ABSTRACT
BACKGROUND/PURPOSE: Dentin bonding agents (DBAs) are cytotoxic to dental pulp cells. This study aimed to evaluate the effects of three DBAs (Optibond Solo Plus, Op; Clearfil SE Bond, SE; and Xeno III, Xe) after diffusion through 0.2-mm or 0.5-mm dentin slices on reactive oxygen species (ROS) production and apoptosis in dental pulp cells. METHODS: The amounts of DBAs diffusing through 0.2-mm or 0.5-mm dentin slices were quantified using a UV-Vis spectrophotometer. The effects of diffused DBAs on ROS production and viability of dental pulp cells were investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay on Days 1 and 2. Flow cytometric analysis and double staining of treated dental pulp cells with Annexin V-fluorescein isothiocyanate (V-FITC) and propidium iodide (PI) were performed on Day 2. RESULTS: Xe showed greatest diffusion through dentin slices after 8-hour period, followed by SE and Op. Dental pulp cells produced a lesser amount of ROS, when treated with DBAs diffusing through a 0.5-mm dentin slice than through a 0.2-mm dentin slice for the same period of time. A small proportion of cells were TUNEL-positive after treatment with any of the three diffused DBAs. Annexin V-FITC/PI staining identified apoptotic cells; cell survival was higher in those cells treated with DBAs diffusing through a 0.5-mm dentin slice than through a 0.2-mm dentin slice. CONCLUSION: The three DBAs after diffusion through 0.2- or 0.5-mm dentin slice still exhibit cytotoxicity to dental pulp cells. However, the 0.5-mm dentin slice is found to be a better barrier than the 0.2-mm dentin slice to protect dental pulp cells from DBA-induced cytotoxicity.
Subject(s)
Apoptosis/drug effects , Bisphenol A-Glycidyl Methacrylate/toxicity , Dental Pulp/pathology , Dentin-Bonding Agents/toxicity , Reactive Oxygen Species/metabolism , Resin Cements/toxicity , Adolescent , Adult , Dental Pulp/cytology , Dentin/chemistry , Humans , Taiwan , Young AdultABSTRACT
OBJECTIVE: To assess the effects of epigallocatechin-3-gallate (EGCG) on cytokine-induced Cyr61 synthesis in human osteoblastic cells and the associated signalling pathways. The therapeutic effect of EGCG on CIA in rats was also studied. METHODS: The expression of Cyr61 and NF-κB pathway molecules was examined by western blotting. CCL2 expression was assessed by northern blotting and ELISA. Interaction between NF-κB and Cyr61 promoter was evaluated by electrophoretic mobility shift assay. In rat CIA, osteoblastic expression of Cyr61 was examined by immunohistochemistry and disease progression was assessed by clinical, radiographic and histological examinations. RESULTS: EGCG inhibited Cyr61 expression stimulated by cytokines in primary human osteoblasts and human osteoblastic cell line U2OS. In U2OS, oncostatin M (OSM) induced IκB-α degradation through the mTOR/rictor/Akt pathway, and EGCG attenuated the action. Electrophoretic mobility shift assay revealed that the OSM-enhanced NF-κB/DNA binding was reduced by EGCG, possibly through abrogating nucleus localization of p65 and p50. Cyr61 enhanced OSM-induced expression of CCL2. Moreover, EGCG diminished OSM-stimulated CCL2 expression at least partially via suppressing Cyr61 induction. Co-distribution of CD68(+) macrophages and Cyr61(+) osteoblasts in osteolytic areas was obvious in the CIA model. Clinical, radiographic and immunohistochemical analyses revealed that administration of EGCG markedly diminished the severity of CIA, macrophage infiltration, and the number of Cyr61-synthesizing osteoblasts. CONCLUSION: By modulating the mTOR/rictor/Akt/NF-κB pathway, EGCG attenuated Cyr61 production in osteoblastic cells and in turn diminished macrophage chemotaxis. Our data support the therapeutic potential of EGCG on arthritis.
Subject(s)
Arthritis/therapy , Catechin/analogs & derivatives , Cysteine-Rich Protein 61/biosynthesis , Cytokines/pharmacology , Osteoblasts/metabolism , Adult , Animals , Arthritis/metabolism , Catechin/pharmacology , Cells, Cultured , Chemokine CCL2/metabolism , Chromones/pharmacology , Cysteine-Rich Protein 61/drug effects , Enzyme Inhibitors/pharmacology , Humans , Inositol Phosphates/pharmacology , Male , Morpholines/pharmacology , NF-kappa B/drug effects , NF-kappa B/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction , Sirolimus/pharmacology , TOR Serine-Threonine Kinases/antagonists & inhibitors , Young AdultABSTRACT
OBJECTIVE: To examine the effects of proinflammatory cytokines on Cyr61 expression in osteoblastic cells and the modulatory action of simvastatin, to assess the role of CREB in Cyr61 induction, and to investigate the relationship of osteoblastic expression of Cyr61 to disease progression in experimental arthritis. METHODS: Cyr61 expression and CREB phosphorylation at serine 133 were examined by Western blotting. Promoter activity of Cyr61 was assessed by luciferase assay with promoter deletion/mutagenesis and forced expression/gene silencing of CREB. Interaction between CREB and the Cyr61 promoter was evaluated by electrophoretic mobility shift assay and chromatin immunoprecipitation. CCL2 expression was examined by Northern blotting and enzyme-linked immunosorbent assay. In rats with collagen-induced arthritis (CIA), osteoblastic expression of Cyr61 was examined by immunohistochemistry, and disease progression was assessed by clinical, radiographic, and histologic examination. RESULTS: In primary human osteoblasts and U2OS cells, Cyr61 expression stimulated by tumor necrosis factor α, interleukin-1ß (IL-1ß), oncostatin M (OSM), and other IL-6-family cytokines was suppressed by simvastatin. In U2OS cells, simvastatin inhibited OSM-induced CREB phosphorylation and CREB-DNA binding. Knockdown of CREB by short hairpin RNA reduced Cyr61 synthesis. OSM-induced Cyr61 promoter activation was dependent on CRE-CREB interaction and inhibited by simvastatin. Cyr61 enhanced CCL2 expression by U2OS cells. Intraarticular injection of simvastatin inhibited CIA progression and diminished the number of Cyr61+ osteoblasts and infiltrating macrophages. CONCLUSION: Simvastatin inhibited cytokine-stimulated Cyr61 expression in osteoblastic cells and suppressed disease progression and osteoblastic expression of Cyr61 in inflammatory arthritis. This finding indicates that simvastatin may have potential as a therapeutic agent for inflammatory arthritis.
