Intracanal Metformin Promotes Healing of Apical Periodontitis via Suppressing Inducible Nitric Oxide Synthase Expression and Monocyte Recruitment.

INTRODUCTION: We have previously shown that intracanal metformin ameliorates apical periodontitis, partially by modulation of osteoblast apoptosis. The action of metformin on other cell types pertinent to the development of apical periodontitis needs to be examined. In the present study, we aimed to analyze whether its effects on the expression of inducible nitric oxide synthase (iNOS) and monocyte recruitment contribute to the therapeutic effect on apical periodontitis. METHODS: Lipopolysaccharide (LPS)-induced expression of iNOS in a human monocytic cell line, Mono-Mac-6, was assessed by Western blot. The amount of nitrite in culture medium was assessed to quantify nitric oxide (NO) production. C-C motif chemokine ligand-2 (CCL-2) synthesis was measured by enzyme-linked immunosorbent assay. Experimental apical periodontitis in rats was treated with root canal debridement with or without intracanal metformin medication. Lesion progression was assessed by conventional radiography and micro-computed tomographic imaging. Cellular expression of iNOS and the number of monocytes/macrophages were assessed by immunohistochemistry. RESULTS: Metformin suppressed LPS-induced iNOS and NO production by monocytes. More importantly, metformin inhibited LPS-enhanced CCL-2 synthesis through modulation of the iNOS/NO pathway. Intracanal metformin reduced bone resorption associated with apical periodontitis and suppressed iNOS expression and monocyte recruitment. CONCLUSIONS: Our results confirmed the therapeutic efficacy of intracanal metformin for apical periodontitis. Suppression of monocyte recruitment through modulation of iNOS expression and NO production is an important mechanism underlying the beneficial effect of metformin.

The potential effect of Bruton's tyrosine kinase in refractory periapical periodontitis.

To determine the expression of Bruton's tyrosine kinase (BTK) in refractory periapical periodontitis and analyze the relationship between BTK and bone resorption in refractory periapical periodontitis. The mechanism of bone resorption is also discussed. The OneArray Plus expression microarray was used to screen for genes related to refractory periapical periodontitis. Real-time PCR was used to detect the expression of BTK in refractory periapical periodontitis tissues. A model of periapical periodontitis was established by sealing E.faecalis into the pulp of rats. To establish a model of E.faecalis LTA infection of osteoclasts, the relationship between BTK and bone destruction during refractory periapical periodontitis was analyzed. OneArray Plus expression microarray results showed that we found that the expression of 1787 genes in the two samples was different. After validating these samples, we found that BTK was closely related to refractory periapical periodontitis. The results showed that the expression of BTK in refractory periapical periodontitis tissues was higher than that in normal tissues. Immunohistochemistry, enzyme histochemistry and real-time PCR showed that the BTK expression curve in the experimental model resembled a reverse V shape from week 1 to week 4. Osteoclasts were cultured in vitro and treated with E. faecalis LTA. The expression of BTK in the E. faecalis model was greater than that in the control group. BTK played an important role in the progression of refractory periapical periodontitis.

MMP2 and MMP9 are Associated with Apical Periodontitis Progression and Might be Modulated by TLR2 and MyD88

Abstract The aim of this study was to evaluate the expression of MMP2 and MMP9 during apical periodontitis (AP) progression in TLR2 (TLR2 KO) and in MyD88 (MyD88 KO) knockout mice compared to wild type (WT) mice. AP was induced in mandibular first molars of TLR2 KO (n= 18), MyD88 KO (n= 18), and WT mice (n= 18). After 7, 21, and 42 days, the animals were euthanized and the jaws were dissected and subjected to histotechnical processing. Subsequent sections were stained by immunohistochemistry and evaluated for detection of MMP2 and MMP9. Statistical analysis of the semi-quantitative analysis of immunohistochemistry was performed using chi-square test (α = 0.05). In the initial periods of AP progression, an increased expression of MMP9 in the TLR2 KO and MyD88 KO mice was observed. In the final periods of AP progression, a reduction of MMP2 expression and an increase of MMP9 expression in the TLR2 KO mice were observed. MMP2 and MMP9 production was modulated for TLR2 and MyD88 during apical periodontitis progression.

