Influence of rotational speed on torque/force generation and shaping ability during root canal instrumentation of extracted teeth with continuous rotation and optimum torque reverse motion.

AIM: To evaluate how different rotational speeds affect the torque/force generation and shaping ability of rotary root canal instrumentation using JIZAI (MANI, Utsunomiya, Japan) nickel-titanium instruments in continuous rotation and optimum torque reverse (OTR) motion. METHODOLOGY: Mesial root canals of extracted mandibular molars were instrumented up to size 25, 0.04 taper using JIZAI instruments, and anatomically matched canals were selected based on geometric features of the canal [canal volume (mm3 ), surface area (mm2 ), length, 15°-20° curvature and radius of curvature (4-8 mm)] after micro-computed tomographic scanning. An automated root canal instrumentation and torque/force analysing device was programmed to permit a simulated pecking motion (2 s downward and 1 s upward at 50 mm min-1 ). The selected canals were prepared with size 25, 0.06 taper JIZAI instruments using continuous rotation or OTR motion and further subdivided according to the rotational speed (300 or 500 rpm, n = 10 each). Real-time clockwise/counterclockwise torque and downward/upward force were recorded using a custom-made torque/force analysing device. Then, the registered pre- and post-operative micro-computed tomographic datasets were examined to evaluate the canal volume changes and centring ratios at 1, 3, 5 and 7 mm from the apical foramen. Data were analysed using two-way analysis of variance or the Kruskal-Wallis test and Mann-Whitney U test with Bonferroni correction (α = 5%). RESULTS: Maximum upward force and clockwise torque were significantly smaller in 500 rpm groups than in 300 rpm groups (P < .05); however, no significant difference was found between continuous rotation and OTR motion (P > .05). OTR motion developed higher maximum counterclockwise torque than continuous rotation (P < .05). Maximum downward force, canal volume changes and centring ratios were not significantly different among all groups (P > .05). There was no file fracture in any of the groups. CONCLUSIONS: Under laboratory conditions using JIZAI instruments, a rotational speed of 500 rpm generated significantly lower maximum screw-in forces and torque values than rotational speed of 300 rpm. Continuous rotation and OTR motion performed similarly in shaping the canals.

Hypoxia-inducible factor 1α promotes interleukin 1ß and tumour necrosis factor α expression in lipopolysaccharide-stimulated human dental pulp cells.

AIM: To elucidate the role of HIF1α in pro-inflammatory cytokine mRNA expression from lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). METHODOLOGY: mRNA expression of interleukin (IL) 1ß and tumour necrosis factor (TNF) α in LPS-stimulated hDPCs was determined by quantitative RT-PCR. Expression of nuclear factor kappa B (NFκB) p65 and phospho-NFκB p65 was analysed by Western blotting. Activation of NFκB signalling was measured by luciferase assay using a reporter vector containing an NFκB response element. Enforced expression of HIF1α was induced by transfection of expression vectors with native or constitutively active forms of HIF1α. Expression of HIF1α protein in hDPCs was evaluated by immunocytochemistry and Western blotting. One-way analysis of variance and the Tukey-Kramer test were performed to determine a significant difference (P < 0.05). RESULTS: mRNA expression of IL1ß and TNFα, protein expression of phospho-NFκB p65 and LPS-induced NFκB signalling activity were promoted in low oxygen conditions (1% O2 ; P < 0.05). These findings were replicated following enforced expression and stabilization of HIF1α in hDPCs. Dimethyloxalylglycine, an inhibitor of prolyl hydroxylase (a HIF1α degrading enzyme), promoted IL1ß and TNFα mRNA expression and NFκB signalling in LPS-stimulated hDPCs (P < 0.05). HIF1α expression was detected in hDPCs cultured in low oxygen conditions (1% O2 ). LPS stimulation further enhanced HIF1α expression in hDPCs, especially within their nuclei. CONCLUSION: HIF1α promoted mRNA expression of IL1ß and TNFα via NFκB signalling in LPS-stimulated hDPCs, suggesting that HIF1α is involved in the progress of inflammation in dental pulp.

Mineral trioxide aggregate suppresses pro-inflammatory cytokine expression via the calcineurin/nuclear factor of activated T cells/early growth response 2 pathway in lipopolysaccharide-stimulated macrophages.

