Comparison of 4 sinus augmentation techniques for implant placement with residual alveolar bone height ≤3 mm.

This study compared implant outcomes following maxillary sinus floor augmentation (MSFA) in edentulous patients with a residual alveolar bone height ≤3 mm. Four techniques were evaluated: 1-stage bone-added osteotome sinus floor elevation procedure (BAOSFE) with simultaneous implant placement; 2-stage BAOSFE with delayed implant placement; 1-stage lateral window sinus floor elevation with simultaneous implant placement; and 2-stage lateral window sinus floor elevation with delayed implant placement. Patients were followed for 18 to 72 months (mean: 52.5 months) after prosthesis placement. Data were analyzed with cone-beam computed tomography. A total of 96 implants from 71 patients were analyzed; pretreatment, there were no significant differences between patients. Total implant survival was 98.9%. The mean residual bone height was significantly higher in the 1-stage BAOSFE group than the other groups (P < .01); 1 implant in this group failed at 3 months. There was no significant difference in total bone height gain between groups. However, the bone height gain of 1st sinus lifting with 2-stage BAOSFE was significantly lower than the 2-stage lateral window procedure (P < .01). There was no prosthesis failure. The favorable implant outcomes suggest these 1-stage and 2-stage MSFA procedures should be considered as alternative treatment options for patients with extremely atrophic posterior maxilla.

Cone-beam computed tomography assessment of Schneiderian membranes: Non-infected and infected membranes, and membrane resolution following tooth extraction: A retrospective clinical trial.

BACKGROUND: Cone-beam computed tomography (CBCT) presurgical assessment on the maxillary sinus can reduce the possibility of Schneiderian membrane perforation. This study examined Schneiderian membrane thickness (SMT) and its relationship with neighboring hard tissues for patients with and without membrane thickening. For patients with sinus infections, we evaluated dimensional changes of the SMT post-extraction relative to pre-extraction SMT and residual bone height (RBH). METHODS: CBCT images from 93 patients needing single-tooth implant reconstruction without (n = 83) and with (n = 14) odontogenic infected maxillary sinuses were assessed. SMT, RBH, and lateral wall thickness (LWT) were measured. Causes of extraction, RBH in the infection site, and retrospective post-extraction record of SMT were recorded for the thickened SMT group. RESULTS: Mean SMT for normal SMT group was 1.13 ± 0.43 mm, RBH was 6.26 ± 2.38 mm; upper and lower LWT was 1.85 ± 0.95 mm, and 3.07 ± 2.26 mm, respectively. RBH and LWT had no significant relationships with SMT. For thickened SMT group, mean values for SMT and RBH prior to extraction were 4.53 ± 2.46 mm and 1.97 ± 1.43 mm, respectively. Pre-extraction SMT had a moderately negative correlation with pre-extraction RBH. SMT resolution in thickened SMT group was observed by 2.80 ± 1.37 months post-extraction; post-extraction SMT was not significantly different from normal SMT group (p = .187). CONCLUSIONS: Within the limitation of the sample size, thickened SMT induced by odontogenic infection subsides about 3 months following tooth extraction, and further sinus lifting implant surgery may be considered.

Nonsurgical periodontal treatment and prosthetic rehabilitation of a renal transplant patient with gingival enlargement: a case report with 2-year follow-up.

BACKGROUND: Drug-induced gingival enlargement is a common condition which can be observed in patients taking immunosuppressive medications following organ transplant surgery. The disfiguring excessive tissue often hinders proper oral hygiene practices, therefore accompanied by periodontitis, tooth mobility, and even pathological tooth migration in extreme cases. This case report presents a conservative treatment protocol for a patient with the aforementioned conditions involving neither surgical nor orthodontic intervention. Few related studies have reported such a noninvasive protocol for managing these kinds of conditions. CASE PRESENTATION: A 51-year-old woman presented with bleeding gingiva, mobile teeth and complained of chewing difficulties. She had undergone renal transplant surgery 16 years prior to this dental visit and had been taking immunosuppressive drugs including cyclosporine ever since. After clinical and radiographic examinations, the patient was diagnosed with drug-induced gingival enlargement, pathological tooth migration, severe periodontitis, and missing teeth. Through careful and meticulous nonsurgical debridement, oral hygiene instruction, tooth extraction, and occlusal adjustment, the patient's periodontium was restored to a healthy state without surgical intervention. Moreover, the patient's chewing function was restored by means of removable partial dentures. Good adaptation of prostheses and satisfaction with overall treatment outcomes were reported. CONCLUSIONS: Through proper diagnosis, treatment, and with good patient cooperation, complex systemic and dental problems can be managed conservatively without invasive surgeries to attain a stable periodontium and eventually, occlusal function could be restored.

