Effects of transforming growth factor-ß1 on plasminogen activation in stem cells from the apical papilla: role of activating receptor-like kinase 5/Smad2 and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling.

AIM: To study the effects of TGF-ß1 on the plasminogen activation (PA) system of stem cells from the apical papilla (SCAP) and its signalling. METHODOLOGY: SCAP cells were isolated from the apical papilla of immature permanent teeth extracted for orthodontic reasons. They were exposed to various concentration of TGF-ß1 with/without pretreatment and coincubation by SB431542 (ALK/Smad2/3 inhibitor), or U0126 (MEK/ERK inhibitor). MTT assay, Western blotting and enzyme-linked immunosorbent assay (ELISA) were used to detect their effects on cell viability, and the protein expression of plasminogen activator inhibitor-1 (PAI-1), urokinase-type plasminogen activator (uPA), uPA receptor (uPAR) and their secretion. The paired Student's t-test was used for statistical analysis. RESULTS: TGF-ß1 significantly stimulated PAI-1 and soluble uPAR (suPAR) secretion of SCAP cells (P < 0.05), whereas uPA secretion was inhibited. Accordingly, TGF-ß1 induced both PAI-1 and uPAR protein expression of SCAP cells. SB431542 (an ALK5/Smad2/3 inhibitor) pretreatment and coincubation prevented the TGF-ß1-induced PAI-1 and uPAR of SCAP. U0126 attenuated the TGF-ß1-induced expression/secretion of uPAR, but not PAI-1 in SCAP. SB431542 reversed the TGF-ß1-induced decline of uPA. CONCLUSIONS: TGF-ß1 may affect the repair/regeneration activities of SCAP via differential increase or decrease of PAI-1, uPA and uPAR. These effects induced by TGF-ß1 are associated with ALK5/Smad2/3 and MEK/ERK activation. Elucidation the signalling pathways and effects of TGF-ß1 is useful for treatment of immature teeth with open apex by revascularization/revitalization procedures and tissue repair/regeneration.

Horizontal root fractures in posterior teeth without dental trauma: tooth/root distribution and clinical characteristics.

AIM: To describe the clinical characteristics and radiographic findings of horizontal root fractures (HRF) in posterior teeth without a history of dental trauma. METHODOLOGY: A total 24 patients and 31 HRF cases in 28 posterior teeth were collected from 2006 to 2015. Clinical examinations and radiographic imaging were evaluated. Value of confidence intervals of the proportions was calculated for data presentation. RESULTS: The number of males (54%) was similar to females (46%). The patients were predominantly between 50 and 70 years of age (75%). Most HRF cases were found in nonendodontically treated teeth (79%), without crown and bridge restorations (82%), and maxillary molars (54%). Many roots of maxillary molars had developed HRF, and the probability was nearly equal. Fractured teeth usually presented with periodontal and apical bone loss, and most patients (92%) were diagnosed with full mouth chronic periodontitis. Tooth wear was another common clinical feature amongst these patients. CONCLUSIONS: HRF in posterior teeth without dental trauma occurred mainly in patients aged between 50 and 70, in nonendodontically treated teeth, teeth with attrition but without crown and bridge restorations, maxillary molars and with periodontal and periapical bony destruction. Periodontal condition, occlusal wear and patients' age at diagnosis were the possible related factors. HRF in posterior teeth without dental trauma is a diagnostic challenge and even misdiagnosed. A thorough clinical examination, radiographic analysis and recognition of the clinical characteristics are helpful in the early diagnosis and treatment of HRF.