AIM: To elucidate mechanisms by which mineral trioxide aggregate (MTA) suppresses pro-inflammatory cytokine mRNA expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. METHODOLOGY: Mineral trioxide aggregate extracts were prepared by immersing set ProRoot MTA in culture medium. RAW264.7 cells were cultured in the presence of LPS and MTA extracts. mRNA expression levels of interleukin (IL)-1α, IL-6, early growth response 2 (Egr2), suppressor of cytokine signalling 3 (Socs3) and IL-10 were quantified with reverse transcription-quantitative polymerase chain reaction. Phosphorylation of nuclear factor-kappa B (NF-κB) p65 in RAW264.7 cells was analysed by Western blotting. Intracellular calcium imaging was performed with Fluo-4 AM. The activity of nuclear factor of activated T cells (NFAT) was determined by luciferase assays. Enforced expression and silencing of Egr2 in RAW264.7 cells were carried out using an expression vector and specific RNAi, respectively. In vivo kinetics of Egr2+ cells in MTA-treated rat molar pulp tissues were examined using immunohistochemistry. Data were analysed by one-way analysis of variance, followed by the Tukey-Kramer test (P < 0.05). RESULTS: Exposure to MTA extracts resulted in reduced mRNA expression levels of IL-1α and IL-6, as well as reduced expression of phosphorylated NF-κB, in LPS-stimulated RAW264.7 cells. Exposure to MTA extracts induced Ca2+ influx, which was blocked by NPS2143, an antagonist of calcium-sensing receptor (CaSR); Ca2+ influx then triggered activation of calcineurin/NFAT signalling and enhanced mRNA expression of Egr2. Enforced expression of Egr2 in RAW264.7 cells promoted the expression of both IL-10 and Socs3. In vivo application of MTA onto rat molar pulp tissue resulted in the appearance of Egr2-expressing cells that coexpressed CD163, a typical M2 macrophage marker. CONCLUSIONS: Mineral trioxide aggregate extracts induced downregulation of IL-1α and IL-6 in LPS-stimulated RAW264.7 cells via CaSR-induced activation of calcineurin/NFAT/Egr2 signalling and subsequent upregulation of IL-10 and Socs3.

Macrophage populations show an M1-to-M2 transition in an experimental model of coronal pulp tissue engineering with mesenchymal stem cells.

AIM: To assess M1/M2 macrophage phenotypes in a coronal pulp regeneration model in rats, under the hypothesis that there are dynamic M1/M2 phenotype changes during the different stages of the pulp regeneration. METHODOLOGY: The maxillary first molars of Wistar rats were pulpotomized, and biodegradable hydrogel-made scaffolds carrying rat bone marrow mesenchymal stem cells were implanted in the pulp chamber. After 3, 7 and 14 days, samples were processed for (i) histological analysis and double immunoperoxidase staining for CD68 (a general macrophage marker) and one of either CCR7 (an M1 marker), CD163 (an M2 marker) or CD206 (an M2 marker); (ii) real-time PCR for AIF1 (an M1 marker), CD163, CD206, IL-10 and TNF-α mRNA expression; and (iii) Western blotting for the detection of CD68, CCR7 and CD206 proteins. RESULTS: Histological analysis of the implanted region revealed sparse cellular distribution at 3 days, pulp-like tissue with a thin dentine bridge-like structure at 7 days, and dentine bridge-like mineralized tissue formation and resorption of most scaffolds at 14 days. CCR7+ macrophages had the highest density at 3 days, and then significantly decreased until 14 days (P < 0.05). In contrast, M2 marker (CD163 or CD206) expressing macrophages had the lowest density at 3 days and significantly increased until 14 days (P < 0.05). AIF1 and TNF-α mRNA levels, and CD68 and CCR7 protein levels were highest at 3 days. CD163 and CD206 mRNA levels, and CD206 protein levels increased with time and showed the highest at 14 days. IL-10 mRNA was highest at 3 days, decreased at 7 days and increased at 14 days. CONCLUSIONS: Macrophages in the regenerating pulp tissue underwent a distinct transition from M1-dominant to M2-dominant, suggesting that the M1-to-M2 transition of macrophages plays an important role in creating a favourable microenvironment necessary for pulp tissue regeneration.

Effect of lipopolysaccharide stimulation on stem cell-associated marker-expressing cells.

