Role of PI3K in the bone resorption of apical periodontitis.

BACKGROUND: Phosphoinositide 3-kinase (PI3K) is located within cells, and is involved in regulating cell survival, proliferation, apoptosis and angiogenesis. The purpose of this study was to investigate the role of PI3K in the process of bone destruction in apical periodontitis, and provide reference data for the treatment of this disease. METHODS: The relative mRNA expression of PI3K, Acp5 and NFATc1 in the normal human periodontal ligament and in chronic apical periodontitis were analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). A mouse model of apical periodontitis was established by root canal exposure to the oral cavity, and HE staining was used to observe the progress of apical periodontitis. Immunohistochemical staining was used to detect the expression of PI3K and AKT in different stages of apical periodontitis, while enzymatic histochemical staining was used for detection of osteoclasts. An Escherichia coli lipopolysaccharide (LPS)-mediated inflammatory environment was also established at the osteoclast and osteoblast level, and osteoclasts or osteoblasts were treated with the PI3K inhibitor LY294002 to examine the role of PI3K in bone resorption. RESULTS: The expression of PI3K, Acp5 and NFATc1 genes in chronic apical periodontitis sample groups was significantly increased relative to healthy periodontal ligament tissue (P < 0.05). Mouse apical periodontitis was successfully established and bone resorption peaked between 2 and 3 weeks (P < 0.05). The expression of PI3K and Akt increased with the progression of inflammation, and reached a peak at 14 days (P < 0.05). The gene and protein expression of PI3K, TRAP and NFATc1 in osteoclasts were significantly increased (P < 0.05) in the E. coli LPS-mediated inflammatory microenvironment compared to the normal control group. Meanwhile in osteoblasts, the gene and protein expression of PI3K, BMP-2 and Runx2 were significantly reduced (P < 0.05) in the inflammatory microenvironment. With the addition of LY294002, expressions of bone resorption-related factors (TRAP, NFATc1) and bone formation-related factors (BMP-2, Runx2) significantly decreased (P < 0.05). CONCLUSIONS: Under the inflammatory environment induced by LPS, PI3K participates in the occurrence and development of chronic apical periodontitis by regulating the proliferation and differentiation of osteoclasts and osteoblasts.

JAK2-STAT3 signaling pathway is involved in rat periapical lesions induced by Enterococcus faecalis.

OBJECTIVES: This study aimed to investigate the role of JAK2-STAT3 (Janus kinase 2/signal transducer and activator of transcription 3) in periapical disease caused by Enterococcus faecalis, as well as the correlation between lipoteichoic acid (LTA) in E. faecalis and the activity of the JAK2-STAT3 signaling pathway and osteoclast formation. MATERIALS AND METHODS: A rat model of periapical periodontitis induced by E. faecalis was established. Periapical bone resorption was confirmed by HE staining. The expression of JAK2, p-JAK2, STAT3, and p-STAT3 was assessed with immunohistochemical staining. Osteoclasts were observed through enzyme histochemical staining. LTA acted on mouse osteoclast precursor cells (RAW264.7 cells); a JAK2 inhibitor (AG490) was used to inhibit the JAK2-STAT3 pathway in RAW264.7 cells. The expression of proteins in the JAK2-STAT3 pathway and TRAP (tartrate resistant acid phosphatase) in RAW264.7 cells was also detected. RESULTS: Rat periapical periodontitis was successfully established and bone resorption peaked at day 21. The expression of critical components in the JAK2-STAT3 pathway increased with the progression of inflammation. LTA promoted the differentiation of RAW264.7 cells into osteoclasts. NFATc1 was highly expressed and was inhibited by AG490. CONCLUSIONS: JAK2-STAT3 signaling pathway plays an important role in the process of periapical bone resorption and osteoclastogenesis.

Role of the Btk-PLCγ2 Signaling Pathway in the Bone Destruction of Apical Periodontitis.