Butyrate induces reactive oxygen species production and affects cell cycle progression in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Short-chain fatty acids, such as butyric acid and propionic acid, are metabolic by-products generated by periodontal microflora such as Porphyromonas gingivalis, and contribute to the pathogenesis of periodontitis. However, the effects of butyrate on the biological activities of gingival fibroblasts (GFs) are not well elucidated. MATERIAL AND METHODS: Human GFs were exposed to various concentrations of butyrate (0.5-16 mm) for 24 h. Viable cells that excluded trypan blue were counted. Cell cycle distribution of GFs was analyzed by propidium iodide-staining flow cytometry. Cellular reactive oxygen species (ROS) production was measured by flow cytometry using 2',7'-dichlorofluorescein (DCF). Total RNA and protein lysates were isolated and subjected to RT-PCR using specific primers or to western blotting using specific antibodies, respectively. RESULTS: Butyrate inhibited the growth of GFs, as indicated by a decrease in the number of viable cells. This event was associated with an induction of G0/G1 and G2/M cell cycle arrest by butyrate (4-16 mm) in GFs. However, no marked apoptosis of GFs was noted in this experimental condition. Butyrate (> 2 mm) inhibited the expression of cdc2, cdc25C and cyclinB1 mRNAs and reduced the levels of Cdc2, Cdc25C and cyclinB1 proteins in GFs, as determined using RT-PCR and western blotting, respectively. This toxic effect of butyrate was associated with the production of ROS. CONCLUSION: These results suggest that butyrate generated by periodontal pathogens may be involved in the pathogenesis of periodontal diseases via the induction of ROS production and the impairment of cell growth, cell cycle progression and expression of cell cycle-related genes in GFs. These events are important in the initiation and prolongation of inflammatory processes in periodontal diseases.

Differential regulation of collagen, lysyl oxidase and MMP-2 in human periodontal ligament cells by low- and high-level mechanical stretching.

BACKGROUND AND OBJECTIVE: Mechanical stretching modulates extracellular matrix (ECM) protein synthesis by periodontal ligament (PDL) cells. However, the mechanoregulation of lysyl oxidase (LOX), a key enzyme for collagen cross-linking, is not fully understood. In the present study, we hypothesized that low-level and high-level mechanical stretching differentially regulates collagen deposition and the expression of LOX and the enzymes responsible for ECM degradation, such as MMP-2 in PDL cells. MATERIAL AND METHODS: Human PDL cells were cultured on flexible-bottom culture plates and subjected to cyclic mechanical stretching (3% and 10% elongation at 0.1 Hz) for 24 and 48 h in a Flexercell FX-4000 strain unit. The levels of expression of type I collagen alpha 1 (COL1A1), type III collagen alpha 1 (COL3A1), lysyl oxidase (LOX), MMP2 and TIMP2 mRNAs were analyzed using an RT-PCR technique. The cell layer and the culture medium were separately collected and processed for detection of the following ECM-related molecules: (i) total collagen content using a Sircol dye-binding method; (ii) LOX protein expression by western blotting; (iii) LOX activity using a fluorometric assay; and (iv) MMP-2 enzyme activity by gelatin zymography. RESULTS: Low-level (3%) mechanical stretching of PDL cells upregulated the expression of COL1A1, COL3A1 and LOX mRNAs, enhanced the production of collagen and increased the LOX activity but did not change the level of expression of MMP2 or TIMP2 mRNA. The collagen content and LOX activity showed obvious elevation in the medium, but not in the cell layer. High-level (10%) mechanical stretching downregulated COL1A1 mRNA but upregulated COL3A1 mRNA; however, the effect on COL3A1 was smaller, and occurred earlier, compared with the effect on the COL1A1 gene. High-level mechanical stretching upregulated the expression of MMP2 and TIMP2 mRNAs but did not change collagen production or LOX activity. Moreover, high-level mechanical stretching increased the level of pro-MMP-2, especially in the cell layer. CONCLUSIONS: This study substantiates the mechanoregulation of the expression of ECM-related molecules in PDL cells. High-level mechanical stretching upregulated the expression of MMP2 and TIMP2 mRNAs, but did not affect collagen production or LOX activity. In addition to increasing the transcription of COL1A1, COL3A1 and LOX genes, low-level mechanical stretching enhanced total collagen production and LOX activity, which should favor ECM stabilization. As an effective regulator of ECM remodeling, mechanical stretching can be exploited in periodontal regeneration and ligament tissue engineering via application of appropriate mechanical stimulation.

A fractal dimensional approach to successful evaluation of apical healing.