AIM: To examine the effect of inflammatory stimuli on the proliferation/migration of dental pulp stem cells by assessing the responses of stem cell-associated marker-expressing cells in rat incisors to lipopolysaccharide (LPS) stimulation in vivo. METHODOLOGY: The crowns of rat incisors were removed, and the coronal pulp chamber was instrumented. After haemostasis, an absorbent point soaked in LPS was inserted into the cavity, which was then sealed. At 3, 12, and 48 h after LPS application, pulp tissues were subjected to double-immunoperoxidase labelling using two of the antibodies against microtubule-associated protein 1B (MAP1B), CD146 and STRO-1. For gene expression analysis, total RNA was extracted, and mRNA expression levels of stem cell factor (SCF), stromal-derived factor 1 (SDF-1), CD146 and MAP1B were analysed with real-time polymerase chain reaction. SCF and SDF-1 protein levels were also assessed by Western blot. Statistical analysis was performed by Kruskal-Wallis nonparametric analysis of variance, followed by Mann-Whitney U-tests with Bonferroni correction. RESULTS: The density of MAP1B+ CD146+ cells and STRO-1+ CD146+ cells in LPS-stimulated pulp tissue increased significantly at 3 h and exhibited a four- to sixfold increase at 48 h as compared with the density observed in normal pulp tissue (P < 0.05). The expression of CD146 mRNA in LPS-stimulated pulp showed significant upregulation at 3 h as compared with that observed in normal pulp tissue (P < 0.05). MAP1B, SCF and SDF-1 mRNA levels also showed significant upregulation at 3 and 72 h (P < 0.05), and Western blot analysis revealed increases in SCF and SDF-1 following LPS stimulation. CONCLUSIONS: LPS-stimulated pulp tissue exhibited upregulation of stem cell differentiation/migration markers and showed increases in the number of MAP1B+ CD146+ and STRO-1+ CD146 stem-like cells.

Evaluation of the Ca ion release, pH and surface apatite formation of a prototype tricalcium silicate cement.

AIM: To evaluate the Ca2+ -releasing, alkalizing and apatite-like surface precipitate-forming abilities of a prototype tricalcium silicate cement, which was mainly composed of synthetically prepared tricalcium silicate and zirconium oxide radiopacifier. METHODOLOGY: The prototype tricalcium silicate cement, white ProRoot MTA (WMTA) and TheraCal LC (a light-cured resin-modified calcium silicate-filled material) were examined. The chemical compositions were analysed with a wavelength-dispersive X-ray spectroscopy electron probe microanalyser with an image observation function (SEM-EPMA). The pH and Ca2+ concentrations of water in which the set materials had been immersed were measured, and the latter was assessed with the EDTA titration method. The surface precipitates formed on the materials immersed in phosphate-buffered saline (PBS) were analysed with SEM-EPMA and X-ray diffraction (XRD). Kruskal-Wallis tests followed by Mann-Whitney U-test with Bonferroni correction were used for statistical analysis (α = 0.05). RESULTS: The prototype cement contained Ca, Si and Zr as major elemental constituents, whereas it did not contain some metal elements that were detected in the other materials. The Ca2+ concentrations and pH of the immersion water samples exhibited the following order: WMTA = prototype cement > TheraCal LC (P < 0.05). All three materials produced Ca- and P-containing surface precipitates after PBS immersion, and the precipitates produced by TheraCal LC displayed lower Ca/P ratios than those formed by the other materials. XRD peaks corresponding to hydroxyapatite were detected in the precipitates produced by the prototype cement and WMTA. CONCLUSION: The prototype tricalcium silicate cement exhibited similar Ca2+ -releasing, alkalizing and apatite-like precipitate-forming abilities to WMTA. The Ca2+ -releasing, alkalizing and apatite-like precipitate-forming abilities of TheraCal LC were lower than those of the other materials.

Bioactivity and biomineralization ability of calcium silicate-based pulp-capping materials after subcutaneous implantation.