Chronic apical periodontitis is characterized by alveolar bone absorption in the apical region and is the result of the participation of various inflammatory mediators. Studies have shown that the Bruton tyrosine kinase- (Btk-) phospholipase Cγ2 (PLCγ2) signaling pathway plays an important role in bone absorption, but it is unknown whether it plays a role in apical periodontitis bone destruction. Therefore, this study verified the role of Btk and PLCγ2 in bone resorption of apical periodontitis by in vivo and in vitro experiments. In the in vivo experiment, a mice model of apical periodontitis was established; apical bone resorption was confirmed by the numbers of osteoclasts and HE staining. Btk, PLCγ2, and nuclear factor of activated T-cells 1 (NFATc-1) were detected by immunohistochemical staining. In the in vitro experiment, lipopolysaccharides (LPS) were used to stimulate osteoclast precursor cell RAW264.7 to establish an inflammatory microenvironment and detect osteoclast differentiation. By silencing Btk, the expression of Btk, PLCγ2, and NFATc-1 was detected by real-time qPCR and Western blot, and osteoclastogenesis was detected by enzyme histochemical staining to further confirm the role of Btk in bone resorption. It was found that the expression of Btk, PLCγ2, and NFATc-1 changed significantly with the progression of inflammation and bone destruction, indicating that Btk and PLCγ2 may be involved in the progression of inflammation in apical periodontitis and bone absorption. In vitro experiments confirmed that the differentiation of osteoclasts and the expression of PLCγ2 and NFATc-1 were significantly inhibited after silencing Btk expression, but osteoclast precursor cells could be differentiated due to the proinflammatory factor lipopolysaccharide. This study demonstrates that Btk and PLCγ2 are key factors involved in the apical inflammatory response and bone destruction.

Osteopontin Promotes Bone Destruction in Periapical Periodontitis by Activating the NF-κB Pathway.

BACKGROUND/AIMS: Periapical periodontitis is caused by bacterial infection and results in both one destruction and tooth loss. Osteopontin (OPN) is a secreted phosphorylated glycoprotein that participates in bone metabolism. METHODS: Thirty-three patients with chronic periapical periodontitis and 10 patients who had undergone the orthodontic removal of healthy tooth tissue (control) at the periodontal ligament were investigated, and an animal model of mouse periapical periodontitis was established for an in vivo analysis. The relationship between OPN and bone destruction during periapical periodontitis was analyzed. Osteoblasts and osteoclasts were cultured in vitro and treated with lipopolysaccharide. An inhibitor of NF-κB was used to pretreat the transfected cells. RESULTS: OPN increased osteoclast proliferation and differentiation, but reduced osteoblasts proliferation and differentiation. OPN activated the NF-κB pathway during periapical periodontitis and accelerated the transfer and phosphorylation of P65 from the cytoplasm to the nucleus. CONCLUSION: This study demonstrated that OPN played important roles in the progression of periapical periodontitis, and a dual role in bone metabolism during periapical periodontitis, linking osteoclasts and osteoblasts. The underlying mechanism may be related to the NF-κB pathway.

Comparison of oral microbiota in orthodontic patients and healthy individuals.

Orthodontic treatments often include dental displacement using a fixed retainer such as braces, which may result in the accumulation of plaque that provides a suitable environment for microorganisms to cause oral infection. So, this study was designed to investigate the microbial diversity among orthodontic patients and healthy individuals. METHODS: Fifty individuals i.e. 30 orthodontic patients and 20 normal individuals were included in this study. Samples were collected during the midterm of orthodontic treatment (10-12 months). Saliva samples were collected and total DNA was isolated. Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) with universal primers targeting the V3 region of 16s rRNA was used to study the bacterial diversity among different orthodontic and control groups. After DGGE profile analysis, the predominant product bands from the gel were excised, cloned, and sequenced to confirm the taxonomic identity followed by its quantification by using real-time PCR with gene-specific primers. RESULTS: Both orthodontic treatment and control groups formed two distinct clustering profiles, but the Shannon-Weaver index (H') indicated greater microbial diversity in the orthodontic group (P = 0.08). Sequence analysis and real-time PCR revealed a greater number of Pseudomonas spp. in the orthodontic group, while there was no significant difference in Streptococcal spp. CONCLUSION: This study suggested alterations in the oral microbiota following orthodontic treatment would provide diagnostic tools to identify prevalent microbes associated with oral infections that may prove useful for developing future therapies.

The novel protein complex FAPα/ITGA5 is involved in the bone destruction of apical periodontitis.

The bacteria that invade the periapical tissue of teeth can directly damage tissue cells such as periapical fibroblasts, leading to an inflammatory response in the periapical tissue and ultimately resulting in bone destruction. We investigated the role of fibroblast activation protein α (FAPα) and integrin α5 (ITGA5) in periapical bone destruction. This study found that FAPα and ITGA5 were highly expressed in human tissues from patients with chronic apical periodontitis. Osteoclast differentiation decreased when FAPα or ITGA5 was silenced and inhibited. The results of protein molecular docking showed that FAPα had good binding affinity to ITGA5, and its free energy was -14.5 kcal/mol. Immunofluorescence staining and co-immunoprecipitation showed that FAPα and ITGA5 formed protein complexes in the inflammatory microenvironment. In conclusion, this study proved that FAPα and ITGA5 participate in the regulation of osteoclast differentiation by forming protein complexes in the inflammatory microenvironment, which then regulates the occurrence and development of chronic apical periodontitis.