Resumo O objetivo deste estudo foi avaliar a expressão de MMP2 e MMP9 durante a progressão da periodontite apical (AP) em camundongos knockout para TLR2 (TLR2 KO) e MyD88 (MyD88 KO) comparados aos camundongos wild type (WT). A AP foi induzida nos primeiros molares inferiores dos camundongos TLR2 KO (n = 18), MyD88 KO (n = 18) e WT (n = 18). Após 7, 21 e 42 dias, os animais foram eutanaziados e as mandíbulas foram dissecadas e submetidas a processamento histotécnico. As lâminas foram coradas por imuno-histoquímica e analisadas para a detecção de MMP2 e MMP9. A análise estatística semi-quantitativa da imuno-histoquímica foi realizada pelo teste qui-quadrado (α = 0,05). Nos períodos iniciais de progressão AP, foi observada uma expressão aumentada de MMP9 nos camundongos TLR2 KO e MyD88 KO. Nos períodos finais de progressão AP, observou-se uma redução da expressão de MMP2 e um aumento da expressão de MMP9 nos camundongos TLR2 KO. A produção de MMP2 e MMP9 foi modulada por TLR2 e MyD88 durante a progressão da periodontite apical.

MMP2 and MMP9 are Associated with Apical Periodontitis Progression and Might be Modulated by TLR2 and MyD88.

The aim of this study was to evaluate the expression of MMP2 and MMP9 during apical periodontitis (AP) progression in TLR2 (TLR2 KO) and in MyD88 (MyD88 KO) knockout mice compared to wild type (WT) mice. AP was induced in mandibular first molars of TLR2 KO (n= 18), MyD88 KO (n= 18), and WT mice (n= 18). After 7, 21, and 42 days, the animals were euthanized and the jaws were dissected and subjected to histotechnical processing. Subsequent sections were stained by immunohistochemistry and evaluated for detection of MMP2 and MMP9. Statistical analysis of the semi-quantitative analysis of immunohistochemistry was performed using chi-square test (α = 0.05). In the initial periods of AP progression, an increased expression of MMP9 in the TLR2 KO and MyD88 KO mice was observed. In the final periods of AP progression, a reduction of MMP2 expression and an increase of MMP9 expression in the TLR2 KO mice were observed. MMP2 and MMP9 production was modulated for TLR2 and MyD88 during apical periodontitis progression.

5-Lipoxygenase Knockout Aggravated Apical Periodontitis in a Murine Model.

5-Lipoxygenase (5-LO) plays a vital role in the host innate immune response, including bacteria-induced inflammation. Apical periodontitis (AP) is due to immune disorders caused by imbalances between bacterial invasion and subsequent host defense response. In this work, we investigated the role of 5-lipoxygenase in AP by using 5- lo knockout mice (5- lo-/- mice). Results showed that 5- lo-/- mice had greater periapical bone loss and more osteoclasts positive for tartrate-resistant acid phosphatase staining than did wild-type mice, as determined by micro-computed tomography and histologic staining. The inflammation- and osteoclastogenesis-related factors IL-1ß, TNF-α, RANK, and RANKL were also significantly elevated in 5- lo-/- mice, whereas osteoprotegerin was reduced. Furthermore, peritoneal macrophages from 5- lo-/- mice revealed an obviously impaired ability to phagocytose the AP pathogenic bacteria Fusobacterium nucleatum. In vivo experiments confirmed that 5- lo knockout led to decreased macrophage recruitment and increased F. nucleatum infection around the periapical area due to decreased leukotriene B4 and LXA4 production. All these results showed that 5- lo knockout impaired the host innate immune system to promote the release of bone resorption-related factors. Therefore, 5- lo deficiency aggravated AP in an experimental murine AP model.

Decreased levels of matrix metalloproteinase-2 in root-canal exudates during root canal treatment.