AIM: To evaluate whether the initial healing of apical radiolucencies 1 year after root canal treatment could be quantitatively identified by the change in fractal dimension (FD) values for the eventually completely healed cases. METHODOLOGY: Twenty-six patients with successful root canal treatment were recruited. All teeth were associated with complete healing either before or at 1 year following treatment (six of 26) or still undergoing healing at 1 year after treatment but completely healed thereafter (20 of 26). Two radiographs were selected for the same patient, one taken before treatment and the other taken 1 year after treatment. Eight regions of interests (ROIs) were selected from each radiograph, two as the experimental group located close to the infected root apex, two as the control group in the healthy bone and the other four in the healthy bone ensuring the image quality. RESULTS: Based on the FD values of the four ROIs in the healthy bone, the two radiographs were confirmed to have been taken with similar projection angles and exposure. The FD values were shown to significantly increase (P = 0.006) and decrease (P = 0.000) around the root apex and the neighbouring region of the apical lesion, respectively. CONCLUSION: Changes in fractal dimension values may serve as a necessary condition to quantitatively indicate the initial healing status 1 year after root canal treatment.

Effect of triethylene glycol dimethacrylate on the cytotoxicity, cyclooxygenase-2 expression and prostanoids production in human dental pulp cells.

AIM: To evaluate the effect of TEGDMA on cell cycle progression as well as alterations of cell cycle-related gene and protein expression. METHODOLOGY: Human dental pulp cells were exposed to 0-5 mmol L(-1) TEGDMA for 24 h. Cytotoxicity was evaluated by 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Cell cycle progression was analysed by propidium iodide (PI) flow cytometry. Cell death pathway was surveyed by annexin V/PI dual-staining flow cytometry. The mRNA expression of cell cycle-related genes (cdc2, cyclinB1 and p21) and COX-2 was evaluated by reverse transcriptase-polymerase chain reaction, and their protein expression was evaluated by Western blotting. The production of PGE(2) and PGF(2α) in the culture medium was determined by enzyme-linked immunosorbent assay. RESULTS: Triethylene glycol dimethacrylate inhibited cellular growth and induced cell cycle deregulation in dental pulp cells. High-dose exposure provoked both necrotic and apoptotic cell death. The gene and protein expression of cdc2, cyclin B1 and cdc25C declined obviously whilst cells treated with 2.5 mmol L(-1) TEGDMA concurrent with the elevated expression of p21. The mRNA and protein expression of COX-2, along with production of PGE(2) and PGF(2α), are drastically raised by 2.5-5 mmol L(-1) TEGDMA. CONCLUSIONS: Triethylene glycol dimethacrylate induced cytotoxicity, cell cycle arrest and apoptosis in dental pulp cells, which was associated with the decline of cdc2, cyclin B1, cdc25C expression and elevation of p21 expression. Concomitantly, COX-2 expression, PGE(2) and PGF(2α) production increased. These effects may contribute to explain the pulpal damage and inflammation induced by TEGDMA after operative procedures.

Carboxylesterase expression in human dental pulp cells: role in regulation of BisGMA-induced prostanoid production and cytotoxicity.

Biocompatibility of dentin bonding agents (DBA) and composite resin may affect the treatment outcome (e.g., healthy pulp, pulpal inflammation, pulp necrosis) after operative restoration. Bisphenol-glycidyl methacrylate (BisGMA) is one of the major monomers present in DBA and resin. Prior studies focused on salivary esterase for metabolism and degradation of resin monomers clinically. This study found that human dental pulp cells expressed mainly carboxylesterase-2 (CES2) and smaller amounts of CES1A1 and CES3 isoforms. Exposure to BisGMA stimulated CES isoforms expression of pulp cells, and this event was inhibited by catalase. Exogenous addition of porcine esterase prevented BisGMA- and DBA-induced cytotoxicity. Interestingly, inhibition of CES by bis(p-nitrophenyl) phosphate (BNPP) and CES2 by loperamide enhanced the cytotoxicity of BisGMA and DBA. Addition of porcine esterase or N-acetyl-l-cysteine prevented BisGMA-induced prostaglandin E(2) (PGE(2)) and PGF(2α) production. In contrast, addition of BNPP and loperamide, but not mevastatin, enhanced BisGMA-induced PGE(2) and PGF(2α) production in dental pulp cells. These results suggest that BisGMA may induce the cytotoxicity and prostanoid production of pulp cells, leading to pulpal inflammation or necrosis via reactive oxygen species production. Expression of CES, especially CES2, in dental pulp cells can be an adaptive response to protect dental pulp against BisGMA-induced cytotoxicity and prostanoid release. Resin monomers are the main toxic components in DBA, and the ester group is crucial for monomer toxicity.

