[Show abstract][Hide abstract] ABSTRACT: The NLRP3/caspase-1 inflammasome pathway plays an important role in cellular immune defence against bacterial infection; however, its function in human dental pulp tissue and human dental pulp fibroblasts remains poorly understood. We demonstrate that NLRP3 protein expression occurs to a greater extent in pulp tissue with irreversible pulpitis than in normal pulp tissue and in tissue with reversible pulpitis. Caspase-1 is present in its active (cleaved) form only in pulp tissue with irreversible pulpitis. NLRP3 and caspase-1 are expressed in the odontoblast layers in normal human dental pulp tissue, whereas in inflamed pulp tissue, the odontoblast layers are disrupted and dental pulp cells are positive for NLRP3 and caspase-1. Additionally, we investigate the role of the NLRP3/caspase-1 inflammasome pathway in human dental pulp fibroblasts and show that ATP activates the P2X7 receptor on the cell membrane triggering K(+) efflux and inducing the gradual recruitment of the membrane pore pannexin-1. Extracellular lipopolysaccharide is able to penetrate the cytosol and activate NLRP3. Furthermore, the low intracellular K(+) concentration in the cytosol triggers reactive oxygen species generation, which also induces the NLRP3 inflammasome. Thus, the NLRP3/caspase-1 pathway has a biological role in the innate immune response mounted by human dental pulp fibroblasts.
Cell and Tissue Research 02/2015; 361(2). DOI:10.1007/s00441-015-2118-7 · 3.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Stem cells from the apical papilla (SCAPs) are important for tooth root development and may be candidates for regenerative endodontic procedures involving immature teeth. The potential use of SCAPs for clinical applications requires a better understanding of their responses to bacterial challenge. We have investigated the effects of exposure of these cells to lipopolysaccharide (LPS). Inflammatory responses arising from bacterial challenges can constrain postinjury tissue regeneration and the effects of nuclear factor I C (NFIC), which plays a critical role in tooth root development. NFIC has been explored for its anti-inflammatory action in the context of endodontic treatment of immature teeth where continued root development is an important outcome.
SCAPs were exposed to LPS, and the expression of Toll-like receptor-4 (TLR4), interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF-α) were assessed by real-time polymerase chain reaction (RT-PCR). The pLenti6.3/v5-NFIC plasmid encoding the full-length NFIC or NFIC silencing by si-RNA (small interfering RNA) in SCAPs were measured by Western blotting or RT-PCR; the effects of NFIC on IL-6, IL-8, and TNF-α were analyzed by RT-PCR. The protein levels were subsequently measured by enzyme-linked immunoassay.
LPS induced the synthesis of IL-6, IL-8, and TNF-α in SCAPs in a time-dependent manner. Pretreatment with a TLR4 inhibitor significantly inhibited LPS-induced IL-6, IL-8, and TNF-α expression. Knockdown of NFIC increased the expression of IL-6, IL-8, and TNF-α, whereas the overexpression of NFIC resulted in the suppression of the inflammatory response stimulated by 1 μg/mL LPS, especially for IL-8. Together, these data show that LPS is recognized by the transmembranous receptor TLR4 to mediate inflammatory responses in SCAPs and NFIC overexpression can suppress LPS-initiated innate immune responses.
The anti-inflammatory effects of NFIC overexpression provide a valuable target to dampen inflammatory responses in the infected pulp to allow tissue regeneration to occur.
Journal of endodontics 11/2013; 39(11):1416-22. DOI:10.1016/j.joen.2013.07.018 · 3.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aim:
To observe the antimicrobial effect of strong acid electrolytic water (SAEW) against an Enterococcus faecalis (E. faecalis) biofilm when used as a root canal irrigant.