OBJECTIVE: To determine the matrix metalloproteinase-2 (MMP-2) levels in root-canal exudates from teeth undergoing root-canal treatment. MATERIAL AND METHODS: The root-canal exudates from six teeth with normal pulp and periradicular tissues that required intentional root canal treatment for prosthodontic reasons and from twelve teeth with pulp necrosis and asymptomatic apical periodontitis (AAP) were sampled with paper points for bacterial culture and aspirated for the detection of proMMP-2 and active MMP-2 by gelatin zymography and the quantification of MMP-2 levels by ELISA. RESULTS: By gelatin zymography, both proMMP-2 and active MMP-2 were detected in the first collection of root-canal exudates from teeth with pulp necrosis and AAP, but not from teeth with normal pulp, and their levels gradually decreased and disappeared at the last collection. Consistently, ELISA demonstrated a significant decrease in MMP-2 levels in the root-canal exudates of teeth with pulp necrosis and AAP following root canal procedures (p<0.05). Furthermore, the MMP-2 levels were significantly lower in the negative bacterial culture than those in the positive bacterial culture (p<0.001). CONCLUSIONS: The levels of MMP-2 in root-canal exudates from teeth with pulp necrosis and AAP were gradually reduced during root canal procedures. Future studies are required to determine if MMP-2 levels may be used as a biomolecule for the healing of apical lesions, similar to the clinical application of MMP-8 as a biomarker.

Dual Role of 5-Lipoxygenase in Osteoclastogenesis in Bacterial-induced Apical Periodontitis.

INTRODUCTION: The aim of this study was to evaluate the role of 5-lipoxigenase (5-LO) in the signaling for osteoclast formation and bone resorption in apical periodontitis (AP) after root canal contamination with oral bacteria. METHODS: AP was experimentally induced in C57BL/6 mice because of contamination of the root canals left open to the oral environment. MK886 was used as a systemic inhibitor of 5-LO (5 mg/kg, daily). After 7, 14, 21, and 28 days, the animals were euthanized, and tissues were removed for gene evaluation by quantitative reverse transcriptase polymerase chain reaction, histologic analysis, and tartrate-resistant acid phosphatase staining. RESULTS: Root canal contamination induced the expression of messenger RNA for 5-LO and leukotriene B4 receptors BLT1 and BLT2. The administration of the 5-LO inhibitor reduced early receptor activator of nuclear factor kappa-B and receptor activator of nuclear factor kappa-B ligand synthesis but augmented late receptor activator of nuclear factor kappa-B ligand and osteoprotegerin expression during the course of AP development. Interestingly, long-term inhibition of 5-LO resulted in increased bone resorption and induced tartrate-resistant acid phosphatase-positive osteoclast formation. The divergent findings related to 5-LO inhibition in osteoclastogenesis signaling, osteoclast formation, and bone resorption were accompanied by differently regulated inflammatory gene expression. Il1b, Il11, Ccl3, Ccl7, and Spp were down-regulated by the 5-LO inhibitor in early AP, but later on Il11, Ccl3, Cxcl9, Cxcl15, and Spp were up-regulated. CONCLUSIONS: 5-LO presented a dual role in osteoclastogenesis during the course of AP development. Early on, osteoclastogenesis signaling was down-regulated by the inhibition of 5-LO, but long-term inhibition failed to prevent synthesis of catabolic mediators that resulted in increased bone loss.

Gene-expression analysis of matrix metalloproteinases 1 and 2 and their tissue inhibitors in chronic periapical inflammatory lesions.

BACKGROUND: Periapical inflammatory lesions have been investigated previously, but understanding of pathogenesis of these lesions (granulomas and radicular cysts) at the molecular level is still questionable. Matrix metalloproteinases (MMPs) are enzymes involved in the development of periapical pathology, specifically inflammation and tissue destruction. To elucidate pathogenesis of periapical granulomas and radicular cysts, we undertook a detailed analysis of gene expression of MMP-1, MMP-2 and their tissue inhibitors, TIMP-1 and TIMP-2. METHODS: A total of 149 samples were analyzed using real-time PCR (59 radicular cysts, 50 periapical granulomas and 40 healthy gingiva samples as controls) for expression of MMP-1, MMP-2, TIMP-1 and TIMP-2 genes. The determination of best reference gene for expression analysis of periapical lesions was done using a panel of 12 genes. RESULTS: We have shown that ß-actin and GAPDH are not the most stable reference controls for gene expression analysis of inflammatory periapical tissues and healthy gingiva. The most suitable reference gene was determined to be SDHA (a succinate dehydrogenase complex, subunit A, flavoprotein [Fp]). We found that granulomas (n = 50) and radicular cysts (n = 59) exhibited significantly higher expression of all four examined genes, MMP-1, MMP-2, TIMP-1, and TIMP-2, when compared to healthy gingiva (n = 40; P < 0.05). CONCLUSION: This study has confirmed that the expression of MMP-1, MMP-2, TIMP-1, and TIMP-2 genes is important for the pathogenesis of periapical inflammatory lesions. Since the abovementioned markers were not differentially expressed in periapical granulomas and radicular cysts, the challenge of finding the genetic differences between the two lesions still remains.