Subject(s)
Arthritis, Experimental/drug therapy , Cysteine-Rich Protein 61/metabolism , Cytokines/pharmacology , Osteoblasts/drug effects , Osteoblasts/metabolism , Simvastatin/pharmacology , Simvastatin/therapeutic use , Animals , Anticholesteremic Agents/administration & dosage , Anticholesteremic Agents/pharmacology , Anticholesteremic Agents/therapeutic use , Cell Line, Tumor , Cells, Cultured , Chemokine CCL2/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Disease Models, Animal , Disease Progression , Humans , Injections, Intra-Articular , Male , Rats , Rats, Sprague-Dawley , Simvastatin/administration & dosage , Treatment OutcomeABSTRACT
AIM: To explore the relationship between general joint hypermobility (GJH) and displacement of the temporomandibular joint (TMJ) disc as evident from magnetic resonance imaging (MRI). METHODS: Fifth finger extension, thumb apposition, elbow extension, knee extension, trunk flexion, and ankle dorsiflexion were measured in 66 young female patients with MRI-evident TMJ internal derangement (ID) and in 30 age-matched female controls. The Beighton score of each subject was measured quantitatively. The possible association between TMJ ID and mobility of a single joint or index of GJH, ie, the Beighton score, were assessed with one-way ANOVA with post-hoc Bonferroni and chi-square test, respectively. Correlations of the mobility of every measured joint were also explored. RESULTS: Very few of the TMJ ID patients and control subjects were diagnosed with GJH according to the Beighton score. The Beighton score did not differentiate between subjects with and without TMJ ID. Subjects with TMJ ID, especially patients with MRI-evident disc displacement without reduction, seemed to have a stiffer trunk than controls, but this may not be of clinical relevance. The mobilities of paired joints were significantly correlated; however, the mobilities of different anatomical joints seemed to be independent. CONCLUSION: Based on the Beighton score, GJH does not seem to be a reliable indicator of the presence of TMJ ID.
Subject(s)
Joint Dislocations/complications , Joint Instability/complications , Temporomandibular Joint Disc/physiopathology , Temporomandibular Joint Disorders/complications , Adolescent , Adult , Ankle Joint/physiopathology , Arthrometry, Articular , Case-Control Studies , Elbow Joint/physiopathology , Female , Finger Joint/physiopathology , Humans , Knee Joint/physiopathology , Magnetic Resonance Imaging , Mandibular Condyle/physiopathology , Range of Motion, Articular/physiology , Spine/physiopathology , Thumb/physiopathology , Young AdultABSTRACT
Sanguinarine is a plant alkaloid present in the root of Sanguinaria canadensis and Poppy fumaria species. Sanguinarine has been used as an antiseptic mouth rinse and a toothpaste additive to reduce dental plaque and gingival inflammation. In this study, we investigated the antiplatelet effects of sanguinarine, aiming to extend its potential pharmacological applications. Sanguinarine inhibited platelet aggregation induced by arachidonic acid (AA), collagen, U46619 and sub-threshold concentration of thrombin (0.05 U/ml) with IC(50) concentrations of 8.3, 7.7, 8.6 and 4.4 microM, respectively. Sanguinarine (5-10 microM) inhibited 10-31% of platelet TXB(2) production, but not platelet aggregation induced by higher concentration of thrombin (0.1 U/ml). SQ29548, a thromboxane receptor antagonist, inhibited the AA-induced platelet aggregation but not TXB(2) production. Sanguinarine suppressed cyclooxygenase-1 (COX-1) activity (IC(50)=28 microM), whereas its effect on COX-2 activity was minimal. Sanguinarine (8, 10 microM) further inhibited the AA-induced Ca(2+) mobilization by 27-62%. In addition, SQ22536, an adenylate cyclase inhibitor, attenuated the inhibitory effect of sanguinarine toward AA-induced platelet Ca(2+) mobilization and aggregation. These results suggest that sanguinarine is a potent antiplatelet agent, which activates adenylate cyclase, inhibits platelet Ca(2+) mobilization, TXB(2) production as well as suppresses COX-1 enzyme activity. Sanguinarine may have therapeutic potential for treatment of cardiovascular diseases related to platelet aggregation.