Changes in peripheral blood lymphocyte phenotypes distribution in patients with oral cancer/oral leukoplakia in Taiwan.

Oral squamous cell carcinoma (OSCC) is common in many Asian countries. The immunopathogenesis of OSCC is unclear. The authors analyzed the lymphocyte subtypes and surface activation markers in healthy Taiwanese people (n=130) and patients with OSCC (n=97)/oral leukoplakia (OL, n=28) using flow cytometry. Univariate analysis found an elevation in the percentage of CD56+ NK cells, CD4+/CD69+ T cells, CD19+/CD69+ B cells and CD56+/CD69+ NK cells in OSCC patients relative to healthy people. The CD19+ and CD19+/CD25+ lymphocyte subtypes decreased in OSCC patients. CD56+ NK cells increased in OL patients. CD56+/CD69+ NK cells were elevated in recurrent and advanced OSCC. Multivariate analysis revealed an increase in CD56+ NK and CD19+/CD69+ cells in OL patients relative to controls. CD19+ B cells declined during progression from OL to OSCC. Betel quid chewing, alcohol, smoking, tumour location and staging showed little effect on lymphocyte subtypes. These results suggest that alterations and activation of NK cells, T and B cells are important and associated with disease status in oral carcinogenesis.

Prostaglandin F(2alpha) stimulates MEK-ERK signalling but decreases the expression of alkaline phosphatase in dental pulp cells.

AIM: To study prostaglandin F(2alpha) (PGF(2alpha)) receptor expression and downstream signalling in cultured human dental pulp cells and the effect of PGF(2alpha) on the alkaline phosphatase (ALP) activity of dental pulp cells. METHODOLOGY: Human dental pulp cells were cultured and exposed to PGF(2alpha). The expression of PGF(2alpha) (FP) receptors was analysed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The activation of extracellular regulated kinase (ERK) and cAMP responsive element binding protein/activating transcription factor-1 (CREB/ATF-1) signalling was determined by Western blotting. The expression of ALP in pulp cells after exposure to PGF(2alpha) was evaluated by ALP staining and PCR. RESULTS: Dental pulp cells expressed FP receptor mRNA and protein. Exposure to PGF(2alpha) revealed little cytotoxicity to pulp cells. PGF(2alpha) induced both ERK and CREB/ATF-1 phosphorylation in pulp cells. Exposure to PGF(2alpha) (>1 micromol L(-1)) further decreased the ALP activity and mRNA expression. However, U0126 (an inhibitor of MEK1) showed little preventive effect on the decline of ALP activity in dental pulp cells by PGF(2alpha). CONCLUSION: PGF(2alpha) may potentially activate FP receptors leading to ERK/CREB-ATF-1 activation during its production in inflamed dental pulp. PGF(2alpha) attenuated the ALP activity of pulp cells possibly via pathways not solely by MEK/ERK activation. PGF(2alpha) is a contributing factor of pulpal inflammation by regulating the activities of pulp cells.

Comparative cytotoxicity of five root canal sealers on cultured human periodontal ligament fibroblasts.