AIM: To evaluate the abilities of three calcium silicate-based pulp-capping materials (ProRoot MTA, TheraCal LC and a prototype tricalcium silicate cement) to produce apatite-like precipitates after being subcutaneously implanted into rats. METHODOLOGY: Polytetrafluoroethylene tubes containing each material were subcutaneously implanted into the backs of Wistar rats. At 7, 14 and 28 days post-implantation, the implants were removed together with the surrounding connective tissue, and fixed in 2.5% glutaraldehyde in cacodylate buffer. The chemical compositions of the surface precipitates formed on the implants were analysed with scanning electron microscopy-electron probe microanalysis (SEM-EPMA). The distributions of calcium (Ca) and phosphorus (P) at the material-tissue interface were also analysed with SEM-EPMA. Comparisons of the thicknesses of the Ca- and P-rich areas were performed using the Friedman test followed by Scheffe's test at a significant level of 5%. RESULTS: All three materials produced apatite-like surface precipitates containing Ca and P. For each material, elemental mapping detected a region of connective tissue in which the concentrations of Ca and P were higher than those in the surrounding connective tissue. The thickness of this Ca- and P-rich region exhibited the following pattern: ProRoot MTA > prototype tricalcium silicate cement ≥ TheraCal LC. ProRoot MTA had a significantly thicker layer of Ca and P than the other materials at all time-points (P < 0.05), and a significant difference was detected between the prototype cement and TheraCal LC at 28 days (P < 0.05). CONCLUSION: After being subcutaneously implanted, all of the materials produced Ca- and P-containing surface precipitates and a Ca- and P-rich layer within the surrounding tissue. The thickness of the Ca- and P-rich layer of ProRoot MTA was significantly thicker than that of the other materials.

GaAlAs laser-induced pulp mineralization involves dentin matrix protein 1 and osteopontin expression.

OBJECTIVES: GaAlAs lasers induce pulp mineralization by promoting reparative dentinogenesis. This study analyzed the expression of dentin matrix protein 1 (DMP1) and osteopontin in GaAlAs laser-irradiated rat molars, to examine the hypothesis that these proteins play a role in the laser-induced reparative dentinogenic process. MATERIALS AND METHODS: The mesial surfaces of the upper first molars of 8-week-old Wistar rats were irradiated with a pulsed GaAlAs laser. After 1-14 days, mRNA expression of DMP1 and osteopontin in the coronal pulp was analyzed using real-time PCR. DMP1, osteopontin, and heat shock protein 25 (HSP25) were immunolocalized at 1-21 days. RESULTS: The pulp exhibited a degenerative zone in its mesial portion on days 1-3, and progressive formation of reparative dentin lined with HSP25-immunoreactive odontoblast-like cells, from day 7 onwards. DMP1 and osteopontin mRNA expression were significantly upregulated on days 1-7 and 3-7, respectively. From day 7 onwards, DMP1 and osteopontin immunoreactivity colocalized along the boundary between the primary and reparative dentin. CONCLUSION: GaAlAs laser irradiation of rat molars induced upregulated DMP1 and osteopontin mRNA expression in the coronal pulp, followed by the formation of reparative dentin and the colocalization of DMP1 and osteopontin immunoreactivity at the site at which this tissue first appeared.

Evaluation of calcium-releasing and apatite-forming abilities of fast-setting calcium silicate-based endodontic materials.

AIM: To evaluate two fast-setting calcium silicate-based endodontic materials (Endocem mineral trioxide aggregate (MTA) and Endocem Zr) with regard to their ability to release calcium ions (Ca(2+)) and produce apatite-like precipitates after immersion in phosphate-buffered saline (PBS). METHODOLOGY: Endocem MTA, Endocem Zr and white ProRoot MTA (WMTA) were used. Chemical composition of the powder of each material was analysed with a wavelength-dispersive X-ray spectroscopy electron probe microanalyser with image observation function (SEM-EPMA). The amount of Ca(2+) released from water-immersed set cements was measured with an EDTA titration method. Morphology and chemical composition of surface precipitates formed on the surface of PBS-immersed cements were analysed with the SEM-EPMA. Data obtained were analysed using one-way analysis of variance and Tukey's honestly significant difference test with a significance level of 5%. RESULTS: Endocem MTA and WMTA contained calcium (Ca), silicon (Si) and bismuth as the major elemental constituents, whereas Endocem Zr contained zirconium as the most abundant element, followed by Ca and Si. The amount of Ca(2+) release was WMTA >Endocem MTA ≥Endocem Zr. After immersion in PBS for 14 days, the three materials produced Ca- and phosphorus (P)-containing apatite-like surface precipitates. WMTA showed higher Ca/P ratio of the precipitates compared with the other cements, with statistical significance between WMTA and Endocem Zr (P < 0.05). CONCLUSION: Compared with WMTA, Endocem MTA and Endocem Zr were associated with significantly less Ca ions release and, when immersed in PBS, produced apatite-like crystalline precipitates of significantly lower Ca/P ratios.