Evaluation of the bond strength of chlorhexidine incorporated into the adhesive system composition: A PRISMA guided meta-analysis.

BACKGROUND/PURPOSE: Composite resin is currently the most widely used dental restoration material. Previous studies have demonstrated that the application of Chlorhexidine (CHX) on the dentin surface after acid etching can result in an improvement in the integrity and stability of tooth restoration through time. In order to better understand whether CHX can help improve the stability of the resin-dentin bond strength, in this study, a comprehensive review of the effect of adding CHX to the adhesive system on the stability of immediate and long-term resin-dentin bond strength was conducted. MATERIALS AND METHODS: This article was written in accordance with the PRISMA Statement and is registered on the International Prospective Register of Systematic Reviews (registration number CRD42018084962). Six electronic databases including PubMed, Embase, Cochrane library, ISI Web of Science, ClinicalTrials.gov and China National Knowledge Infrastructure (CNKI) were searched up to October, 2018. Ten articles were selected from 340 possible eligible articles for meta-analysis, and 41 sets of data were analyzed in the meta-analysis. RESULTS: The results indicated that the use of 0.1% and 0.2% CHX does not adversely affect the immediate bond strength (p > 0.05), but both 0.1% and 0.2% CHX increased bond strength compared with the control group over 12 months (p < 0.05). However, this trend does not represent a longer period of aging. CONCLUSION: In these in vitro clinical trials, CHX incorporated into the bonding systems maintained the stability of bond strength.

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.

Enterococcus faecalis Lipoteichoic Acid-induced NLRP3 Inflammasome via the Activation of the Nuclear Factor Kappa B Pathway.

INTRODUCTION: We wished to examine the effects of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome on periapical periodontitis induced by Enterococcus faecalis and to investigate the molecular mechanisms of lipoteichoic acid (LTA) derived from E. faecalis on the expression of the NLRP3 inflammasome. METHODS: A model of periapical periodontitis by sealing E. faecalis into the pulp chambers of rats was established. We then examined the relationship between the expression, location, distribution, and concentration of NLRP3, caspase-1, and interleukin 1ß with the inflammatory progression by immunohistochemistry and undertook correlation analyses. RAW264.7 cells were cultured in the absence or presence of LTA together with or without nuclear factor kappa B (NF-κB) inhibitor BAY 11-7082; NLRP3 inflammasome expression was measured by Western blotting, the enzyme-linked immunosorbent assay, and real-time quantitative polymerase chain reaction. An immunofluorescence study was conducted to further detect whether NF-κB can be completely inhibited by BAY 11-7082 or activated by LTA. RESULTS: An animal model of periapical periodontitis was established successfully. Expression of NLRP3, caspase-1, and interleukin 1ß protein was observed in the inflamed area. The expression of these 3 proteins had a significant positive correlation (P < .05). Overall, our results showed that, compared with the negative control group, LTA could directly activate expression of messenger RNA and protein of the NLRP3 inflammasome (P < .05), whereas BAY 11-7082 inhibited it (P < .05). CONCLUSIONS: Our results suggested that LTA can act as a directly stimulating factor associated with expression of the NLRP3 inflammasome during periapical periodontitis, which is mainly linked with the NF-κB signaling activation pathway.

[The correlation analysis on Enterococcus faecalis biofilm and clinical manifestation in retreatment of root canal].

OBJECTIVE: To detect the ability of Enterococcus faecalis (E. faecalis) biofilm formation and explore the relationship between E. faecalis biofilm formation ability and clinical manifestation. METHODS: 96 well plate with the establishment of 53 E. faecalis in vitro biofilm model, combined with crystal violet staining, was used to test the biofilm formation ability of the clinical isolates E. faecalis and analyze the relationship between biofilm formation capacity and clinical manifestation. RESULTS: In total 53 E. faecalis strains, 40 strains(75.47%) had biofilm forming ability. Statistical analysis revealed that the capacities of biofilm formation between E. faecalis isolated from with fistula and without fistula was significantly different (P<0.05). CONCLUSION: In the retreatment of root canal, the ability of biofilm formation of E. faecalis separated from the teeth without fistula is better than those separated from the teeth with fistula.