Effects of TGF-ß1 on plasminogen activation in human dental pulp cells: Role of ALK5/Smad2, TAK1 and MEK/ERK signalling.

Transforming growth factor-ß1 (TGF-ß1) plays an important role in the pulpal repair and dentinogenesis. Plasminogen activation (PA) system regulates extracellular matrix turnover. In this study, we investigated the effects of TGF-ß1 on PA system of dental pulp cells and its signalling pathways. Dental pulp cells were treated with different concentrations of TGF-ß1. MTT assay, reverse transcription-polymerase chain reaction, Western blotting and enzyme-linked immunosorbant assay (ELISA) were used to detect the effect of TGF-ß1 on cell viability, mRNA and protein expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1) as well as their secretion. The phosphorylation of Smad2 and TAK1 was analysed by Pathscan ELISA or Western blotting. Cells were pretreated with SB431542 (ALK5/Smad2/3 inhibitor), 5z-7-oxozeaenol (TAK1 inhibitor) and U0126 (MEK/ERK inhibitor) for examining the related signalling. TGF-ß1 slightly inhibited cell growth that was reversed by SB431542. TGF-ß1 upregulated both RNA and protein expression of PAI-1 and uPAR, whereas it downregulated uPA expression. Accordingly, TGF-ß1 stimulated PAI-1 and soluble uPAR (suPAR) secretion of pulp cells, whereas uPA secretion was inhibited. TGF-ß1 induced the phosphorylation of Smad2 and TAK1. In addition, SB431542, 5z-7-oxozeaenol and U0126 attenuated the TGF-ß1-induced secretion of PAI-1 and suPAR. These results indicate that TGF-ß1 is possibly involved in the repair/regeneration and inflammatory processes of dental pulp via regulation of PAI-1, uPA and uPAR. These effects of TGF-ß1 are related to activation of ALK5/Smad2, TAK1 and MEK/ERK signalling pathways. Clarifying the signal transduction for the effects of TGF-ß1 is helpful for pulpo-dentin regeneration and tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.

Strontium ranelate treatment in a postmenopausal woman with osteonecrosis of the jaw after long-term oral bisphosphonate administration: a case report.

Bisphosphonates (BPs) suppress bone resorption and increase bone strength, thus reducing the risk of fracture. Oral BPs are widely used for the prevention and treatment of osteoporosis and osteopenia. Here, we describe the case of a postmenopausal woman who took oral alendronate for >3 years for osteoporosis. The patient presented at the clinic with sharp jaw pain and swelling on the left mandible 4 months after extraction of the third molar. Clinical examinations identified an inflamed mucosal opening with pus over an area of necrotic bone. Initial images of cone beam computed tomography revealed a sequestrum at the extracted socket. The condition did not improve after 1 week of antibiotic treatment; therefore, the alendronate treatment was terminated and the patient was prescribed strontium ranelate instead. The patient gradually recovered and, at the 2-year follow-up, the site of BP-related osteonecrosis of the jaw healed completely as determined by both clinical and cone beam computed tomography measures. The bone mineral densities in the femoral neck and lumbar spine improved after 1 year, and were maintained at the 3-year follow-up. The serum C-terminal cross-linking telopeptide values also gradually increased from the initial 130 pg/mL to 320 pg/mL at the 3-year follow-up. Taken together, this case supports the use of strontium ranelate as an alternative treatment for postmenopausal women who receive long-term oral BP treatments and are at risk for serious complications of BP-related osteonecrosis of the jaw.

Evaluation of the Mandibular Lingual Canal and Anterior Loop Length to Minimize Complications Associated With Anterior Mandibular Surgeries: A Cone-Beam Computed Tomography Study.