AIM: To evaluate the cytotoxicity of current root canal sealers to periodontal ligament (PDL) fibroblasts. METHODOLOGY: Five root canal sealers (Canals, Canals-N, Topseal, Sealapex, Tubliseal) were prepared and placed into transwells. After initial setting for 1 h, the transwells with sealers were placed into cultured PDL fibroblasts. They were cultured for further 3 or 18 h. Morphological changes were observed. Cell viability was estimated by 3-(4,5-dimethyl-thiazol-2-yl)-2,5- diphenyl-tetrazolium bromide (MTT) assay. RESULTS: Marked retraction and death of PDL fibroblasts were observed after exposure to Canals or Topseal for 3 h. A 3-h exposure of PDL fibroblasts to Tubliseal stimulated MTT reduction. Canals-N showed little cytotoxicity even after an exposure of 18 h. CONCLUSION: Canals was the most toxic sealer, followed by Topseal. Sealapex and Tubliseal had comparable and moderate cytotoxicity to PDL fibroblasts, whereas Canals-N showed little cytotoxicity. Exposure to Tubliseal may modulate MTT reduction in PDL fibroblasts. Canals-N had good biocompatibility.

Post-tooth extraction sepsis without locoregional infection--a population-based study in Taiwan.

OBJECTIVE: To investigate the incidence and risk factors of post-tooth extraction sepsis in patients without locoregional infection. SUBJECTS AND METHODS: We assessed all claim records of the Taiwanese National Health Insurance program in 2005. Admissions for patients aged > or =16 years containing a discharge diagnosis of sepsis, and who received tooth extraction within 14 days before the admission were identified. Patient charts were reviewed to confirm the diagnosis of sepsis and rule out other infection sources. The relationship between postextraction sepsis (PES) and clinical parameters was analyzed. RESULTS: Thirty-three of the 2 223 971 extraction cases met the criteria of PES, an incidence of 1.48 per 100 000, and seven patients (21.2%) died of the disease. Aging significantly increased the risk of PES (P < 0.001). Pre-existing comorbidities were found in 20 of the 33 cases, with diabetes mellitus and hematologic diseases the most common. The method, number, and position of extraction had no influence on PES incidence. Blood cultures were positive in 25 patients (75.8%) and isolates included species of the Streptococcus, Actinomyces, Klebsiella, Bacteroides, Prevotella, and Enterococcus genera. CONCLUSION: Tooth extraction is associated with a low but significant risk of postoperative sepsis, especially in the elderly and patients with underlying diseases.

Preliminary study on wilcoxon learning machines.

As is well known in statistics, the resulting linear regressors by using the rank-based Wilcoxon approach to linear regression problems are usually robust against (or insensitive to) outliers. This motivates us to introduce in this paper the Wilcoxon approach to the area of machine learning. Specifically, we investigate four new learning machines, namely Wilcoxon neural network (WNN), Wilcoxon generalized radial basis function network (WGRBFN), Wilcoxon fuzzy neural network (WFNN), and kernel-based Wilcoxon regressor (KWR). These provide alternative learning machines when faced with general nonlinear learning problems. Simple weights updating rules based on gradient descent will be derived. Some numerical examples will be provided to compare the robustness against outliers for various learning machines. Simulation results show that the Wilcoxon learning machines proposed in this paper have good robustness against outliers. We firmly believe that the Wilcoxon approach will provide a promising methodology for many machine learning problems.

Horizontal/oblique root fractures in the palatal root of maxillary molars with associated periodontal destruction: case reports.

AIM: To report two cases of palatal root fracture in maxillary molars that were successfully managed in the short term by root canal treatment and root amputation. SUMMARY: In the first case, a 48-year-old woman with bony destruction and a deep periodontal pocket on the palatal root of tooth 26 (FDI) underwent root canal treatment. Bleeding into the palatal canal and radiolucent lines over the root suggested a fracture. Further evidence was provided by an electronic apex locator. Subsequent surgery confirmed the presence of a horizontal root fracture and the fractured root was removed. In the second case, a 75-year-old woman presented with pain from the left posterior teeth. Clinical examination revealed an oblique root fracture of tooth 27 palatal roots with abscess formation and a deep periodontal pocket. Palatal root amputation and odontoplasty were performed. This was followed by root canal treatment. Both teeth were preserved in the short term and early healing of these two cases was uneventful. KEY LEARNING POINTS: Horizontal/oblique root fracture of the palatal root in molars is rare. A combination of periodontal and root canal treatment and palatal root amputation may allow short-term preservation of functional teeth.

Antioxidant and pro-oxidant properties of chlorhexidine and its interaction with calcium hydroxide solutions.