Subject(s)
Alkaloids/pharmacology , Benzophenanthridines/pharmacology , Blood Platelets/drug effects , Calcium/metabolism , Cyclic AMP/metabolism , Cyclooxygenase Inhibitors/pharmacology , Isoquinolines/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , Thromboxane B2/metabolism , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Adenylyl Cyclases/metabolism , Animals , Arachidonic Acid/pharmacology , Blood Platelets/metabolism , Collagen/pharmacology , Cyclooxygenase 1/metabolism , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , In Vitro Techniques , Rabbits , Receptors, Thromboxane/drug effects , Receptors, Thromboxane/metabolism , Thrombin/pharmacologyABSTRACT
AIMS: To analyze the bone mineral density (BMD) in a group of young female patients with a disc displacement in at least 1 temporomandibular joint (TMJ) as well as in a group of age-matched young females with a normal condyle-disc relationship. METHODS: Fifty-six young female patients with anterior disc displacement based on magnetic resonance imaging (MRI) and 40 age- and gender-matched controls with asymptomatic TMJs were recruited for this study. Subjects between 18 and 30 years were recruited. Based on the MRI findings, 10 of the 40 subjects in the control group also had anterior disc displacement. In all, 16 subjects had an anterior disc displacement with reduction (DDwR), 50 had an anterior disc displacement without reduction (DDw/oR), and 30 had a normal condyle-disc relationship. BMD was measured in the lumbar area by means of dual-energy x-ray absorptiometry. The relationship between the 3 types of condyle-disc relationship and BMD was then analyzed. RESULTS: Patients with a DDw/oR had a significantly lower mean BMD value in the lumbar area than the subjects with a normal condyle-disc relationship (P < .05, analysis of variance, post-hoc with Bonferroni test). Twenty-two (44%) of 50 patients with DDw/oR had osteopenia. CONCLUSION: Low BMD is often associated with DDw/oR in young Taiwanese female patients.
Subject(s)
Bone Density , Temporomandibular Joint Disorders/physiopathology , Absorptiometry, Photon , Adolescent , Adult , Analysis of Variance , Case-Control Studies , Female , Humans , Joint Dislocations/physiopathology , Lumbar Vertebrae/physiopathology , Magnetic Resonance ImagingABSTRACT
INTRODUCTION: Recently, we have shown that tissue hypoxia stimulates the progression of periapical lesions by up-regulating glycolysis-dependent apoptosis of osteoblasts. Other facets of hypoxia-induced metabolic reprogramming in disease pathogenesis require further investigation. In this study, we examined the connection between hypoxia-augmented glutamine catabolism in osteoblasts and the development of periapical lesions. METHODS: Primary human osteoblasts were cultured under hypoxia. The expression of glutaminase 1 (GLS1) was examined using Western blot analysis. The production of glutamate was measured by colorimetric assay. Knockdown of GLS1 was performed with small interfering RNA technology. C-C motif chemokine ligand 2 (CCL2) secretion and chemotaxis of J774 macrophages were examined by enzyme-linked immunosorbent assay and transwell migration assay, respectively. In a rat model of induced periapical lesions, the relations between disease progression and osteoblastic expression of GLS1 or macrophage recruitment were studied. RESULTS: Hypoxia enhanced GLS1 expression and subsequent glutamate production in osteoblasts. Glutamate induced chemoattraction of macrophages by osteoblasts through up-regulation of CCL2 synthesis. Hypoxia promoted CCL2 secretion and macrophage recruitment through augmentation of glutaminolysis. Knockdown of GLS1 abolished hypoxia-induced effects. In rat periapical lesions, progressive bone resorption was significantly related to elevated GLS1 expression in osteoblasts and increased macrophage recruitment. CONCLUSIONS: In addition to the rise in glycolytic activity, the progression of periapical lesions is also associated with enhanced glutamine catabolism in osteoblasts. GLS1 may be a potential therapeutic target in the management of periapical lesions.
Subject(s)
Glutaminase/metabolism , Macrophages/physiology , Osteoblasts/enzymology , Periapical Periodontitis/pathology , Animals , Blotting, Western , Cells, Cultured , Disease Progression , Glutaminase/physiology , Glutamine/metabolism , Humans , Osteoblasts/physiology , Rats , Rats, Sprague-DawleyABSTRACT
1.--Thrombin is activated during gingival tissue injury and inflammation. Thrombin (platelet)-rich plasma has been used for periodontal regeneration with success. Thrombin and other bacterial proteases also affect the functions of adjacent periodontal cells via stimulation of protease-activated receptors (PARs). 2.--We noted that thrombin (0.1-2 U ml(-1)), human, and frog PAR-1 agonist peptide (20-240 microM) induced the gingival fibroblast (GF)-populated collagen gel contraction within 2 h of exposure. However, PAR-2, PAR-3, and PAR-4 agonist peptide (20-240 microM) showed little effect on collagen gel contraction. U73122 (phospholipase C inhibitor) and 2-APB (IP3 antagonist) were effective in inhibition of GF contraction. 3.--Thrombin-induced GF contraction was inhibited by 5 mM EGTA (an extracellular calcium chelator) and verapamil (an L-type calcium channel blocker). In addition, W7 (10 and 25 microM, a calcium/calmodulin (CaM) inhibitor), ML-7 (50 microM, myosin light chain kinase (MLCK) inhibitor), and HA1077 (100 microM, Rho kinase inhibitor) completely inhibited the thrombin-induced collagen gel contraction. Thrombin also induced the phosphorylation of ERK1/ERK2 and elevated the Rho-GTP levels in GF. 4.--However, U0126 only partially inhibited the thrombin-induced GF contraction. Similarly, wortmannin (100 nM), LY294002 (20 microM) (two PI3K inhibitor) and genistein also showed partial inhibition. Moreover, NAC was not able to suppress the GF contraction, as supported by the slight decrease in reactive oxygen species production in GF by thrombin. 5.--Thrombin also stimulated metalloproteinase-2 (MMP-2) and MMP-3 production in GF. But addition of GM6001 or 1,10-phenanthroline, two MMP inhibitors, could not inhibit the thrombin-induced GF contraction. 6.--These results indicate that thrombin is crucial in the periodontal inflammation and wound healing by promoting GF contraction. This event is mainly mediated via PAR-1 activation, PLC activation, extracellular calcium influx via L-type calcium channel, and the calcium/CaM-MLCK and Rho kinase activation pathway.