PURPOSE: This retrospective study evaluated the localization, incidence, and dimensions of the mandibular lingual canal and the anterior loop in the Taiwanese population using the simulation and visual interpretation of cone-beam computed tomography to minimize complications during symphysis block surgical procedures. MATERIALS AND METHODS: The sample population consisted of 215 patients (105 men and 110 women; mean age, 57 yr). The median lingual canal, symphysis bone thickness, and anterior loop length were defined and calculated using cone-beam computed tomography and 3-dimensional reconstructed images. The correlation of all data for men and women was assessed and analyzed statistically using unpaired t tests. RESULTS: All patients exhibited at least 1 median lingual canal in the symphysis, and the diameter of the main branch ranged from 0.21 to 1.48 mm (mean, 0.85 mm), with relevant differences between genders (longer in men than in women). A harvesting depth of 4 mm for the distance from the buccal bone to the terminal end of the median lingual canal resulted in a risk of neurovascular injury (13.0%); this risk was notably higher in women (19.1%) than in men (6.7%). The right and left anterior loop lengths ranged from 0 to 5.46 mm (mean, 2.60 mm) and from 0 to 5.57 mm (mean, 2.61 mm), respectively, with no relevant differences between genders or sides. CONCLUSIONS: The results suggest that routine cone-beam computed tomographic examinations before surgical interventions in the symphysis region are necessary because of the numerous complicated anatomic variations.

Basic Fibroblast Growth Factor Regulates Gene and Protein Expression Related to Proliferation, Differentiation, and Matrix Production of Human Dental Pulp Cells.

INTRODUCTION: Basic fibroblast growth factor (bFGF) plays differential effects on the proliferation, differentiation, and extracellular matrix turnover in various tissues. However, limited information is known about the effect of bFGF on dental pulp cells. The purposes of this study were to investigate whether bFGF influences the cell differentiation and extracellular matrix turnover of human dental pulp cells (HDPCs) and the related gene and protein expression as well as the role of the mitogen-activated protein kinase (MEK)/extracellular-signal regulated kinase (ERK) signaling pathway. The expression of fibroblast growth factor receptors (FGFRs) in HDPCs was also studied. METHODS: The expression of FGFR1 and FGFR2 in HDPCs was investigated by reverse-transcription polymerase chain reaction. HDPCs were treated with different concentrations of bFGF. Cell proliferation was evaluated using the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Cell differentiation was evaluated using alkaline phosphatase (ALP) staining. Changes in messenger expression of cyclin B1 and tissue inhibitor of metalloproteinase (TIMP) 1 were determined by reverse-transcription polymerase chain reaction. Changes in protein expression of cdc2, TIMP-1, TIMP-2, and collagen I were determined by Western blotting. U0126 was used to clarify the role of MEK/ERK signaling. RESULTS: HDPCs expressed both FGFR1 and FGFR2. Cell viability was stimulated by 50-250 ng/mL bFGF. The expression and enzyme activities of ALP were inhibited by 10-500 ng/mL bFGF. At similar concentrations, bFGF stimulates cdc2, cyclin B1, and TIMP-1 messenger RNA and protein expression. bFGF showed little effect on TIMP-2 and partly inhibited collagen I expression of pulp cells. U0126 (a MEK/ERK inhibitor) attenuated the bFGF-induced increase of cyclin B1, cdc2, and TIMP-1. CONCLUSIONS: bFGF may be involved in pulpal repair and regeneration by activation of FGFRs to regulate cell growth; stimulate cdc2, cyclin B1, and TIMP-1 expression; and inhibit ALP. These events are partly associated with MEK/ERK signaling.

Clinical and Radiographic Characteristics of Vertical Root Fractures in Endodontically and Nonendodontically Treated Teeth.

INTRODUCTION: A vertical root fracture (VRF) is a root fracture extending along the longitudinal axis of roots and is often noted in endodontically treated teeth. However, the clinical and radiographic characteristics of VRFs are not completely known. METHODS: A total of 65 teeth with 68 vertical fractured roots in 58 Chinese patients were investigated. The clinical examination records and radiographic images were reviewed in detail. RESULTS: A total of 24 male (41.38%) and 34 female (58.62%) patients aged 25-90 years (average = 57 years) were included; 51 (87.93%) and 7 (12.07%) patients exhibited 1 tooth and 2 teeth with VRFs, respectively, in the dentition. VRFs occurred mainly in the mesial root (20 roots, 57.14%) of the mandibular molars (29 teeth, 44.62%). Clinically, teeth with VRFs usually presented a periodontal probing depth >5 mm (44 teeth, 91.67%, P < .001) with a prosthesis (55 teeth, 84.62%, P < .001) and a relatively intact dentition (42 patients exhibited <4 missing teeth in the dentition, 77.78%, P < .001). Most of the nonendodontically treated VRFs exhibited attrited occlusal surfaces. Radiographic characteristics of the teeth with VRFs were typically associated with prior root canal treatment (56 teeth, 86.15%, P < .001), periodontal bone loss (62 teeth, 95.38%, P < .001), apical bone loss (52 teeth, 80.00%, P < .001), and periodontal ligament widening (61 teeth, 93.85%, P < .001). The mesial roots of the mandibular molars were most susceptible to VRFs in both endodontically and nonendodontically treated teeth. CONCLUSIONS: These results elucidated some clinical and radiographic and diagnostic features that facilitate VRF identification.