AIM: To evaluate the antioxidant and pro-oxidant properties of chlorhexidine (CHX). METHODOLOGY: The scavenging and generation of reactive oxygen species (ROS) by CHX in the presence or absence of saturated Ca(OH)(2) solutions was evaluated. The reaction emitted chemiluminescence in the presence of lucigenin thus was determined by a luminometer to evaluate the levels of ROS production. Changes in DNA conformation were analysed by agarose gel electrophoresis. Paired Student's t-test was used to compare the difference between groups. RESULTS: Chlorhexidine (0.00002-0.02%) effectively scavenged 56-88% of the superoxide radicals generated by the xanthine/xanthine oxidase reaction. Through analysis of PUC18 DNA conformation changes, CHX was shown to be a mild scavenger of hydroxyl radicals generated by H(2)O(2) plus FeCl(2). However, CHX (>0.083%) decreased the mobility of PUC18 plasmid DNA with potential production of DNA-DNA cross-link and severe DNA breaks (presence of DNA smear) at further higher concentrations. Furthermore, CHX induced ROS production including H(2)O(2) and superoxide radicals in 0.1N NaOH (pH = 12.76) or Ca(OH)(2) (pH = 12.5) solutions. CONCLUSION: Chlorhexidine exhibited both antioxidant and pro-oxidant properties under different conditions. These events are possibly involved in the killing of root canal and periodontal microorganisms when CHX and Ca(OH)(2) were used in combination or separately. Potential genotoxicity and tissue damage when extruded into the periradicular tissue and at higher concentrations should be considered during periodontal and endodontic practice.

Hydroxychavicol, a novel betel leaf component, inhibits platelet aggregation by suppression of cyclooxygenase, thromboxane production and calcium mobilization.

BACKGROUND AND PURPOSE: Platelet hyperactivity is important in the pathogenesis of cardiovascular diseases. Betel leaf (PBL) is consumed by 200-600 million betel quid chewers in the world. Hydroxychavicol (HC), a betel leaf component, was tested for its antiplatelet effect. EXPERIMENTAL APPROACH: We tested the effect of HC on platelet aggregation, thromboxane B(2) (TXB(2)) and reactive oxygen species (ROS) production, cyclooxygenase (COX) activity, ex vivo platelet aggregation and mouse bleeding time and platelet plug formation in vivo. The pharmacokinetics of HC in rats was also assessed. KEY RESULTS: HC inhibited arachidonic acid (AA) and collagen-induced platelet aggregation and TXB(2) production. HC inhibited the thrombin-induced TXB(2) production, but not platelet aggregation. SQ29548, suppressed collagen- and thrombin-induced TXB(2) production, but not thrombin-induced platelet aggregation. HC also suppressed COX-1/COX-2 enzyme activity and the AA-induced ROS production and Ca(2+) mobilization. HC further inhibited the ex vivo platelet aggregation of platelet-rich plasma (>100 nmole/mouse) and prolonged platelet plug formation (>300 nmole/mouse) in mesenteric microvessels, but showed little effect on bleeding time in mouse tail. Moreover, pharmacokinetics analysis found that more than 99% of HC was metabolized within 3 min of administration in Sprague-Dawley rats in vivo. CONCLUSIONS AND IMPLICATIONS: HC is a potent COX-1/COX-2 inhibitor, ROS scavenger and inhibits platelet calcium signaling, TXB(2) production and aggregation. HC could be a potential therapeutic agent for prevention and treatment of atherosclerosis and other cardiovascular diseases through its anti-inflammatory and antiplatelet effects, without effects on haemostatic functions.

Cytokine-induced prostaglandin E2 production and cyclooxygenase-2 expression in dental pulp cells: downstream calcium signalling via activation of prostaglandin EP receptor.