Matrix Matalloproteinase-8 Gene Polymorphism in Chronic Periapical Lesions.

BACKGROUND/AIM: Increased levels of matrix metalloproteinase-8 (MMP-8) have been associated with risk of developing chronic periapical lesion (CPL). Gene polymorphisms are contributing factors in the pathogenesis of the inflammation. The aim of this study was to analyze polymorphism in the gene MMP-8 and its association and influence on clinical manifestation of CPL. METHODS: A total of 240 unrelated Macedonian subjects were included in the present study. Polymorphism -799 C/T in the gene MMP-8 detected with restriction enzyme BgIII was studied in 120 patients with CPL and 120 controls without any signs of chronic or acute inflammatory process in the jaw. The amplification of the region of selected gene was made with polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP). RESULTS: Our results showed that there were differences in the allele and genotype frequencies of the MMP8 -799 C/T polymorphisms between patients with CPL and controls (p < 0.05). Also this study suggests that MMP-8 polymorphism -799 C/T was a risk for expression of CPL (OR=8.27<3.44

Actividad de metaloproteinasa de matriz extracelular-9 en fluido crevicular gingival durante la respuesta reparativa en periodontitis apical asintomática / Activity of matrix metalloproteinase-9 in gingival crevicular fluid during the reparative phase in asymptomatic apical periodontitis

OBJETIVO: Los procesos destructivos del periodonto apical están en su mayoría mediados por proteasas específicas. Existe evidencia de que los niveles de metaloproteinasa de matriz extracelular-9 (MMP-9) en el fluido crevicular gingival (FCG) podrían reflejar la presencia de periodontitis apical asintomática (PAA). El objetivo de este estudio fue evaluar la actividad de MMP-9 durante la respuesta reparativa periapical en controles postendodóncicos entre una semana y 6 meses y en controles sanos. MATERIALES Y MÉTODOS: En este estudio prospectivo se incluyeron 28 pacientes con periodontitis apical asintomática, y se tomaron muestras de FCG a partir de dientes con PAA en línea base y controles postendodóncicos de una semana, uno, 3 y 6 meses. Adicionalmente se incluyeron controles contralaterales sanos. Las muestras se eluyeron y analizaron mediante gelatinogramas y densitometría. RESULTADOS: En pacientes con periodontitis apical asintomática se identificaron tanto la proforma como la forma activa de la MMP-9. Estas presentaron aumentos significativos en controles postendodóncicos de 3 y 6 meses con signos de reparación periapical. CONCLUSIONES: Los niveles de MMP-9 aumentaron significativamente en dientes con diagnóstico de PAA durante la fase reparativa. Estos resultados sugieren que MMP-9 también cumpliría un papel fisiológico durante la reparación periapical, que es susceptible de ser evaluado a través del análisis del FCG.

OBJECTIVE: Apical destructive periodontal processes are largely mediated by specific proteases. Evidence supports that the levels of extracellular matrix metalloproteinase-9 (MMP-9) could reflect the presence of asymptomatic apical periodontitis (AAP) in gingival crevicular fluid (GCF). The aim of this study was to evaluate the activity of MMP-9 during reparative response in periapical post endodontic controls between 1 week and 6 months later and in healthy controls. MATERIALS AND METHODS: A prospective study was performed on 28 patients with a diagnosis of AAP. GCF samples were taken from AAP teeth at baseline and post-endodontic controls at 1 week, 1, 3 and 6 months. Additional healthy contralateral controls were obtained, and samples were eluted and analyzed by densitometric scanning and gelatin zymography. RESULTS: In patients with asymptomatic apical periodontitis, both the pro form and the active form of MMP-9 were identified. These showed significant increases in post-endodontic controls at three and six months, with signs of periapical repairing. CONCLUSIONS: MMP-9 levels significantly increased in teeth diagnosed with AAP during the reparative phase. These results suggest that MMP-9 might be involved in the healing of apical tissues that might be reflected in GCF.

MMP9 deficiency increased the size of experimentally induced apical periodontitis.