Transforming growth factor beta 1 increases collagen content, and stimulates procollagen I and tissue inhibitor of metalloproteinase-1 production of dental pulp cells: Role of MEK/ERK and activin receptor-like kinase-5/Smad signaling.

BACKGROUND/PURPOSE: In order to clarify the role of transforming growth factor beta 1 (TGF-ß1) in pulp repair/regeneration responses, we investigated the differential signaling pathways responsible for the effects of TGF-ß1 on collagen turnover, matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of metalloproteinase-1 (TIMP-1) production in human dental pulp cells. METHODS: Pulp cells were exposed to TGF-ß1 with/without pretreatment and coincubation by 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenyl mercapto)butadiene (U0126; a mitogen-activated protein kinase kinase [MEK]/extracellular signal-regulated kinase [ERK] inhibitor) and 4-(5-benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H- imidazol-2-yl)-benzamide hydrate (SB431542; an activin receptor-like kinase-5/Smad signaling inhibitor). Sircol collagen assay was used to measure cellular collagen content. Culture medium procollagen I, TIMP-1, and MMP-3 levels were determined by enzyme-linked immunosorbent assay. RESULTS: TGF-ß1 increased the collagen content, procollagen I, and TIMP-1 production, but slightly decreased MMP-3 production of pulp cells. SB431542 and U0126 prevented the TGF-ß1-induced increase of collagen content and TIMP-1 production of dental pulp cells. CONCLUSION: These results indicate that TGF-ß1 may be involved in the healing/regeneration processes of dental pulp in response to injury by stimulation of collagen and TIMP-1 production. These events are associated with activin receptor-like kinase-5/Smad2/3 and MEK/ERK signaling.

Lysophosphatidylcholine induces cytotoxicity/apoptosis and IL-8 production of human endothelial cells: Related mechanisms.

Increased levels of oxidized low-density lipoprotein oxLDL) are shown to elevate the risk of cardiovascular diseases such as atherosclerosis, thrombosis, stroke, and myocardial infarction. This is possibly due to the toxic effects of oxLDLs on vascular cells. Various oxLDLs including lysophosphatidylcholine (LPC) and 7-ketocholesterol injure vascular endothelial cells and stimulate inflammatory reaction. However the toxicity of LPC on endothelial cells is not clear. In this study, human endothelial cells were exposed to LPC. Cytotoxicity was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. Propidium iodide (PI) staining or PI/Annexin V dual staining flow cytometry were used to determine cell cycle progression and apoptosis. Reactive oxygen species (ROS) level was analyzed by DCFH-DA labeling flow cytometry. RNA and protein expression of endothelial cells was studied by reverse transcriptase-polymerase chain reaction and western blotting. IL-8 secretion was measured by enzyme-linked immunosorbant assay. LPC showed cytotoxicity to endothelial cells (>50 µg/ml). LPC induced cell cycle arrest and apoptosis with concomitant inhibition of cdc2 and cyclin B1 expression. LPC stimulated intracellular ROS production and ATM/Chk2, ATR/Chk1 and Akt activation. IL-8 expression and secretion in endothelial cells were induced by LPC. LPC-induced apoptosis, and IL-8 expression/secretion was attenuated by LY294002, a PI3K/Akt inhibitor. These results reveal that LPC is involved in the pathogenesis of atherosclerosis and vascular diseases by stimulation of inflammation and injury to endothelial cells. These events are related to ROS, ATM/Chk2, ATR/Chk2 and PI3K/Akt signaling. Understanding the toxic mechanisms of LPC is useful for future prevention and treatment atherosclerosis.

Effect of Butyrate on Collagen Expression, Cell Viability, Cell Cycle Progression and Related Proteins Expression of MG-63 Osteoblastic Cells.