Subject(s)
Collagen/physiology , Fibroblasts/physiology , Gingiva/cytology , Thrombin/physiology , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cell Size , Cells, Cultured , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , Gels , Humans , Metalloproteases/biosynthesis , Myosin-Light-Chain Kinase/antagonists & inhibitors , Oxidation-Reduction , Peptides/pharmacology , Reactive Oxygen Species/metabolism , Receptors, Proteinase-Activated/agonists , Signal Transduction , Type C Phospholipases/physiology , rho GTP-Binding Proteins/metabolismABSTRACT
PURPOSE: This study investigated the effect of implant design and bone quality on insertion torque (IT), implant stability quotient (ISQ), and insertion energy (IE) by monitoring the continuous change in IT and ISQ while implants were inserted in artificial bone blocks that simulate bone of poor or poor-to-medium quality. MATERIALS AND METHODS: Polyurethane foam blocks (Sawbones) of 0.16 g/cm³ and 0.32 g/cm³ were respectively used to simulate low density and low- to medium-density cancellous bone. In addition, some test blocks were laminated with a 1-mm 0.80 g/cm³ polyurethane layer to simulate cancellous bone with a thin cortical layer. Four different implants (Nobel Biocare Mk III-3.75, Mk III-4.0, Mk IV-4.0, and NobelActive-4.3) were placed into the different test blocks in accordance with the manufacturer's instructions. The IT and ISQ were recorded at every 0.5-mm of inserted length during implant insertion, and IE was calculated from the torque curve. The peak IT (PIT), final IT (FIT), IE, and final ISQ values were statistically analyzed. RESULTS: All implants showed increasing ISQ values when the implant was inserted more deeply. In contrast to the ISQ, implants with different designs showed dissimilar IT curve patterns during the insertion. All implants showed a significant increase in the PIT, FIT, IE, and ISQ when the test-block density increased or when the 1-mm laminated layer was present. Tapered implants showed FIT or PIT values of more than 40 Ncm for all of the laminated test blocks and for the nonlaminated test blocks of low to medium density. Parallel-wall implants did not exhibit PIT or FIT values of more than 40 Ncm for all of the test blocks. NobelActive-4.3 showed a significantly higher FIT, but a significantly lower IE, than Mk IV-4.0. CONCLUSIONS: While the existence of cortical bone or implant designs significantly affects the dynamic IT profiles during implant insertion, it does not affect the ISQ to a similar extent. Certain implant designs are more suitable than others if high IT is required in bone of poor quality. The manner in which IT, IE, and ISQ represent the implant primary stability requires further study.
Subject(s)
Bone Density/physiology , Dental Implantation, Endosseous/methods , Dental Implants , Dental Prosthesis Design , Biomechanical Phenomena , Humans , Materials Testing , Models, Anatomic , Polyurethanes/chemistry , Stress, Mechanical , Torque , VibrationABSTRACT
INTRODUCTION: Osteoblast apoptosis is important in the regulation of inflammatory bone resorption. Hypoxia resulting from inflammation enhances glycolysis and apoptosis. Sirtuin 6 (SIRT6) is a modulator of glucose metabolism and apoptosis. In the study we assessed the role of SIRT6 in hypoxia-induced glycolysis and apoptosis in osteoblasts, with special attention on the significance of these cellular processes in periapical lesions. METHODS: Human bone marrow-derived osteoblasts were cultured under hypoxia. Expression of lactate dehydrogenase A was examined by Western blot, and production of lactate was measured by colorimetric assay. Cleavage of poly (adenosine diphosphate ribose) polymerase was used as an apoptosis marker and assessed by Western blot. SIRT6 was overexpressed in osteoblasts by lentiviral gene transduction, and then glycolytic and apoptotic responses were studied. In a rat model of bacteria-induced periapical lesions, expressions of SIRT6 and markers of glycolysis and apoptosis in osteoblasts were examined. RESULTS: Hypoxia enhanced lactate dehydrogenase A expression and lactate production in osteoblasts. Poly (adenosine diphosphate ribose) polymerase cleavage was induced by hypoxia or lactate treatment. SIRT6 suppressed hypoxia-augmented glycolysis and inhibited apoptosis induced by hypoxia or lactate treatment. Expression of SIRT6 in osteoblasts was downregulated by hypoxia and inflammatory mediators. Development of periapical lesions in rats was associated with decreased expression of SIRT6 and increased glycolysis and apoptosis in osteoblasts. CONCLUSIONS: Our study suggested that hypoxia-induced apoptosis of osteoblasts is dependent on glycolytic activity. SIRT6 is a negative regulator of inflammation and may alleviate periapical lesions by suppressing osteoblastic glycolysis and apoptosis.