INTRODUCTION: 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]). METHODS: 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-1ß), tumor necrosis factor alpha (TNF-α), MMP-2, and MMP-8. RESULTS: 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-1ß, TNF-α, MMP-2, and MMP-8 were found up-regulated in MMP-9 KO mice (P < .05). CONCLUSIONS: 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.

MMP-7 and TIMP-1, new targets in predicting poor wound healing in apical periodontitis.

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.

Expression levels of matrix metalloproteinase-9 and gram-negative bacteria in symptomatic and asymptomatic periapical lesions.

INTRODUCTION: The aim of this study was to test the hypothesis that the expression of matrix metalloproteinase (MMP)-9 is significantly elevated in patients with symptomatic apical periodontitis and to correlate this with the detected amount of gram-negative bacteria. METHODS: Twenty-six patients with periapical lesions involving at least 2 teeth were included in this study. The patients were divided into 2 groups: the symptomatic (SYM) group included 13 patients expressing pain with periapical lesions, and the asymptomatic (ASYM) group included 13 patients expressing no pain. Root canal treatment was performed followed by endodontic surgery and periapical lesion collection. Periapical lesions were serially cut into 4-µ sections. Some sections were processed for histologic examination using hematoxylin-eosin stain. Other sections were processed for immunohistochemical examination. For MMP-9, the area fraction of the positive cells was measured, and the percentage of the MMP-9-immunopositive area to the total area of the microscopic field was calculated. For gram-negative stain cells, the number of cells showing the pink-red color was counted per microscopic field. The Student's t test was used to compare the SYM and ASYM groups. The Pearson correlation coefficient was used to determine a significant correlation between the number of cells and the MMP-9 level. The significance level was set at P ≤ .05. RESULTS: The SYM group showed a statistically significantly higher mean number of gram-negative cells (P = .001) and MMP-9 area percent (P < .001) than the ASYM group. There was a statistically significant positive (r = .927) correlation between the number of gram-negative cells and the MMP-9 area percent (P< .001). CONCLUSIONS: There is good evidence to suspect a significant role of gram-negative bacteria and MMP-9 in symptomatic periapical lesions.

Sexual dimorphism in periapical inflammation and bone loss from mitogen-activated protein kinase phosphatase-1 deficient mice.

INTRODUCTION: Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) has been shown to be a key negative regulator of the MAPK pathways of the innate immune system. The impact of MKP-1 in an endodontic model has yet to be studied. Thus, the purpose of this study was to determine the role of MKP-1 in a bacterial-driven model of pathologic endodontic bone loss. METHODS: Pulps were exposed in both lower first molars of 10-week-old mkp-1(+/+) and mkp-1(-/-) mice and left open to the oral environment for either 3 or 8 weeks. At death, mandibles were harvested and scanned by micro-computed tomography (µCT) to determine periapical bone loss. Histopathologic scoring was then performed on the samples to determine the amount of inflammatory infiltrate within the periapical microenvironment. RESULTS: Significant bone loss and inflammatory infiltrate were found in all experimental groups when compared with control. No statistical difference was found between mkp-1(+/+) and mkp-1(-/-) at either time point with respect to bone loss or inflammatory infiltrate. At 8 weeks, male mkp-1(-/-) mice were found to have significantly more bone loss and inflammatory infiltrate when compared with female mkp-1(-/-) mice. There was also a significant correlation between an increase in bone loss and increase in inflammatory infiltrate. CONCLUSIONS: A sexual dimorphism exists in the periapical inflammatory process, where male mkp-1(-/-) mice have more inflammation than female mkp-1(-/-) mice. The increase in inflammatory infiltrate correlates to more bone loss in the male mice.

Genetic susceptibility to periapical disease: conditional contribution of MMP2 and MMP3 genes to the development of periapical lesions and healing response.

INTRODUCTION: It 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. METHODS: Sixteen 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. RESULTS: An 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. CONCLUSIONS: Variations 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.

Use of aminoguanidine, a selective inducible nitric oxide synthase inhibitor, to evaluate the role of nitric oxide in periapical inflammation.