Relationship of biofilm formation and gelE gene expression in Enterococcus faecalis recovered from root canals in patients requiring endodontic retreatment.

INTRODUCTION: Enterococcus faecalis is known to be the most frequently detected species in root canals with failed endodontic treatment. Many studies are available on biofilm formation and the expression of virulence factors such as gelatinase (gelE) in E. faecalis. However, the relationship of biofilm formation and the expression of gelE in E. faecalis recovered from root canals undergoing orthograde retreatment is not well understood. METHODS: E. faecalis was isolated from clinical samples of root canal retreatment, and the expression of gelE in E. faecalis was assessed. Automatic microplate reader and scanning electron microscopy were used to investigate the biofilm formation ability of E. faecalis isolates. Real-time quantitative polymerase chain reaction was used for detecting the expression of gelE in biofilm-positive and biofilm-negative E. faecalis isolates. RESULTS: The detection rate of E. faecalis in the root canal retreatment cases was 39.26%. An automatic microplate reader showed that most isolates were able to form biofilms, and the biofilm formation ability of strains isolated from the teeth without a sinus tract was better than that with a sinus tract (P < .05). The expression of gelE was stronger in the cases of apical radiolucency than in those without the symptom (P < .05). The expression of gelE was higher in the biofilm-positive than in biofilm-negative strains (P < .05). CONCLUSIONS: Biofilm formation in E. faecalis was facilitated in the cases without a sinus tract. In the cases of apical radiolucency and in the biofilm-positive strains, the expression of gelE was higher.

[Analysis of relationship between the detection of Enterococcus faecalis in post-treatment endodontic disease and clinical symptom or sign].

OBJECTIVE: To detect the Enterococcus faecalis (E. faecalis) in post-treatment endodontic disease, and to analyze the relationship between the occurrence of E. faecalis and clinical symptom. METHODS: 108 teeth which need root canal retreatment were collected, and the clinical symptoms and physical signs were recorded. Bacterium samples from root canal were taken, and genome DNA from bacterial samples were extracted. The occurrence of E. faecalis by means of the polymerase chain reaction was investigated. RESULTS: The detection rate of E. faecalis in cases of root canal retreatment was 47.2%, while in cases with symptoms or signs, or cases with both symptoms and signs, the root canal E. faecalis detection rates were 52.6%, 57.9%, 62.5%. The detection rates of E. faecalis between cases with clinical symptom and without clinical symptom demonstrated statistical significance (P < 0.05). The detection rates between cases with both clinical symptom and manifestly aneretic root and cases without clinical symptom and manifestly aneretic root had statistical significance (P < 0.05). In the group of clinical symptom, the detection rate of E. faecalis in cases with biting pain was 66.7%, clearly higher than those without biting pain (P < 0.05). CONCLUSION: The occurrence of E. faecalis in cases of root canal retreatment correlates with clinical symptoms.

[Effect of different decalcification condition on tooth-clearing technique].

OBJECTIVE: To evaluate shrinkage range of cleared teeth caused by nitric acid with different temperature and concentration. METHODS: 48 human teeth were root canal-prepared and filled, then randomly and averagely divided into six groups on the basis of temperature and density of nitric acid and the condition of whether or not added the oscillate. Group A was 20 degrees C with 6% nitric acid, group B was 20 degrees C with 6% nitric acid and oscillate, group C was 20 degrees C with 3% nitric acid, group D was 20 degrees C with 3% nitric acid and oscillate, group E was 30 degrees C with 6% nitric acid and oscillate, group F was 30 degrees C with 3% nitric acid and oscillate. After achieving the standard of the decalcification, all the specimens were gradually dehydrated, and then cleared and conserved using methyl salicylate. Time-consumed and shrinkage range of all the specimens were recorded and analyzed. RESULTS: The time of decalcification in group E was the fastest, then was group F, group B. Group C was the last one. The anastole of the specimens was group E > group B > group A, group F > group D > group C, group B > group D, group E > group D, there was significant difference (P < 0.05). Group C had significant difference with other groups (P < 0.05). The anastole rate of the specimens had no significant difference between group A and group B, group C and group D, group B and group F, group D and group F. CONCLUSION: In 20 degrees C, 3% nitric acid with oscillate to carry out the decalcification can use less time and get less anastole. The result of the tooth-clearing technique is the best.