The effect of SAEW, sodium hypochlorite (5.25%; NaOCl) and sodium chloride (0.9%; normal saline) on E. faecalis biofilm vitality on coverslips was observed by confocal laser scanning microscopy (CLSM). Thirty-five root canals from extracted human teeth were sterilized prior to contamination with E. faecalis for four weeks. Bacterial samples were collected with sterile paper points and plated onto BHI agar plates for 48 h. Root canal walls were observed by scanning electron microscopy before and after instrumentation, together with root canal irrigation with SAEW, NaOCl or normal saline, with or without ultrasonic vibration. Antimicrobial effectiveness was established by counting colony-forming units and analysed by two-way anova.
Confocal laser scanning microscopy revealed that SAEW decreased E. faecalis biofilm vitality, and the proportion of dead bacteria increased in accordance with increasing treatment time. Most bacteria in the biofilms were killed after 10-min treatment. No significant difference was observed between SAEW and NaOCl groups at the same treatment time (P > 0.05) or in the susceptibility of E. faecalis to SAEW and NaOCl (P > 0.05) in extracted human teeth with or without ultrasonic activation. SAEW and NaOCl were more effective against E. faecalis biofilm than normal saline, and antimicrobial efficacy was significantly enhanced by ultrasonic vibration (P < 0.05).
Strong acid electrolytic water effectively killed E. faecalis in a biofilm both on coverslips and in the root canals of extracted human teeth.
International Endodontic Journal 02/2013; 46(10). DOI:10.1111/iej.12084 · 2.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the effects of lipopolysaccharide (LPS) on interleukin-8 (IL-8) and related intracellular signalling pathways in human dental pulp stem cells (hDPSCs).
Human pulp tissues were isolated from human impacted third molars, and the hDPSCs were cultured and characterized. The effects of LPS on IL-8 and Toll-like receptor 4 (TLR4) gene expression in hDPSCs were investigated using real-time quantitative RT-PCR and ELISA. Whether TLR4/MyD88/NF-кB was involved in the LPS-induced up-regulation of IL-8 in hDPSCs was determined using transient transfection, luciferase assay and ELISA. The involvement of MAPKs in the LPS-induced up-regulation of IL-8 in hDPSCs was investigated via transient transfection, luciferase assay, ELISA and western blot. The data were statistically analysed using Student's t-test or one-way anova followed by the Student–Neumann–Keuls test.
Cells exposed to LPS not only displayed an enhanced expression of TLR4 but also showed an elevated IL-8 gene expression; exposure to LPS also resulted in the induction of IL-8 gene transcription via promoter activation. The LPS-induced IL-8 promoter activation was inhibited through dominant-negative mutations in TLR4 and MyD88, but not in TLR2. The LPS-induced IL-8 protein release was attenuated through the administration of TLR4-neutralizing antibody or MyD88 inhibitory peptide and a dominant-negative mutation in IκBα. In contrast, IL-8 protein release was enhanced through the expression of NF-κB p65. Treatment with PDTC, TPCK or Bay117082 effectively antagonized LPS-induced IL-8 protein release. Moreover, both the promoter activity and the LPS-induced release of IL-8 were diminished upon the administration of U0126 and SB203580, but not SP600125. Moreover, the exposure to LPS activated ERK1/2 and p38 MAPK phosphorylation in cells.
This study reports the LPS-mediated transcriptional and post-translational up-regulation of IL-8, which is a process that also involves TLR4, MyD88, NF-κB and MAPK.
International Endodontic Journal 06/2012; 46(2). DOI:10.1111/j.1365-2591.2012.02096.x · 2.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many pathogenic microorganisms were found in an infected root canal. The object of this study was to evaluate the effect of MTAD in combination with nisin on the pathogens associated with root canal infection.
The survival rates of 9 pathogenic bacteria were determined after 1-, 5-, and 10-minute treatment with MTAD, MTAN (substitution of doxycycline with nisin), and MTADN (nisin in combination with doxycycline). The survival rates of Enterococcus faecalis in the starvation phase and pretreatment alkalization as well as in the normal physiological state under MTAD, MTAN, and MTADN challenge for 1, 5, and 10 minutes were evaluated and compared. Furthermore, scanning electron microscopy was used to observe the morphologic modification of Actinomyces naeslundii, Lactobacillus paracasei, and Porphyromonas gingivalis after MTAD and MTADN treatment.