AIM: To determine whether (i) proinflammatory cytokines stimulate prostaglandin E(2) (PGE(2)) production and cyclooxygenase (COX) gene expression in dental pulp cells, and (ii) pulp cells that express different prostaglandin E(2) receptor (EP) isoforms and their activation by PGE(2) leads to downstream Ca(2+) signalling. METHODOLOGY: Cultured human dental pulp cells were exposed to interleukin (IL)-1beta and tumour necrotic factor-alpha (TNF-alpha). The expression of COX-1 and COX-2 was measured with reverse transcriptase-polymerase chain reaction (RT-PCR). The production of PGE(2) was measured using an enzyme-linked immunosorbent assay. Expression of prostaglandin EP receptor isoforms was studied by RT-PCR, whereas fura-2 fluorescence was used to measure calcium mobilization. The Kruskal-Wallis test and Wilcoxon sum rank test with Bonferroni correction were used for statistical analysis. RESULTS: Interleukin-1beta and TNF-alpha stimulate PGE(2) production of human dental pulp cells (P < 0.05). IL-1beta stimulated the COX-2 but not COX-1 mRNA expression. Pulp cells express mainly EP2, EP3 and EP1 receptors as analysed by RT-PCR. PGE(2) (0.25-2 micromol L(-1)) stimulated the Ca(2+) mobilization as indicated by increase in fura-2 fluorescence. CONCLUSIONS: Interleukin-1beta and TNF-alpha may stimulate PGE(2) production in dental pulp cells. Activation of prostaglandin EP receptors in dental pulp cells by PGE(2) may induce Ca(2+) signalling to regulate cellular biological activity during inflammation.

Cell-mediated immunity and head and neck cancer: with special emphasis on betel quid chewing habit.

Betel quid (BQ) chewing is popular in Taiwan, India, and many southeast-Asian countries. BQ chewing has strong association with the risk of oral leukoplakia (OL), oral submucous fibrosis (OSF), and oral cancer (OC). BQ components exhibit genotoxicity and may alter the structure of DNA, proteins and lipids, resulting in production of antigenicity. BQ ingredients are also shown to induce keratinocyte inflammation by stimulating the production of prostaglandins, TNF-alpha, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in keratinocytes. These events may provoke tissue inflammation, early cell-mediated immunity (CMI), and immune surveillance in BQ chewers. However, BQ components also directly affect the functional activities of immunocompotent cells, and moreover tumor cells may hypo-respond to the CMI via diverse mechanisms such as induction of apoptosis of lymphocytes, induction of production of suppressor T cells, downregulation of MHC molecules in tumor cells, etc. Clinically, an alteration in lymphocyte subsets, a decrease in total number of lymphocytes, and a reduction in functional activities of CMI have been observed in isolated peripheral blood mononuclear cells (PBMC) and tumor infiltrated lymphocytes (TIL) in patients with OSF, OL or OC. Adaptation of tumor cells to immune system may promote clonal selection of resistant tumor cells, leading to immune tolerance. Future studies on effects of BQ components on CMI and humoral immunity in vitro and in vivo can be helpful for chemoprevention of BQ-related oral mucosal diseases. To elucidate how virus infection, tobacco, alcohol and BQ consumption, and other environmental exposure affect the immune status of patients with oral premalignant lesions or OC will help us to understand the immunopathogenesis of OC and to develop immunotherapeutic strategies for OC.

Effects of TGF-beta s on the growth, collagen synthesis and collagen lattice contraction of human dental pulp fibroblasts in vitro.

Transforming growth factor-beta (TGF-beta) is important in regulating the repair and regeneration of damaged dental pulp. For further elucidating the roles of different isoforms of TGF-beta in the healing and inflammatory processes of human dental pulp, we found that TGF-beta1, TGF-beta2 and TGF-beta3 inhibited the growth of two human dental pulp cell strains in vitro by 19-29, 18-25 and 23-26%, respectively, at a concentration of 0.5 ng/ml. TGF-beta also differentially stimulated the collagen synthesis of pulp cells. Collagen synthesis increased by 1 ng/ml of TGF-beta1 and TGF-beta2 by 42 and 51%, respectively. TGF-beta3 (0.1-1 ng/ml) lacked of stimulatory effect on collagen synthesis of pulp cells. Pulp cells have the intrinsic capacity to contract collagen lattice, leading to decreasing of lattice diameter. An 8 h exposure to TGF-beta1 and TGF-beta2 enhanced the pulp cell-populated collagen lattice contraction at concentrations ranging from 0.2 to 3 ng/ml. At similar concentrations, TGF-beta3 lacked of this stimulatory effect. When collagen lattice were detached after 24 h of exposure, TGF-beta1 and TGF-beta2 (0.6-3 ng/ml) induced the pulp cells-populated collagen lattice contraction within 4-8h of gel detachment. These results indicate that TGF-beta-induced collagen lattice contraction is a late cellular event. These in vitro results indicate that effects of TGF-beta isoforms on the growth, collagen synthesis and collagen lattice contraction of pulp cells may play crucial roles in the pathobiological processes of dental pulp.