The purpose of this study was to evaluate the effects of aminoguanidine (AG) as a selective inhibitor of inducible nitric oxide synthase (iNOS) on the degree of inflammatory response in periapical lesions in the canine teeth of cats. Root canals from 52 cat canine teeth were exposed to the oral cavity and sealed after 7 days. One day before pulp exposure, cats were administered either AG (experimental group) or normal saline (control group), which was continued on a daily basis until the day of sacrifice. Animals were sacrificed at 28 days after pulp exposure. Inflammatory response in the periapical zones was analyzed histologically. The degree of periapical inflammation in the AG group was significantly lower than that in the control group (P < 0.05). Selective iNOS inhibitors such as AG thus reduce the intensity of inflammatory responses in periapical lesions.

Inmunodetección de metaloproteinasas de matriz extracelular (MMPs)-2, -9, -13 y -14 en lesiones apicales asociadas con periodontitis apical asintomática / Immunodetection of matrix metalloproteinases (MMPs)-2, -9, -13 and -14 in periapical lesions associated with asymptomatic apical periodontitis

La periodontitis apical asintomática (PAa) es una patología infecciosa caracterizada por destrucción ósea perirradicular asociada a un proceso inflamatorio crónico y producción de mediadores inflamatorios, entre los cuales se encuentran las metaloproteinasas de matriz extracelular (MMPs). Entre éstas, las MMPs-13, -14, -2 y -9, son producidas por el tejido óseo y degradan sinérgicamente el colágeno tipo I, principal componente de los tejidos periodontales, y gelatina, producto de la degradación y desnaturación del colágeno. El objetivo de este estudio fue determinar el patrón de expresión de las MMPs-2, -9, -13 y -14 en granulomas periapicales (GPAs), quistes radiculares inflamatorios (QRIs) y ligamento periodontal sano (LS). Materiales y Métodos: Se seleccionaron 12 pacientes con diagnóstico clínico de PAa e indicación de exodoncia a partir de los cuales se obtuvieron biopsias de lesiones periapicales (LPAs). Como controles, se seleccionaron 7 individuos con indicación de exodoncia de premolares por ortodoncia, obteniéndose biopsias de LS. Se efectuó el diagnóstico anátomo-patológico de los especímenes y se caracterizó la expresión de las MMPs en estudio mediante inmunohistoquímica. Resultados: Las MMPs en estudio sólo se detectaron en GPAs y QRIs, y se inmunolocalizaron principalmente en el infiltrado inflamatorio de éstos. Adicionalmente, la MMP-2 se identificó en fibroblastos del tejido conectivo. Conclusiones: MMPs-2, -9, -13 y -14 se expresan predominantemente en el infiltrado inflamatorio de las LPAs y no en LS, y por tanto se sugiere la participación de estos mediadores en la patogénesis de la PAa.

Asymptomatic apical periodontitis (aAP) is an infectious disease characterized by perirradicular bone destruction associated with chronic inflammation and release of inflammatory mediators, such as matrix metalloproteinases (MMPs). MMPs-13, -14 and -2, -9 are bone-expressed enzymes that can synergistically degrade collagen I, the main component of periodontal extracellular matrix, and gelatin, the product of degradation and denaturation of collagen. The aim of this study was to characterize the expression pattern of MMPs-2, -9, -13, and -14 in periapical granulomas (PGs), radicular cysts (RCs) and healthy periodontal ligament (PDL). Materials and Methods: Individuals with clinical diagnosis of aAP and indication of extraction were selected (N=12), and biopsies of periapical lesions (PLs) were obtained. For controls, 7 subjects with indication of premolar extraction for orthodontic reasons were selected, and PDL biopsies were obtained. Samples were diagnosed by anatomopathological examination and immunohistochemical staining was carried out to characterize MMPs expression. Results: MMPs-2, -9, -13 and -14 detection was limited to PLs and were localized mainly to inflammatory infiltrate on both, PGs and RCs. Additionally, MMP-2 was immunolocalized to fibroblasts from the connective tissue. Conclusions: Whereas MMPs-2, -9, -13 and -14 were not detected in healthy periodontal ligament, they were highly expressed on inflammatory infiltrate from PGs and RCs, suggesting a role of these mediators in aAP pathogenesis.

Expression of MMP-8 and MMP-13 in the development of periradicular lesions.

