent apical periodontitis condition. Matrix metalloproteinases (MMPs) are associated with levels of inflammation and are involved in caries, pulpal, and periapical tissue destruction. MMPs also play a major role in bone resorption. In this study, we hypothesized that polymorphisms in MMP genes and their regulators may contribute to an individual’s increased susceptibility to apical tissue destruction in response to deep carious lesions. MethodsSixteen hundred radiographic records obtained through the University of Pittsburgh School of Dental Medicine Dental Registry and DNA Repository were screened for subjects with deep carious lesions in dentin with or without periapical lesions (≥3 mm). DNA samples of 268 patients were sorted into 2 groups: 158 cases with deep carious lesions but no periapical lesions (controls) and 110 cases with periapical lesions and deep carious lesions (cases). Sixteen SNP markers in MMP2, MMP3, MMP9, MMP13, MMP14, and TIMP2, were selected for genotyping. Genotypes were generated by endpoint analysis in a real-time polymerase chain reaction instrument. Analyses were performed comparing cases and controls. Allele and genotypic frequencies and haplotype analysis were calculated using the PLINK program. ResultsAn association was found for MMP3 rs639752 (P = .03) and rs679620 (P = .004) genotypes in individuals with periapical lesions. We also observed altered transmission of MMP2 marker haplotypes (P = .000004) in these individuals. ConclusionsVariations in MMP2 and MMP3 are associated with periapical lesion formation in individuals with untreated deep carious lesions. Future studies could help predict host susceptibility to developing periapical lesions.

Expresión de Factor Estimulante de Colonias-1 (CSF-1) y Catepsina-K en Lesiones Periapicales

Periapical Lesions (PLs) represent an immunoinflammatory disease of infectious origin, characterized by periapical tissue destruction. PLs are linked to the expression of various inflammatory mediators: Colony Stimulating Factor-1 (CSF-1) that stimulates differentiation of osteoclast precursor cells and Cathepsin K, an enzyme associated with bone destruction, could be related to osteolysis during PL. The aim of this study was to characterize the expression of these mediators in inflammatory radicular cyst, periapical granuloma and healthy periodontal tissue. Materials and MethodsWe selected 13 individuals with clinical diagnosis of Apical Periodontitis (AP) and indication of extraction. Healthy subjects having indication of extraction for orthodontic reasons were also included. After extraction, specimens were biopsied and histological diagnosis was made. Subsequently, immunohistochemical staining was carried out to identify the mediators in study, and finally the samples were examined in a light microscope. ResultsCSF-1 and Cathepsin K expression was distributed similarly in apical granulomas and radicular cysts. They were mainly immunolocalized to inflammatory infiltrates. Also lining epithelium, cyst capsule, and vascular endothelium were mildly immunopositive. No immunoreactivity was observed in healthy periodontium. ConclusionsCSF-1 and Cathepsin K are expressed in PLs and may participate in the pathogenesis of AP

PII: S1043-4666(09)00567-5

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 α (TNF-α); 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-α, 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-α, SOCS1 and RANKL, and SOCS3 and TNF-α. 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.

The Activation of Matrix Metalloproteinases by a Whole-cell Extract from Prevotella nigrescens

Periradicular lesions are primarily evoked as a response to a bacterial challenge emanating from an infected root canal. Many bacteria such as those of the genera Porphyromonas, Prevotella, and others have been isolated from infected root canals. The cause of periradicular lesions is related to the destruction of the extracellular matrix (ECM). Matrix metalloproteinases (MMPs) such as interstitial collagenase (MMP-1), gelatinase A (MMP-2), gelatinase B (MMP-9), and so on are products of inflammatory cells and, once activated, are intimately involved in the degradation of the ECM. However, there are no reports regarding the destruction of the ECM by bacterial extracts from Prevotella nigrescens ( P. nigrescens). The present study was conducted to evaluate the activating effect of a whole-cell extract (WCE) of P. nigrescens on proMMP-2 and proMMP-9. P. nigrescens WCE was mixed with proMMP-2 or proMMP-9 under many conditions, and the activation of these MMPs was determined by gelatin zymography. A band indicating a lower molecular weight of 66 kd or 84 kd, which migrated faster than the band of proMMP-2 (72 kd) or proMMP-9 (92 kd) respectively, was detected, which could be the active form of either MMP. The present study suggests that P. nigrescens might be able to activate proMMP-2 and proMMP-9 in vivo and that this activation might be related to the destruction of periapical tissues.