Reactive oxygen species are crucial for hydroxychavicol toxicity toward KB epithelial cells.

Betel quid (BQ) chewing shows a strong correlation to the incidence of oral submucous fibrosis (OSF), leukoplakia and oral cancer. BQ contains mainly areca nut, lime, Piper betle leaf (PBL) and the inflorescence of P. betle (IPB). Hydroxychavicol (4-allyl-catechol, HC), as a major phenolic compound in PBL and IPB, is shown to induce oxidative stress, glutathione (GSH) depletion and cell cycle deregulation. Using bivariate BrdU/PI flow cytometry, KB cells in DNA synthesis (S phase) are shown to be sensitive to the toxic effect of HC and show cell cycle arrest and apoptosis following exposure to 0.1 and 0.3 mM HC. HC-induced apoptosis and cell cycle arrest are associated with mitochondrial membrane potential (delta Psim) depolarization as revealed by a decrease in rhodamine fluorescence. N-acetyl-L-cysteine (1 mM), superoxide dismutase (100 U/ml) and catalase (1000 U/ml) were effective in prevention of HC-induced GSH depletion (as indicated by chloromethylfluorescein fluorescence), reactive oxygen species (ROS) production (by dichlorofluorescein fluorescence), cell cycle arrest and apoptosis. However, dimethylthiourea (2 mM), neocuproine (1 mM), 1,10-phenanthroline (200 microM) and desferrioxamine (0.5 mM) showed little effect on HC-induced cell changes. HC elevated the cellular and mitochondrial GSH levels at moderate concentrations (0.05-0.1 mM), whereas at a concentration of 0.3 mM, inhibitory effects were noted. These results indicate that HC consumption may be associated with BQ-chewing-related oral mucosal diseases via GSH depletion, ROS production, mitochondrial dysfunction, cell cycle disturbance and the induction of apoptosis. These events are related to the production of superoxide radicals and hydrogen peroxide.

Scavenging property of three cresol isomers against H2O2, hypochlorite, superoxide and hydroxyl radicals.

Formocresol has long been used for pulpotomy of primary teeth and as an intracanal medicament. Little is known, however, about the pharmacological effect of tricresols. This study showed that three cresol isomers, o-cresol, m-cresol and p-cresol, are H2O2 scavengers with a 50% inhibitory concentration (IC50) of 502, 6.7 and 10.16 microM, respectively. o-, m- and p-cresol were also shown to be effective scavengers of superoxide radicals generated by xanthine/xanthine oxidase with an IC50 of 282, 153 and > 4000 microM, respectively, as analyzed by luminometer. o-, m- and p-cresol showed protective effects on the DNA breaks generated by H2O2/FeCl2 and FeCl3/ascorbate/H2O2 systems at concentrations ranging from 70 microM to 1.43 mM, o-, m- and p-cresol also showed differential protective effects against DNA breaks induced by 0.17% NaOCl with 100% inhibitory concentration (IC100) of about 10, 1 and 10 mM, respectively. In addition, reaction with 3% H2O2 and 0.17% NaOCl completely prevented NaOCl-induced DNA breaks. The results indicate that the three cresol isomers are effective ROS scavengers and may prevent ROS induced damage when used as pulpotomy agents or as intracanal medicaments. Owing to the difference in the position of the functional hydroxyl group in the three cresol isomers, m-cresol is the most effective ROS scavenger. Concomitant use of H2O2 for root canal irrigation may diminish both the tissue dissolving capacity of NaOCl and NaOCl-induced DNA damage.