AIMS: Butyric acid is one major metabolic product generated by anaerobic Gram-negative bacteria of periodontal and root canal infection. Butyric acid affects the activity of periodontal cells such as osteoblasts. The purposes of this study were to investigate the effects of butyrate on MG-63 osteoblasts. METHODS: MG-63 cells were exposed to butyrate and cell viability was estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The mRNA and protein expression of type I collagen and cell cycle-related proteins were measured by reverse-transcriptase polymerase chain reaction (RT-PCR), western blotting or immunofluorescent staining. Cellular production of reactive oxygen species (ROS) was analyzed by 2',7'-dichlorofluorescein (DCF) fluorescence flow cytometry. RESULTS: Exposure to butyrate suppressed cell proliferation, and induced G2/M (8 and 16 mM) cell cycle arrest of MG-63 cells. Some cell apoptosis was noted. The mRNA expression of cdc2 and cyclin-B1 decreased after exposure to butyrate. The protein expression of type I collagen, cdc2 and cyclin B1 were decreased, whereas the expression of p21, p27 and p57 was stimulated. Under the treatment of butyrate, ROS production in MG-63 cells markedly increased. CONCLUSIONS: The secretion of butyric acid by periodontal and root canal microorganisms may inhibit bone cell growth and matrix turnover. This is possibly due to induction of cell cycle arrest and ROS generation and inhibition of collagen expression. These results suggest the involvement of butyric acid in the pathogenesis of periodontal and periapical tissue destruction by impairing bone healing responses.

7-Ketocholesterol induces ATM/ATR, Chk1/Chk2, PI3K/Akt signalings, cytotoxicity and IL-8 production in endothelial cells.

Cardiovascular diseases (atherosclerosis, stroke, myocardiac infarction etc.) are the major systemic diseases of elder peoples in the world. This is possibly due to increased levels of oxidized low-density lipoproteins (oxLDLs) such as 7-ketocholesterol (7-KC) and lysophosphatidylcholine (LPC) that damage vascular endothelial cells, induce inflammatory responses, to elevate the risk of cardiovascular diseases, Alzheimer's disease, and age-related macular degeneration. However the toxic effects of 7-KC on endothelial cells are not known. In this study, 7-KC showed cytotoxicity to endothelial cells at concentrations higher than 10 µg/ml. 7-KC stimulated ATM/Chk2, ATR-Chk1 and p53 signaling pathways in endothelial cells. 7-KC also induced G0/G1 cell cycle arrest and apoptosis with an inhibition of Cyclin dependent kinase 1 (Cdk1) and cyclin B1 expression. Secretion and expression of IL-8 in endothelial cells were stimulated by 7-KC. 7-KC further induced intracellular ROS production as shown by increase in DCF fluorescence and Akt phosphorylation. LY294002 attenuated the 7-KC-induced apoptosis and IL-8 mRNA expression of endothelial cells. These results indicate that oxLDLs such as 7-KC may contribute to the pathogenesis of atherosclerosis, thrombosis and cardiovascular diseases by induction of endothelial damage, apoptosis and inflammatory responses. These events are associated with ROS production, activation of ATM/Chk2, ATR/Chk1, p53 and PI3K/Akt signaling pathways.

Prostaglandin E2 Stimulates EP2, Adenylate Cyclase, Phospholipase C, and Intracellular Calcium Release to Mediate Cyclic Adenosine Monophosphate Production in Dental Pulp Cells.

INTRODUCTION: Prostaglandin E2 (PGE2) plays a crucial role in pulpal inflammation and repair. However, its induction of signal transduction pathways is not clear but is crucial for future control of pulpal inflammation. METHODS: Primary dental pulp cells were exposed to PGE2 and 19R-OH PGE2 (EP2 agonist) or sulprostone (EP1/EP3 agonist) for 5 to 40 minutes. Cellular cyclic adenosine monophosphate (cAMP) levels were measured using the enzyme-linked immunosorbent assay. In some experiments, cells were pretreated with SQ22536 (adenylate cyclase inhibitor), H89 (protein kinase A inhibitor), dorsomorphin (adenosine monophosphate-activated protein kinase inhibitor), U73122 (phospholipase C inhibitor), thapsigargin (inhibitor of intracellular calcium release), W7 (calmodulin antagonist), verapamil (L-type calcium channel blocker), and EGTA (extracellular calcium chelator) for 20 minutes before the addition of PGE2. RESULTS: PGE2 and 19R-OH PGE2 (EP2 agonist) stimulated cAMP production, whereas sulprostone (EP1/EP3 agonist) shows little effect. PGE2-induced cAMP production was attenuated by SQ22536 and U73122 but not H89 and dorsomorphin. Intriguingly, thapsigargin and W7 prevented PGE2-induced cAMP production, but verapamil and EGTA showed little effect. CONCLUSIONS: These results indicate that PGE2-induced cAMP production is associated with EP2 receptor and adenylate cyclase activation. These events are mediated by phospholipase C, intracellular calcium release, and calcium-calmodulin signaling. These results are helpful for understanding the role of PGE2 in pulpal inflammation and repair and possible future drug intervention.