Temporospatial localization of dentine matrix protein 1 following direct pulp capping with calcium hydroxide in rat molars.

AIM: To examine the temporospatial expression of dentine matrix protein 1 (DMP1; a noncollagenous protein involved in mineralized tissue formation), osteopontin (another noncollagenous protein detected during reparative dentinogenesis) and nestin (a marker of differentiating/differentiated odontoblasts), following direct pulp capping with calcium hydroxide in rat molars. METHODOLOGY: The maxillary first molars of 8-week-old Wistar rats had their pulps exposed and capped with calcium hydroxide. The pulp-capped teeth were collected from 6 h to 14 days postoperatively and processed for immunohistochemistry for DMP1, osteopontin and nestin. Cell proliferation was monitored using 5-bromo-2'-deoxyuridine (BrdU) labelling. RESULTS: The capped pulps initially exhibited superficial necrotic changes followed by the formation of new matrix and its mineralization. DMP1 immunoreactivity was observed in the matrix beneath the necrotic layer from 6 h onwards and present in the outer portion of the newly formed mineralized matrix from 7 days onwards. Osteopontin displayed a similar expression pattern, although it occupied a narrower area than DMP1 at 6 and 12 h. Nestin-immunoreactive cells appeared beneath the DMP1-immunoreactive area at 1 day, were distributed beneath the newly formed matrix at 5 days and exhibited odontoblast-like morphology by 14 days. BrdU-positive cells significantly increased at 2 and 3 days (P < 0.05) and then decreased. CONCLUSIONS: The deposition of DMP1 at exposed pulp sites preceded the appearance of nestin-immunoreactive cells, active cell proliferation and new matrix formation after pulp capping with calcium hydroxide in rat molars, suggesting that DMP1 acts as a trigger of pulp repair. The colocalization of DMP1 and osteopontin suggests that these two proteins play complementary roles.

Bioactivity evaluation of three calcium silicate-based endodontic materials.

AIM: To compare white ProRoot MTA (WMTA), EndoSequence BC sealer (BC sealer) and Biodentine with regard to their ability to produce apatites and cause Ca and Si incorporation in adjacent human root canal dentine after immersion in phosphate-buffered saline (PBS). METHODOLOGY: Root sections of human single-rooted teeth were filled with one of the materials and immersed in PBS for 1, 7, 30 or 90 days (n = 5 each). Morphology and elemental composition of surface precipitates and interfacial dentine were analysed using a wavelength-dispersive X-ray spectroscopy electron probe microanalyser with image observation function. Ca- and Si-incorporation depths in the interfacial dentine were measured. In addition, the amount of Ca ions released from the test materials was measured by EDTA titration. RESULTS: All materials produced surface precipitates of acicular or lath-like morphology with Ca/P ratio of 1.6 : 2.0. Within dentinal tubules, the three materials formed tag-like structures that were frequently composed of Ca- and P-rich and Si-poor materials, suggesting intratubular precipitation. Ca- and Si-incorporation depths were in the order of Biodentine > WMTA > BC sealer, with a significant difference between BC sealer and the others at several time-points (P < 0.05, anova and Tukey's honestly significant difference test). The concentration of released Ca ions was in the order of Biodentine > WMTA > BC sealer with significant differences between the materials (P < 0.05). CONCLUSIONS: Compared with Biodentine and WMTA, BC sealer showed less Ca ion release and did not show Ca and Si incorporation as deeply in human root canal dentine when immersed in PBS for up to 90 days.

Uptake of calcium and silicon released from calcium silicate-based endodontic materials into root canal dentine.