Subject(s)
Apoptosis , Glycolysis , Hypoxia/pathology , Osteoblasts/pathology , Periapical Periodontitis/metabolism , Periapical Periodontitis/pathology , Sirtuins/metabolism , Adult , Animals , Cells, Cultured , Humans , Isoenzymes/metabolism , L-Lactate Dehydrogenase/metabolism , Lactate Dehydrogenase 5 , Models, Animal , Poly(ADP-ribose) Polymerases/metabolism , Rats, Sprague-Dawley , Young AdultABSTRACT
BACKGROUND: Inflammation is believed to be related to the pathogenesis of nasal polyp (NP). Inducible cyclooxygenase (COX-2) and interleukin (IL) 6 are important mediators of inflammation. However, no information is available regarding the expression of these mediators in nasal polyp fibroblasts (NPFs). The inductive effects of proinflammatory cytokines (IL-1alpha or tumor necrosis factor alpha) alone or in combination with prostaglandin E(2) on IL-6 and COX-2 messenger RNA (mRNA) synthesis in NPFs were investigated. DESIGN: The expressions of IL-6 and COX-2 mRNAs in NPFs and in 34 surgical specimens of NP were detected by Northern blot and in situ hybridization. RESULTS: Significant amounts of constitutive IL-6 and COX-2 mRNAs were produced in NPFs. Cytokines induced IL-6 and COX-2 mRNA synthesis in NPFs. Meloxicam (a specific COX-2 inhibitor) suppressed the induction of cytokines on IL-6 mRNA levels, and these effects could be reversed by exogenous prostaglandin E(2). In situ hybridization revealed that IL-6 and COX-2 mRNAs were detected primarily in fibroblasts, macrophages, and plasma cells. Aggregation of plasma cells as well as collagen deposition in vicinity to IL-6 mRNA-producing fibroblasts was found. Rich vascularity around COX-2 mRNA(+) fibroblasts was also identified. CONCLUSIONS: The pathogenesis of nasal polyposis involves NPFs through synthesizing IL-6 to modulate the activation of immune responses (plasma cell formation) and synthesis of stroma. Inducible cyclooxygenase also contributes to NP development by promoting vasodilatation and modulating the cytokine-induced IL-6 gene expression in NPFs.
Subject(s)
Fibroblasts/pathology , Gene Expression/genetics , Interleukin-6/genetics , Isoenzymes/genetics , Nasal Polyps/genetics , Nasal Polyps/pathology , Prostaglandin-Endoperoxide Synthases/genetics , RNA, Messenger/genetics , Antineoplastic Agents/pharmacology , Cyclooxygenase 2 , Dinoprostone/pharmacology , Fibroblasts/drug effects , Gene Expression/drug effects , Humans , In Vitro Techniques , Interleukin-1/pharmacology , Isoenzymes/drug effects , Membrane Proteins , Oxytocics/pharmacology , Prostaglandin-Endoperoxide Synthases/drug effects , RNA, Messenger/drug effects , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Increased levels of interleukin-1 (IL)-1, tumor necrosis factor-alpha (TNF-alpha), IL-6, and prostaglandin E2 (PGE2) have been detected in inflamed pulp tissue. To gain further insight into the molecular pathogenesis of pulpitis, we investigated the effects of IL-1alpha or TNF-alpha and PGE2, either alone or in combination on IL-6 and cyclooxygenase (COX)-2 messenger RNA (mRNA) production in cultured human dental pulp (HDP) fibroblasts. Exposure of HDP fibroblasts to IL-1alpha or TNF-alpha resulted in elevated levels of IL-6 (approximately 3.4 to approximately 10.4-fold) and COX-2 (approximately 5 to approximately 6.2-fold) mRNA. Simultaneous addition of IL-1alpha and PGE2 or TNF-alpha and PGE2 to the cultures significantly reduced the cytokine-induced IL-6 mRNA synthesis ranging from 45% to 65%. However, indomethacin enhanced the cytokine-stimulated IL-6 mRNA synthesis by approximately 1.7 to approximately 3.4-fold. This action could be reversed by exogenous PGE2. In contrast, PGE2 or indomethacin failed to modify the stimulatory effect of IL-1alpha or TNF-alpha on COX-2 gene expression. Because excessive levels of IL-6 and prostaglandins have been connected with the pathogenesis of several inflammatory diseases, our results suggest the involvement of HDP fibroblasts in the development of pulpitis via producing IL-6 and COX-2. Furthermore, expression of IL-6 and COX-2 genes in this cell seems to be differentially regulated by cytokines through prostaglandin-dependent and -independent pathways.