L. fermenti, L. paracasei, A. viscosus, A. naeslundii, Streptococcus gordonii, and Peptostreptococcus were more sensitive to MTADN and MTAN than to MTAD. MTAD, MTAN, and MTADN showed a rapid antibacterial effect on P. gingivalis, Prevotella intermedia, and Fusobacterium nucleatum. Enterococcus faecalis in the stress state was as sensitive to MTAD, MTAN, and MTADN as the control E. faecalis. Furthermore, in the observation of scanning electron microscopy, the membranes in A. naeslundii and L. paracasei presented significant rupture, and P. gingivalis did not exhibit significant damage after MTADN treatment.
MTAD in combination with nisin improved antibacterial efficacy against pathogens, especially for some gram-positive bacteria associated with persistent intracanal infection. Therefore, the combination had the potential to be used as an effective intracanal irrigation.
Journal of endodontics 04/2012; 38(4):490-4. DOI:10.1016/j.joen.2011.11.015 · 3.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This case series describes observations of 12 patients who developed horizontal root fractures in non-endodontically treated teeth. Using clinical and radiographic examination, horizontal fractures were observed in eight maxillary first molars, one maxillary second molar, one mandibular first premolar and four mandibular second premolars. A total of 12 teeth had clinically intact crowns and 2 had carious. Eight fractures of maxillary molars occurred in the palatal roots, while one fracture was observed in the distobuccal root. Eleven patients experienced pain from the affected teeth during mastication. Within the limits of this case series, it was concluded that these fractures were related to; root morphology, damaging masticatory habits and excessive occlusal forces.
[Show abstract][Hide abstract] ABSTRACT: Objectives: To evaluate the antimicrobial effect of different root canal irrigants and their effects on the repair of apical and periapical tissue of teeth with chronic apical periodontitis in dog.
Methods: After devitalization of the pulp tissue of 80 premolar root canals in 4 dogs, the teeth were divided into 4 groups and respectively filled with lipopolysaccharide(Ⅰ), mixed bacteria(Ⅱ), Enterococcus faecalis(Ⅲ), normal saline(Ⅳ) and sealed. After 3 months, the teeth of each dog were randomly divided into three groups, treated with different combinations of root canal irrigants during the RCT (5.25% NaOCl plus 17%EDTA group, 3% H2O2 plus NS group and NS group as control).The bacteria in each canal were sampled before and after the preparation and counted under light microscope. 3 months later, the animals were sacrificed and the obtained histological sections were stained with hematoxylin and eosin for optical microscopic analysis of apical and periapical repair.
Results: Radiographs showed that LPS group(Ⅰ) had lesions at 30days; mixed bacteria group(Ⅱ) had lesions at 45 days and showed a rapid aggravation; E.faecalis group(Ⅲ) had lesions at 90 days. NaOCl-EDTA group showed better antimicrobial effect against facultative anaerobes than H2O2-NS and the control group (P<0.05). The difference between the NaOCl-EDTA and H2O2-NS groups against obligate anaerobes or E.faecalis was not significant(P>0.05). The inflammatory responses among all the groups in dog Ⅰ were the same (P>0.05); in dog Ⅱ there was better histological repair(P<0.05) in the NaOCl-EDTA group than the H2O2-NS group or the control; in dog Ⅲ there were no significant differences between the NaOCl-EDTA group and the H2O2-NS group(P>0.05).
Conclusion: The mixed bacteria can cause apical periodontitis rapidly and effectively. The root canal irrigants of 5.25% NaOCl plus 17%EDTA have a stronger antimicrobial efficacy on several predominant bacteria, and resulted in better repair.
2nd Meeting of IADR Pan Asian Pacific Federation (PAPF) and the 1st Meeting of IADR Asia/Pacific Region 2009;