AIM: To compare Biodentine and White ProRoot mineral trioxide aggregate (MTA) with regard to Ca and Si uptake by adjacent root canal dentine in the presence of phosphate-buffered saline (PBS). METHODOLOGY: Root canals of bovine incisor root segments were instrumented, filled with either Biodentine or MTA (n = 20 each) and then immersed in Ca-and Mg-free PBS for 1, 7, 30 or 90 days (n = 5 each). Unfilled, unimmersed dentine specimens (n = 5) served as controls. The specimens were sectioned longitudinally, and the ultrastructure of the dentine-material interface and the elemental composition/distribution in the material-adjacent dentine were analysed using a wavelength-dispersive X-ray spectroscopy electron probe microanalyser with image observation function. Data were statistically analyzed using one-way anova and Tukey's honestly significant difference test or the Mann-Whitney U-test. RESULTS: Along the material-dentine interface, both materials formed a tag-like structure that was composed of either Ca- and P-rich crystalline deposits or the material itself. The width of a Ca- and Si-rich layer detected along the dentine layer of the material-dentine interface showed increases over time. The Ca- and Si-rich layer width was significantly larger (P < 0.05) in Biodentine than MTA at 30 and 90 days. CONCLUSIONS: Both Biodentine and MTA caused the uptake of Ca and Si in the adjacent root canal dentine in the presence of PBS. The dentine element uptake was more prominent for Biodentine than MTA.

Immunohistochemical analysis of subcutaneous tissue reactions to methacrylate resin-based root canal sealers.

AIM: To investigate subcutaneous tissue reactions to methacrylate resin-based root canal sealers by immunohistochemical assessment of inflammatory/immunocompetent cell infiltration. METHODOLOGY: Silicone tubes containing freshly mixed Epiphany SE sealer, MetaSEAL, Super-Bond RC sealer, or a zinc oxide-eugenol sealer (Canals) were subcutaneously implanted into the backs of Wistar rats. Solid silicone rods implanted in different animals served as controls. After 7, 14 and 28 days, connective tissue surrounding the implants (n = 8, each) was processed for immunoperoxidase staining using OX6 (reactive to major histocompatibility complex class II molecules), ED1 (reactive to macrophages), and W3/13 (reactive primarily to neutrophils), and the number of positively stained cells within each field (1.2 × 0.8 mm) was enumerated. Statistical differences were analysed with Friedman's test and Scheffe's test (comparisons between test materials) or Mann-Whitney's U-test (test-control comparisons). RESULTS: Canals showed a significantly higher number of W3/13-positive cells (mostly neutrophils) than MetaSEAL at 28 days (P < 0.05). There were no significant differences in the numbers of OX6- or ED1-positive cells between each test material at any time point. Test-control comparisons revealed several significant differences for each antibody. This was most notable for ED1, where all the test materials at each time point, except for Epiphany SE at 28 days, showed significantly larger values than the corresponding controls. CONCLUSIONS: All the methacrylate resin-based sealers tested showed a similar level of inflammatory/immunocompetent cell infiltration. MetaSEAL induced less-intense neutrophil infiltration than Canals. Controls exhibited milder infiltration of inflammatory/immunocompetent cells compared with all the test materials.

Antigen-presenting cells in human radicular granulomas.

Substantial numbers of dendritic cells have been detected in radicular granulomas. To test the hypothesis that local antigen presentation from dendritic cells to T-cells is involved critically in immunological responses within radicular granulomas, we compared characteristics of dendritic cells and macrophages by morphological and biological analyses. Under light microscopy, HLA-DR+ and CD68+ cells showed diverse profiles, including dendritic-shaped cells. Transmission electron microscopy revealed that HLA-DR+ dendritic cells, with long cytoplasmic processes and lacking distinct phagosomes, were concentrated in the lymphocyte-rich area. HLA-DR alpha-chain, CD83, and CD86 mRNAs from HLA-DR+ dendritic cells, and CD28 mRNA from CD28+ T-cells were up-regulated in lymphocyte-rich area. Scanning electron microscopy demonstrated that the density of gold particles on dendritic cells was higher than that on HLA-DR+ macrophages. These results suggest that dendritic cells in radicular granulomas are associated with local defense reactions as stronger antigen-presenting cells, as compared with macrophages.

Efficiency of dual-cured resin cement polymerization induced by high-intensity LED curing units through ceramic material.