Subject(s)
Cytokines/pharmacology , Dental Pulp/metabolism , Interleukin-6/biosynthesis , Isoenzymes/biosynthesis , Prostaglandin-Endoperoxide Synthases/biosynthesis , Pulpitis/metabolism , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Blotting, Northern , Cells, Cultured , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/pharmacology , Dental Pulp/cytology , Dinoprostone/pharmacology , Gene Expression Regulation/drug effects , Humans , Indomethacin/pharmacology , Interleukin-1/pharmacology , Membrane Proteins , RNA, Messenger/biosynthesis , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
Various root-end filling materials have been used to prevent the entry of root-canal pathogens into periapical regions. Five root-end filling materials were compared regarding the cytotoxicity, apoptosis, and mitochondrial dehydrogenase (MDH) activities of human periodontal ligament (PDL) fibroblasts, with the use of a novel transwell culture system. Exposure to IRM (a ZnO eugenol-based intermediate restorative material), a 2-ethoxybenzoic acid cement (Super EBA), and amalgam for 3 days inhibited the MDH activity of PDL fibroblasts as indicated by decrease in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction by 97%, 95%, and 51%, respectively. Evident suppression of MTT reduction by amalgam and glass ionomer cement (GIC) was noted after 5 days of exposure, with 73% and 46% of inhibition, respectively. Mineral trioxide aggregates (MTA) showed little effect on MDH activity. IRM and Super EBA were cytotoxic to PDL fibroblasts as indicated by a trypan blue dye exclusion technique. GIC and amalgam showed mild cytotoxicity. IRM, GIC, and amalgam further induced apoptosis of PDL cells, as revealed by the presence of sub-G0/G1 DNA content in flow cytometric histogram. Twenty-four-hour exposure to IRM and Super EBA elevated the MDH activities to 156% and 117%, correspondingly, of that of control. Eugenol, a phenolic ingredient in Super EBA and IRM, also increases MDH activity of PDL fibroblasts by 45% and 51%, at concentrations of 0.5 and 1 mM. However, at concentrations higher than 0.5 mM, eugenol decreased the number of viable PDL fibroblasts. These results suggest that MTA is a biocompatible root-end filling material, followed by self-curing Fuji II GIC and amalgam. IRM and Super EBA ingredients induced marked cytotoxicity and transiently stimulate MDH activities, which is possibly due to their content of eugenol and induction of cellular adaptive response.
Subject(s)
Eugenol/pharmacology , Fibroblasts/drug effects , Mitochondria/enzymology , Oxidoreductases/metabolism , Periodontal Ligament/cytology , Root Canal Filling Materials/pharmacology , Alkaline Phosphatase/metabolism , Cell Survival/drug effects , DNA/analysis , DNA/biosynthesis , Flow Cytometry , Gingiva/cytology , Humans , Mitochondria/drug effects , Periodontal Ligament/drug effects , Tetrazolium Salts , ThiazolesABSTRACT
"TiGlass" was designed and was known to promote initial adhesion and increase migration of rat calvarial osteoblats. In this article, migration study and a series of epifluorescence microscopic studies were conducted to find out the composition of focal adhesion on titanium surface. The translucent titanium surface was applied in random migration analysis and immunofluorescence cell staining. In the immunofluorescent double staining, phosphorylated focal adhesion kinase was tested with vinculin. Various integrin subunits were then tested with vinculin to study the composition of activated focal adhesions. Integrin subunit α5 and αV were tested against ß3; integrin subunits α5, αV, ß3, and αVß3 were tested with F-actin, respectively. The MG-63 cells began migration earlier and migrated faster on "TiGlass." Immunofluorescent double staining revealed that all focal adhesion kinase in the focal adhesions were activated on both the surfaces. The osteoblast was inferred to made adhesion to titanium and glass through integrins. The focal adhesions on glass were found to be composed of integrin subunits αV and ß3. However, on "TiGlass," integrin subunits α5 might have supplemented the adhesion to titanium. Results from double staining of integrin subunits α5, αV, ß3, and αVß3 with F-actin also supported integrin subunits α5 might have involved in adhesion of titanium.
Subject(s)
Cell Movement/drug effects , Coated Materials, Biocompatible/pharmacology , Focal Adhesions/metabolism , Glass/chemistry , Osteoblasts/cytology , Osteoblasts/metabolism , Titanium/pharmacology , Actins/metabolism , Animals , Cell Line , Fluorescent Antibody Technique , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Focal Adhesions/drug effects , Humans , Integrins/metabolism , Mutant Proteins/metabolism , Osteoblasts/drug effects , Phosphorylation/drug effects , Protein Subunits/metabolism , Rats , Staining and Labeling , Vinculin/metabolismABSTRACT
INTRODUCTION: In this study, the role of transcription factor Forkhead/winged helix box protein O3a (FoxO3a) in Cyr61 expression and its modulation by simvastatin were investigated in cultured murine osteoblasts and a rat model of induced apical periodontitis. We also examined the effects of simvastatin on the synthesis of chemokine CCL2 and chemotaxis of macrophages in vitro. METHODS: We assessed tumor necrosis factor (TNF)-α-stimulated expression of Cyr61 and phosphorylated inactive FoxO3a (p-FoxO3a) in MC3T3-E1 murine osteoblasts by Western analysis. Forced expression of FoxO3a by lentiviral-based gene transduction was performed, and its effect on Cyr61 expression was evaluated. The modulation of CCL2 secretion and macrophage chemotaxis by simvastatin were examined by enzyme-linked immunosorbent assay and transwell migration assay, respectively. In a rat model of induced apical periodontitis, the relation between disease progression and osteoblastic expression of Cyr61, p-FoxO3a, and CCL2 and macrophage recruitment were studied by radiographic and immunohistochemistry analyses. RESULTS: Western blot analysis showed enhanced expression of Cyr61 and p-FoxO3a after TNF-α treatment in a time-dependent manner. Simvastatin significantly counteracted the actions of TNF-α. Forced expression of FoxO3a reduced TNF-α-stimulated Cyr61 synthesis. Simvastatin and FoxO3a diminished TNF-α-induced CCL2 secretion and macrophage recruitment, whereas Cyr61 partially restored the stimulating action. In rat periapical lesions, simvastatin significantly attenuated bone resorption, reduced osteoblastic expressions of Cyr61, p-FoxO3a, and CCL2, and suppressed macrophage recruitment. CONCLUSIONS: Simvastatin may alleviate periapical lesions by enhancing FoxO3a activity to suppress the synthesis of Cyr61 in osteoblasts. Moreover, the downstream effector mechanism of Cyr61 may involve CCL2 production and macrophage recruitment.