Areca nut components stimulate ADAM17, IL-1α, PGE2 and 8-isoprostane production in oral keratinocyte: role of reactive oxygen species, EGF and JAK signaling.

Betel quid (BQ) chewing is an etiologic factor of oral submucous fibrosis (OSF) and oral cancer. There are 600 million BQ chewers worldwide. The mechanisms for the toxic and inflammatory responses of BQ are unclear. In this study, both areca nut (AN) extract (ANE) and arecoline stimulated epidermal growth factor (EGF) and interleukin-1α (IL-1α) production of gingival keratinocytes (GKs), whereas only ANE can stimulate a disintegrin and metalloproteinase 17 (ADAM17), prostaglandin E2 (PGE2) and 8-isoprostane production. ANE-induced EGF production was inhibited by catalase. Addition of anti-EGF neutralizing antibody attenuated ANE-induced cyclooxygenase-2 (COX-2), mature ADAM9 expression and PGE2 and 8-isoprostane production. ANE-induced IL-1α production was inhibited by catalase, anti-EGF antibody, PD153035 (EGF receptor antagonist) and U0126 (MEK inhibitor) but not by α-naphthoflavone (cytochrome p450-1A1 inhibitor). ANE-induced ADAM17 production was inhibited by pp2 (Src inhibitor), U0126, α-naphthoflavone and aspirin. AG490 (JAK inhibitor) prevented ANE-stimulated ADAM17, IL-1α, PGE2 production, COX-2 expression, ADAM9 maturation, and the ANE-induced decline in keratin 5 and 14, but showed little effect on cdc2 expression and EGF production. Moreover, ANE-induced 8-isoprostane production by GKs was inhibited by catalase, anti-EGF antibody, AG490, pp2, U0126, α-naphthoflavone, Zinc protoporphyrin (ZnPP) and aspirin. These results indicate that AN components may involve in BQ-induced oral cancer by induction of reactive oxygen species, EGF/EGFR, IL-1α, ADAMs, JAK, Src, MEK/ERK, CYP1A1, and COX signaling pathways, and the aberration of cell cycle and differentiation. Various blockers against ROS, EGF, IL-1α, ADAM, JAK, Src, MEK, CYP1A1, and COX can be used for prevention or treatment of BQ chewing-related diseases.

Differences in oral habit and lymphocyte subpopulation affect malignant transformation of patients with oral precancer.

BACKGROUND/PURPOSE: In Taiwan, the combination of betel quid chewing, alcohol consumption, and smoking habits increases oral cancer risk by 123-fold compared to persons without these habits. Lymphocyte populations in patients may potentially affect the malignant transformation of oral precancer. METHODS: A total of 28 patients with oral precancer from our previous cohort were enrolled in this study, and their personal information and oral habits were documented. Their lymphocyte populations (CD4+, CD8+, CD19+, and CD56+) and activation markers (CD25 and CD69) were determined by flow cytometry from 1999 to 2004. After follow up till December 2014, data of patients with/without malignant transformation were recorded, and the relation between oral habits and percentage of initial lymphocyte markers was evaluated using the Student t test and Fisher's exact test. RESULTS: Ten precancer patients developed oral squamous cell carcinoma with a mean period of malignant transformation of 6.8 ± 2.1 years. Patients with malignant transformation had a mean age of 48.4 ± 5.0 years (n = 10), relatively more than that of patients without malignant transformation (41.6 ± 6.3 years, n = 18) (p < 0.05). An increase was noted in the population of peripheral blood mononuclear cells expressing CD4+CD69+, CD19+CD69+, and CD56+CD69+ (p < 0.05) in precancer patients with malignant transformation. Alcohol consumption showed an association with the malignant transformation of patients with precancer (p = 0.030), whereas betel quid and smoking showed little effect. CONCLUSION: These results suggest that age, alcohol consumption, and early activation of T cells, B cells, and natural killer cells are crucial in the malignant transformation of oral precancer. Analysis of patient's lymphocyte populations may help predict the malignant transformation of oral precancer.

IL-1ß-induced MCP-1 expression and secretion of human dental pulp cells is related to TAK1, MEK/ERK, and PI3K/Akt signaling pathways.