AIM: To elucidate the expressions of MMP-8 and MMP-13 in experimentally induced rat periradicular lesions by means of the reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical staining. METHODOLOGY: Thirty rats were used and periradicular lesions in mandibular first molar teeth were established following pulp exposure. The animals were sacrificed at 0 (no exposure control), 1, 2, 3, 4 and 6 weeks after pulp exposure. The right molars were used for RT-PCR analysis of MMP-8 and MMP-13. The left molars were subjected to immunohistochemical staining with both MMPs. The areas of these lesions were measured histometrically, and the numbers of both reactive cells in the periapical portion were counted per unit area. Significant differences were analysed by the Mann-Whitney U-test. RESULTS: MMP-8 gene expression gradually increased from 2 to 4 weeks, but slightly decreased at 6 weeks. MMP-13 gene expression gradually increased from 1 to 3 weeks. At 4 and 6 weeks, the level of expression was as high as that at 3 weeks. Immunohistochemically, MMP-8 was first detected at 2 weeks and gradually increased until 4 weeks. MMP-13 gradually increased from 1 to 4 weeks. Both MMPs decreased at 6 weeks. The area of the periradicular lesions gradually increased from 1 to 4 weeks, showing a large increase in week 2 and 3 in particular, but then decreased in week 6. MMP-13-expressing cells were significantly greater than MMP-8-positive cells at week 1 and 2. CONCLUSIONS: These findings indicate that MMP-8 and MMP-13 were related to the development of periradicular lesions. It is suggested that MMP-13 increased from an early stage during their development and that MMP-8 is involved in the progression of tissue destruction including bone resorption.

The role of iNOS and PHOX in periapical bone resorption.

Nitric oxide (NO) and reactive oxygen species (ROS) are key molecules in resistance to pathogens. Little is known about their role in pathogenesis of periapical lesions. To address this issue, we induced periapical lesions in mice lacking nitric oxide synthase (iNOS(-/-)) or phagocyte oxidase (PHOX(-/-)). iNOS(-/-) mice expressed higher levels of IL-1ß, TNF-α, RANK, RANKL, and MCP-1 than C57BL/6 and PHOX(-/-). Apical thickening of the periodontal ligament was also greater in iNOS(-/-) compared with other groups. Interestingly, ROS production did not interfere in periapical lesion progression, but seemed to be essential for the appearance of multinucleated TRAP-positive cells. Thus, periapical lesion progression in iNOS(-/-) was associated with an imbalance of pro-inflammatory cytokines (IL-1ß and TNF-α), bone-resorptive modulators (RANK and RANKL), and MCP-1. We conclude that NO, but not ROS, controls progression of bone resorption in a murine experimental model of apical periodontitis.

Sonic extracts from a bacterium related to periapical disease activate gelatinase A and inactivate tissue inhibitor of metalloproteinases TIMP-1 and TIMP-2.

AIM: To examine the effects of sonicated bacterial extracts (SBEs) from three related to periapical disease bacteria (Porphyromonas gingivalis, P. endodontalis and F. nucleatum) on the activation of matrix metalloproteinase (MMP-2) and the inactivation of tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2). METHODOLOGY: Each SBE was added to cultures of human periodontal ligament (PL) cells or HT1080 cells and their supernatants were analysed by zymography for MMP-2. Each SBE was added to PL cell cultures, and the amount of TIMP-1 was determined by ELISA. P. gingivalis SBE was incubated with HT1080 cell culture supernatants, and the amounts of TIMP-1 and TIMP-2 were determined by ELISA. Statistical analysis was performed with the paired Student's t-test. RESULTS: In extracts of PL cells that had been incubated in the presence of P. gingivalis SBE, one representing pro-MMP-2 (72 kDa) and a band corresponding to the active MMP-2 (66 kDa) were observed; but in the other extracts it was not detected. When HT1080 cells were treated with P. gingivalis SBE, the pro-MMPs was processed into 86- and 66-kDa fragments, but in the other extracts, the processing did not occur when the other SBEs were used. When PL cells were incubated with the same SBEs, the amount of TIMP-1 was markedly decreased (P < 0.01), but in the other extracts, it was not. The amounts of both TIMP-1 and TIMP-2 were decreased in a dose-dependent manner when HT1080 cell culture supernatant was incubated with P. gingivalis SBE. CONCLUSIONS: These findings suggest that P. gingivalis SBE may cause connective tissue to be destroyed, contributing to the process of periapical disease, by activating pro-MMP-2 as well as by inactivating TIMP-1 and TIMP-2.