Subject(s)
Cysteine-Rich Protein 61/antagonists & inhibitors , Forkhead Transcription Factors/drug effects , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Osteoblasts/drug effects , Periapical Periodontitis/physiopathology , Simvastatin/pharmacology , 3T3 Cells , Alveolar Bone Loss/pathology , Alveolar Bone Loss/physiopathology , Animals , Cell Line , Chemokine CCL2/drug effects , Chemotaxis/drug effects , Disease Models, Animal , Disease Progression , Forkhead Box Protein O3 , Macrophages/drug effects , Macrophages/pathology , Mice , Osteoblasts/pathology , Periapical Periodontitis/pathology , Radiography, Dental, Digital , Rats , Rats, Sprague-Dawley , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
PURPOSE: The appropriate surface composition and topography are crucial for osseointegration of titanium dental implants, and surface properties are known to enhance cell adhesion and promote expression of specific osteoblastic genes. In this study, a translucent titanium coating on glass coverslip (TiGlass) was introduced as a potential tool for direct observation of cell behavior on a titanium surface. MATERIALS AND METHODS: Scanning electron microscopy, energy-dispersive x-ray analysis, and atomic force microscopy were performed on TiGlass to provide information about its physical properties. Random migration, osteoblastic gene expression, and immunofluorescence cell staining on TiGlass were also examined and analyzed. RESULTS: The translucent titanium surface offered excellent optical characteristics that facilitated transmitted light observations under an optical microscope, transforming the opaque metal into an observable titanium matrix. Random migration analysis of the primary osteoblasts on TiGlass revealed that the titanium coating enhanced the migration speed of the osteoblasts and significantly shortened the time lag for the initial migration behavior. Further study of osteoblastic gene expression on this smooth titanium surface revealed no significant changes. Co-localization of actin filament and vinculin was found on TiGlass under epifluorescent microscopy. CONCLUSION: The application of a translucent titanium-coated coverslip in vitro altered the migration pattern of osteoblasts. Collectively, the results suggest that titanium promotes initial adhesion and accelerates osteoblast migration.
Subject(s)
Coated Materials, Biocompatible/chemistry , Dental Materials/chemistry , Glass/chemistry , Osteoblasts/physiology , Titanium/chemistry , Actins/analysis , Alkaline Phosphatase/analysis , Animals , Cell Adhesion/physiology , Cell Culture Techniques , Cell Movement/physiology , Collagen Type I/analysis , Core Binding Factor Alpha 1 Subunit/analysis , Fluoresceins , Fluorescent Antibody Technique , Fluorescent Dyes , Integrin-Binding Sialoprotein/analysis , Materials Testing , Microscopy, Atomic Force , Microscopy, Electron, Scanning , Osteopontin/analysis , Phalloidine/analogs & derivatives , Rats , Rats, Wistar , Spectrometry, X-Ray Emission , Surface Properties , Vinculin/analysisABSTRACT
Chemokines are a large group of proteins implicated in migration, activation, and differentiation of leukocytes. They are well-surveyed in mammals, but less is known in lower vertebrates about their spatiotemporal expressions and functions. From an evolutionary point of view, comparative analyses may provide some fundamental insights into these molecules. In mammals, CCL21 and CCL25 are crucial for thymocyte homing. Herein, we identified and cloned the zebrafish orthologues of CCL21 and CCL25, and analyzed their expression in embryos and adult fish by in situ hybridization. We found that CCL21 was expressed in the craniofacial region, pharynx, and blood vessels in embryos. In adult fish, CCL21 transcripts were located in the kidney, spinal cord, and blood cells. In contrast, expression of CCL25 was only detected in the thymus primordia in embryos. In adult fish, transcripts of CCL25 were maintained in the thymus, and they were also found in the brain and oocytes. Furthermore, we performed an antisense oligonucleotide experiment to evaluate the biological function of CCL25. Results showed that the recruitment of thymocytes was impeded by morpholino-mediated knockdown of CCL25, suggesting that CCL25 is essential for colonization of T-cells in the thymus in early development. Together, our results demonstrate the basic profiles of two CCL chemokines in zebrafish. The tissue-specific expression patterns may pave the way for further genetic dissection in this model organism.