OBJECTIVE: This study aimed to evaluate the ability of high-intensity light-emitting diode (LED) and other curing units to cure dual-cured resin cement through ceramic material. METHODS: A halogen curing unit (Jetlite 3000, Morita), a second-generation LED curing unit (Demi, Kerr), and two high-intensity LED curing units (PenCure 2000, Morita; Valo, Ultradent) were tested. Feldspathic ceramic plates (VITABLOCS Mark II, A3; Vita Zahnfabrik) with thicknesses of 1.0, 2.0, and 3.0 mm were prepared. Dual-cured resin cement samples (Clearfil Esthetic Cement, Kuraray Noritake Dental) were irradiated directly or through one of the ceramic plates for different periods (5, 10, 15, or 20 seconds for the high-intensity LED units and 20, 40, 60, or 80 seconds for the others). The Knoop hardness test was used to determine the level of photopolymerization that had been induced in the resin cement. Data were analyzed by one-way analysis of variance and Dunnett's post-hoc test to identify test-control (maximum irradiation without a ceramic plate) differences for each curing unit (p<0.05). RESULTS: For all curing units, the curing conditions had a statistically significant effect on the Knoop hardness numbers (KHNs) of the irradiated cement samples (p<0.001). In general, the KHN decreased with increasing plate thickness and increased as the irradiation period was extended. Jetlite 3000 achieved control-level KHN values only when the plate thickness was 1.0 mm. At a plate thickness ≥2.0 mm, the LED units (except for PenCure 2000 at 3.0 mm) were able to achieve control-level KHN values when the irradiation time was extended. At a plate thickness of 3.0 mm, irradiation for 20 seconds with the Valo or for 80 seconds with the Demi were the only methods that produced KHN values equivalent to those produced by direct irradiation. CONCLUSION: Regardless of the type of curing unit used, indirect irradiation of dual-cured resin cement through a ceramic plate resulted in decreased KHN values compared with direct irradiation. When the irradiation period was extended, only the LED units were able to achieve similar KHN values to those observed under direct irradiation in the presence of plates ≥2.0-mm thick. High-intensity LED units require a shorter irradiation period than halogen and second-generation LED curing units to obtain KHN values similar to those observed during direct irradiation.

Co-increase of nerve fibers and HLA-DR- and/or factor-XIIIa-expressing dendritic cells in dentinal caries-affected regions of the human dental pulp: an immunohistochemical study.

Neuro-immune interaction has been suggested to play some modulatory role in the immunodefense of the dentin/pulp complex. In this study, we performed a simultaneous immunohistochemical observation of neural elements and pulpal dendritic cells (PDCs) on human carious teeth, to obtain morphological evidence for neuro-immune interaction in response to dentinal tubule-derived carious stimuli. Human third molars bearing a pulp-exposure-free caries lesion were studied. Immunoperoxidase staining was performed with anti-HLA-DR, anti-coagulation factor XIIIa, and anti-CD14 as PDC markers, and anti-low-affinity nerve growth factor receptor (NGFR), anti-protein gene products 9.5, and anti-calcitonin gene-related peptide as nerve markers. The carious teeth usually exhibited localized accumulation of both PDCs and nerve fibers immunoreactive to each marker, in the para-odontoblastic region corresponding to the pulpal end of carious dentinal tubules. Semi-quantitative digital densitometry revealed that pixel numbers corresponding to factor-XIIIa- and NGFR-immunoreactivity were significantly higher in the carious regions than those in the non-carious regions of the same teeth as well as those in the corresponding regions of intact teeth. Classification of specimens with respect to caries depth showed that the co-increase was most apparent in teeth with superficial caries. The increase of PDCs was less pronounced in carious teeth with reparative dentin. These findings suggest that both pulpal nerves and PDCs respond promptly and actively to dentinal tubule-derived carious stimuli. The synchronized accumulation of the two structures suggests an increased opportunity for neuro-immune interaction that may be of significance in the modulation of pathological processes in the dental pulp.

The role of leukotriene B4 in neutrophil infiltration in experimentally-induced inflammation of rat tooth pulp.

Inflammation was induced in rat dental pulp by applying bacterial lipopolysaccharide (LPS). Extirpated tissue samples from inflamed pulps were incubated in vitro in a Krebs buffer containing Ca2+ ionophore A23187, and leukotriene (LT) B4 released into the medium was determined by radio-immunoassay. Production of LTB4 could be detected three to 24 h after the application of LPS and showed a maximum at 12 h. Histologically, marked infiltration of neutrophils, but not other leukocytes, was characteristically observed in the LPS-applied pulps, and the temporal change in neutrophil infiltration was almost parallel, but somewhat more delayed than LTB4 production. When BW755C, a dual inhibitor of cyclo-oxygenase and lipoxygenase, was given to the animals before the application of LPS, both the production of LTB4 and the number of infiltrated neutrophils were significantly decreased, whereas administration of indomethacin had no effect. These results suggest that LTB4 may be involved in neutrophil infiltration in pulpal inflammation. It was also suggested that a major early source of LTB4 in experimental pulpitis was leukocytes, primary neutrophils, because the synthesis of LTB4 in the inflammed pulp was diminished by depletion of circulating leukocytes with cyclophosphamide prior to the application of LPS.