OBJECTIVE: Interleukin-1ß (IL-1ß) is an inflammatory molecule of the dental pulp. IL-1ß stimulates cyclooxygenase-2 (COX-2) and prostaglandins production of pulp cells and affects the pulpal inflammation and repair. However, the effects of IL-1ß on Monocyte Chemotactic Factor-1 (MCP-1) of dental pulp cells and its relation to transforming growth factor ß-activated kinase-1 (TAK1), PI3K/Akt, and MEK/ERK signaling and COX activation are not fully clear. DESIGN: Human dental pulp cells were exposed to IL-1ß with/without pretreatment and co-incubation by aspirin (a COX inhibitor), 5z-7-oxozeaenol (a TAK1 inhibitor), LY294002 (a PI3K/Akt inhibitor) or U0126 (a MEK/ERK inhibitor). Viable cell number was evaluated by MTT assay. MCP-1 mRNA expression was tested by reverse transcriptase-polymerase chain reaction (RT-PCR). MCP-1 and COX-2 protein expression was studied by western blot. MCP-1 in the culture medium was measure by ELISA. RESULTS: IL-1ß showed little cytotoxicity to pulp cells. It stimulated MCP-1 mRNA and protein expression and MCP-1 secretion. Aspirin, U0126, LY294002 and 5z-7-oxozeaenol attenuated the IL-1ß-induced MCP-1 expression. In addition, 5z-7-oxozeaenol, LY294002, U0126 and aspirin prevented the IL-1ß-induced MCP-1 secretion of pulp cells. CONCLUSION: These results indicate that IL-1ß may be involved in the pulpal inflammatory and healing processes by inducing MCP-1 expression and secretion. These events are related to differential activation of TAK1, PI3K/Akt and MEK/ERK 1/2 signaling and COX activation. These results are important for future pharmacologic intervention of pulpal inflammatory diseases.

Cytotoxicity and transformation of C3H10T1/2 cells induced by areca nut components.

BACKGROUND/PURPOSE: Betel quid (BQ) chewing is popular in Taiwan and many other countries. There are about 200-600 million BQ chewers in the world. BQ chewing is one major risk factor of oral cancer and oral submucous fibrosis (OSF). While areca nut (AN), a main component of BQ, exhibits genotoxicity, its transformation capacity and its role in the initiation and promotion stages of carcinogenesis are not fully clear. METHODS: Mouse C3H10T1/2 cells were exposed to AN extract (ANE) for 24 hours. Cytotoxicity was evaluated by colony forming efficiency. For the transformation assay, C3H10T1/2 cells were exposed to ANE for 24 hours and then incubated in medium with/without 12-O-tetradecanolylphorbol-13-acetate (TPA; a tumor promoter) for 42 days. Cells were stained with Giemsa and type II and type III transformed foci were counted for analysis of the transformation capacity of ANE. RESULTS: ANE exhibited cytotoxicity to C3H10T/12 cells at concentrations higher than 320 µg/mL as shown by a decrease in colony numbers. ANE (80-640 µg/mL) alone mildly stimulated the transformed foci formation (p > 0.05). In the presence of TPA, ANE (80-640 µg/mL) markedly stimulated the transformed foci formation. The percentage of dishes with foci increased from 0% in controls to 20% in ANE (80 µg/mL and 320 µg/mL)-treated groups and further increased to 65-94% in ANE plus TPA groups. CONCLUSION: These results indicate that ANE is a weak complete carcinogen. ANE is an effective tumor initiator and can induce malignant transformation of C3H10T1/2 cells in the presence of a tumor promoter. ANE may be involved in multistep chemical carcinogenesis by its malignant transformation capacity.

Effects of Camphorquinone on Cytotoxicity, Cell Cycle Regulation and Prostaglandin E2 Production of Dental Pulp Cells: Role of ROS, ATM/Chk2, MEK/ERK and Hemeoxygenase-1.