Subject(s)
Chemokine CCL21/genetics , Chemokines, CC/genetics , Transcriptome , Zebrafish Proteins/genetics , Zebrafish/genetics , Amino Acid Sequence , Animals , Chemokine CCL21/chemistry , Chemokine CCL21/immunology , Chemokine CCL21/metabolism , Chemokines, CC/chemistry , Chemokines, CC/immunology , Chemokines, CC/metabolism , Cloning, Molecular , Embryo, Nonmammalian/metabolism , Models, Molecular , Molecular Sequence Data , Oocytes/metabolism , Phylogeny , Sequence Alignment , Thymus Gland/embryology , Thymus Gland/metabolism , Zebrafish/embryology , Zebrafish/immunology , Zebrafish/metabolism , Zebrafish Proteins/chemistry , Zebrafish Proteins/immunology , Zebrafish Proteins/metabolismABSTRACT
INTRODUCTION: Osteoblastic expression of cysteine-rich 61 (Cyr61) correlates with the severity of periapical lesion-associated bone loss, but the regulatory mechanism of Cyr61 expression was not known. METHODS: In the study we examined the effect of major histocompatibility complex class II transactivator (CIITA) on tumor necrosis factor (TNF)-alpha-induced Cyr61 synthesis in U2OS human osteoblastic cells by Western blot analysis. In a rat model of bacteria-induced apical periodontitis, we assessed the relation between osteoblastic expressions of CIITA/Cyr61 and disease progression by radiographic and immunohistochemistry studies. RESULTS: We found that forced expression of CIITA suppressed Cyr61 synthesis in U2OS cells. In rat periapical lesions, osteoblastic CIITA decreased as the disease progressed, and expression of CIITA is negatively related to Cyr61 synthesis in osteoblasts. CONCLUSIONS: Our data showed that CIITA is a repressor of Cyr61 synthesis in osteoblasts, and it might play a protective role in the pathogenesis of bone resorption in apical periodontitis, possibly through down-regulating the expression of Cyr61 in osteoblasts.
Subject(s)
Cysteine-Rich Protein 61/antagonists & inhibitors , Nuclear Proteins/pharmacology , Osteoblasts/drug effects , Periapical Periodontitis/physiopathology , Trans-Activators/pharmacology , Alveolar Bone Loss/pathology , Alveolar Bone Loss/physiopathology , Animals , Blotting, Western , Cell Line, Tumor , Cysteine-Rich Protein 61/drug effects , Disease Models, Animal , Disease Progression , Electrophoresis, Polyacrylamide Gel , Genes, MHC Class II , Humans , Image Processing, Computer-Assisted , Osteoclasts/pathology , Periapical Periodontitis/microbiology , Radiography, Dental, Digital , Random Allocation , Rats , Rats, Sprague-Dawley , Time Factors , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
OBJECTIVES: Acquired anterior open bites were reported as the consequence of condylar collapse, which was associated with inflammatory TMJ disorders. However, we have seen such malocclusion patients whose condylar changes seemed to be related to TMJ degeneration associated with internal derangement. The aims of this study were to review the clinical history and to study the TMJ MRI of these patients. STUDY DESIGN: TMJ MRIs of patients, who had presented acquired anterior open bite at first visit, were retrieved from the image database for the analysis. Clinical histories focused on internal derangement were collected retrospectively. The soft tissue and hard tissue changes disclosed by MRI were also studied. RESULTS: All patients had experienced common signs/symptoms of TMJ internal derangement. All affected TMJs had anteriorly displaced disks and degenerative changes. Horizontally destructed condylar forms were seen significantly more frequently in these patients. CONCLUSION: TMJ degeneration associated with displaced disks might be a cause leading to the development of acquired anterior open bite.
Subject(s)
Open Bite/etiology , Osteoarthritis/complications , Temporomandibular Joint Disorders/complications , Temporomandibular Joint/pathology , Adolescent , Adult , Female , Humans , Joint Dislocations/complications , Joint Dislocations/pathology , Magnetic Resonance Imaging , Occlusal Splints , Open Bite/pathology , Osteoarthritis/pathology , Retrospective Studies , Temporomandibular Joint Disorders/pathologyABSTRACT
STATEMENT OF PROBLEM: Because of an imagining principle called active triangulation in the Cerec system, a shadow is cast distal to the illuminated objects. This distal shadow may be enlarged when the occlusal-cervical height of the prepared tooth is increased. Depth data of the shadow are unreliable, so the internal fit of Cerec crowns has been questioned. PURPOSE: This study evaluated the influence of different convergence angles and tooth preparation heights on the internal adaptation of Cerec crowns. MATERIAL AND METHODS: Tooth preparations were made on typodont teeth with different combinations of convergence angles and occlusal-cervical heights: Group I = 20 degrees angle, 6 mm height; Group II = 20 degrees angle, 4 mm height; Group III = 12 degrees angle, 6 mm height; and Group IV = 12 degrees angle, 4 mm height. Ten Cerec crowns were fabricated for each type of tooth preparation. Measurements of the internal fit were performed with the cement space replica technique and an image analysis system. Three-way analysis of variance was used to analyze the differences in cement space with different tooth preparations and the number of times that milling tools were used to prepare the Cerec crowns (P<.05). Multiple comparisons were made to evaluate differences between groups (P<.0083). RESULTS: Cerec crowns with a 12 degrees convergence angle demonstrated the best internal fit (cement space in Groups III and IV = 121 +/- 41 microm and 115 +/- 42 microm, respectively). The difference between the 2 convergence types was within the range of the scanning error (25 microm) produced by the Cerec camera. The number of times that milling tools were used had no significant effect on internal fit (P=.78). Tooth preparation height equal to or shorter than 6 mm occlusal-cervically with both 12 degrees and 20 degrees convergence angles also had no significant effect on internal fit (P>.0083). Cement space at distal walls (185 +/- 28 microm) was the thickest among all axial walls (P=.0001) and was twice as thick as that at the facial (90 +/- 14 microm) and palatal walls (92 +/- 15 microm). CONCLUSION: Within the limitations of this study, there was little difference in the internal fit of Cerec crowns prepared with convergence angles of 12 degrees and 20 degrees. Distal shadows influenced the thickness of the cement spaces, particularly at the distal walls. However, tooth preparations with an occlusal-cervical height not greater than 6 mm did not exaggerate the effect of the distal shadows.