An immunohistochemical study of the distribution of immunocompetent cells, especially macrophages and Ia antigen-expressing cells of heterogeneous populations, in normal rat molar pulp.

The precise distribution of various immunocompetent cells in rat molar pulp was immunohistochemically examined by use of seven anti-rat monoclonal antibodies. It was demonstrated that rat molar pulp contained many OX6 (anti-Ia antigen)-positive cells and a large number of ED1 (anti-monocytes, macrophages, and dendritic cells)-positive, ED2 (anti-tissue macrophages)-positive, and/or OX35 (anti-macrophages and CD4+ lymphocytes)-positive cells. Macrophage-like cells predominated in the central portion of the pulp, while cells of dendritic appearance usually existed in the periphery of the pulp. Double-immunoperoxidase staining revealed that these cells showed some heterogeneity, but the majority could be classified as ED1+/OX6-/ED2+ cells, which may be Ia-histiocytes. Findings also suggested that true dendritic cells may be included in the ED1+/OX6+/ED2- category of cells. A small number of T lymphocytes and plasma cells were also detected. These results suggest that the normal dental pulp contains a variety of immunocompetent cells, with macrophages as the most dominating. Following the exogenous invasion of pathogenic stimuli in the pulp, these cells may participate in the defense reaction by acting as phagocytes or antigen-presenting cells, which are essential for the initiation of immune responses.

Structural and functional association between substance P- and calcitonin gene-related peptide-immunoreactive nerves and accessory cells in the rat dental pulp.

Defense mechanisms of the dentin/pulp complex involve a variety of biological systems in which immunocompetent cells, the nervous system, and the vascular supply play important roles. In the present study, pulpal accessory cells were examined regarding (i) their structural relationship to nerves and (ii) how the functional capacities of these cells were affected by neuropeptides. Micro-anatomic association was investigated in the normal rat molar pulp with the use of double-immunofluorescence staining and dual-channel confocal laser scanning microscopy. Examinations of confocal laser scanning microscopic images from single focal planes revealed the presence of apparent contacts between thin, varicose nerve fibers and immunocompetent cells, indicating proximity between these two structures. The close associations were most frequently observed in the para-odontoblastic region of the coronal pulp, where more than 70% of class II antigen-expressing (OX6+) cells showed proximity to nerve fibers immunoreactive to calcitonin gene-related peptide. The corresponding figure for substance P was about 50%. ED2+ macrophages closely associated with nerves were less frequently observed. Functional studies conducted in vitro demonstrated that 10(-9) to 10(-7) mol/L of substance P significantly increased (p < 0.05), while 10(-7) to 10(-6) mol/L of calcitonin gene-related peptide suppressed (p < 0.01) proliferation of purified T-lymphocytes stimulated with sub-optimal concentrations of concanavalin A in the presence of rat incisor pulpal cells as accessory cells. These data suggest that pulpal sensory nerve fibers and their products may have an influence upon the immune defense of the dental pulp.

Kinetics of macrophages and lymphoid cells during the development of experimentally induced periapical lesions in rat molars: a quantitative immunohistochemical study.

Quantitative phenotypical analysis for macrophages and lymphoid cells was made on periapical lesions induced by making unsealed pulp exposures in rat molars. Exudative macrophages of diverse morphology were the most dominating immunocompetent cells during all experimental periods. They exhibited almost complete distribution through the periapical lesions by the onset of active lesion expansion, and their kinetics and morphology were considerably similar to Ia+ non-lymphoid cells. Lymphoid cells started to show a notable increase shortly before the onset of active lesion expansion. CD4+ lymphocytes dominated at first, whereas CD8+ lymphocytes and plasma cells were dominant when the lesion size was stabilized. These findings suggest that macrophages of heterogenous populations play essential roles in the lesion pathogenesis by acting, depending on their functional heterogeneity, as effector cells or as antigen-presenting cells that stimulate CD4+ lymphocytes to induce an immune response. Interactions between plasma cells and CD8+ lymphocytes may be related to lesion chronicity.