The Potential Role of Suppressors of Cytokine Signaling in the Attenuation of Inflammatory Reaction and Alveolar Bone Loss Associated with Apical Periodontitis

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 α (TNF-α); 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-α, 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-α, SOCS1 and RANKL, and SOCS3 and TNF-α. 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.

The Expression of Macrophage and Neutrophil Elastases in Rat Periradicular Lesions

Macrophage elastase and neutrophil elastase are involved in tissue destruction in periradicular lesions. The purpose of this study was to examine these elastases immunohistochemically during development of periradicular lesions induced in rat mandibular first molar by pulpal exposure for 7, 14, 21, 28, and 42 days. Histologically, periapical inflammation developed from 7 to 21 days and then subsided after 28 days. The area of these lesions gradually increased from 7 to 28 days and subsequently decreased at 42 days. Macrophage elastase was first detected at 7 days and predominantly shown from 14 to 28 days, whereas neutrophil elastase gradually increased from 14 to 28 days. Macrophage elastase was significantly greater than neutrophil elastase from 7 to 21 days. These results suggest that macrophage elastase was enhanced from an early stage during the development of these lesions and that neutrophil elastase was related to the expansion of periapical tissue destruction including bone resorption.

The synergistic effect of peptidoglycan and lipopolysaccaride on osteoclast formation.

The purpose of this study was to investigate the mechanisms leading to periapical tissue destruction by the Gram-positive bacterial cell component, peptidoglycan (PGN) and Gram-negative bacterial cell component lipopolysaccaride (LPS). Osteoclast precursor RAW 264.7 cells were cultured with 50 ng/ml receptor activator of NF-kappaB ligand (RANKL) for 72 hours. RANKL was then removed and the cells were treated with various concentrations of PGN in the presence or absence of various concentration of LPS for an additional 48 hours. RT-PCR analysis was performed to examine the presence of receptors on osteoclasts known to be involved in mediating cellular activation in response to PGN (TLR2) and LPS (TLR 4). PGN dose dependently and reproducibly stimulated TRAP-positive multinucleated osteoclast-like (OCL) cell formation. PGN and LPS had synergistic effects on the induction of OCL cell formation. Both unstimulated and RANKL-stimulated RAW 264.7 cells expressed TLR2 and TLR4 mRNA. The results underscore the importance of considering both Gram-positive and Gram-negative bacteria when interpreting findings associated with primary and secondary periapical lesions.

Recovery of lumbar vertbral cancellous bone after the second lactation in rats

Apical periodontitis is an inflammation and destruction of periapical tissues. Matrix metalloproteinase-9 (MMP-9) is thought to be involved in periapical lesion formation and progression. The aim of this study was to evaluate the lesion progression in MMP-9 knockout (KO) mice compared with that in control mice (wild type [WT]).The pulps of mouse mandibular first molars were exposed; animals were killed at 0, 7, 14, 21, and 28 days after surgery. Hematoxylin-eosin–stained sections were observed for the description of pulpal, apical, periapical features, and the periapical lesion size. The periapical lesion size was further measured with micro–computed tomographic imaging. The number of osteoclasts was also counted by tartrate-resistant acid phosphatase histoenzymology. Real-time polymerase chain reaction and immunohistochemistry were used to analyze the expression levels of receptor activator of NF-κB (RANK), receptor activator of NF-κB ligand (RANKL), osteoprotegerin (OPG), interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), MMP-2, and MMP-8.There was a significant difference (P < .05) between the 2 types of animals regarding the periapical lesion size, which was larger in MMP-9 KO animals. No significant differences (P > .05) were found between WT and MMP-9 KO mice related to the osteoclast number as well as the pulpal, apical, and periapical features. More neutrophil cells were observed in MMP-9 KO animals than WT mice (P < .05). The expression levels of RANK, RANKL, OPG, IL-1β, TNF-α, MMP-2, and MMP-8 were found up-regulated in MMP-9 KO mice (P < .05).MMP-9 KO animals developed larger periapical lesions with greater inflammatory response, indicating an important role of MMP-9 in the host's immune and inflammatory response to root canal and periradicular infection.