Camphorquinone (CQ) is a popularly-used photosensitizer in composite resin restoration. In this study, the effects of CQ on cytotoxicity and inflammation-related genes and proteins expression of pulp cells were investigated. The role of reactive oxygen species (ROS), ATM/Chk2/p53 and hemeoxygenase-1 (HO-1) and MEK/ERK signaling was also evaluated. We found that ROS and free radicals may play important role in CQ toxicity. CQ (1 and 2 mM) decreased the viability of pulp cells to about 70% and 50% of control, respectively. CQ also induced G2/M cell cycle arrest and apoptosis of pulp cells. The expression of type I collagen, cdc2, cyclin B, and cdc25C was inhibited, while p21, HO-1 and cyclooxygenase-2 (COX-2) were stimulated by CQ. CQ also activated ATM, Chk2, and p53 phosphorylation and GADD45α expression. Besides, exposure to CQ increased cellular ROS level and 8-isoprostane production. CQ also stimulated COX-2 expression and PGE2 production of pulp cells. The reduction of cell viability caused by CQ can be attenuated by N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD), but can be promoted by Zinc protoporphyin (ZnPP). CQ stimulated ERK1/2 phosphorylation, and U0126 prevented the CQ-induced COX-2 expression and prostaglandin E2 (PGE2) production. These results indicate that CQ may cause cytotoxicity, cell cycle arrest, apoptosis, and PGE2 production of pulp cells. These events could be due to stimulation of ROS and 8-isoprostane production, ATM/Chk2/p53 signaling, HO-1, COX-2 and p21 expression, as well as the inhibition of cdc2, cdc25C and cyclin B1. These results are important for understanding the role of ROS in pathogenesis of pulp necrosis and pulpal inflammation after clinical composite resin filling.

Role of ALK5/Smad2/3 and MEK1/ERK Signaling in Transforming Growth Factor Beta 1-modulated Growth, Collagen Turnover, and Differentiation of Stem Cells from Apical Papilla of Human Tooth.

INTRODUCTION: Transforming growth factor ß1 (TGF-ß1) plays an important role in cell proliferation, matrix formation, and odontogenesis. This study investigated the effects of TGF-ß1 on stem cells from apical papilla (SCAPs) and its signaling by MEK/ERK and Smad2. METHODS: SCAPs were exposed to TGF-ß1 with/without pretreatment and coincubation by SB431542 (an ALK5/Smad 2/3 inhibitor) or U0126 (a MEK/ERK inhibitor). Cell growth was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay or direct counting of viable cells. Collagen content was determined by using the Sircol collagen assay (Biocolor Ltd, Newtownabbey, Northern Ireland). Cell differentiation was evaluated by measuring alkaline phosphatase (ALP) activity. Smad2 and ERK1/2 phosphorylation was analyzed by Western blotting or PathScan phospho-enzyme-linked immunosorbent assay (Cell Signaling Technology Inc, Danvers, MA). RESULTS: TGF-ß1 stimulated the growth and collagen content of cultured SCAPs. TGF-ß1 stimulated ERK1/2 and Smad2 phosphorylation within 60 minutes of exposure. Pretreatment by U0126 and SB431542 effectively prevented the TGF-ß1-induced cell growth and collagen content in SCAPs. TGF-ß1 stimulated ALP activity at lower concentrations (0.1-1 ng/mL) but down-regulated ALP at higher concentrations (>5 ng/mL). U0126 prevented 0.5 ng/mL TGF-ß1-induced ALP activity but showed little effect on 10 ng/mL TGF-ß1-induced decline of ALP in SCAPs. Interestingly, SB431542 attenuated both the stimulatory and inhibitory effects on ALP by TGF-ß1. CONCLUSIONS: TGF-ß1 may affect the proliferation, collagen turnover, and differentiation of SCAPs via differential activation of ALK5/Smad2 and MEK/ERK signaling. These results highlight the future use of TGF-ß1 and SCAP for engineering of pulpal regeneration and apexogenesis.

Effect of interleukin-1ß on ICAM-1 expression of dental pulp cells: role of PI3K/Akt, MEK/ERK, and cyclooxygenase.

OBJECTIVE: Interleukin-1ß (IL-1ß) is an important inflammatory mediator of the dental pulp. IL-1ß stimulates cyclooxygenase-2 (COX-2) expression and prostanoid production of pulp cells and affects the inflammatory and healing processes of the dental pulp. There are two interleukin-1 (IL-1) receptors, IL-1RI and IL-1RII, with opposing effect after activation. However, the expression of IL-1Rs, the effects of IL-1ß on intercellular adhesion molecule-1 (ICAM-1) of dental pulp cells, and its relation to protein kinase B (Akt) signaling and COX activation are not clear. METHOD: Human dental pulp cells were treated with IL-1ß with/without pretreatment and co-incubation by LY294002 (a PI3K/Akt inhibitor), U0126 [a mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) inhibitor], aspirin (a COX inhibitor), or eugenol (a COX inhibitor) for different time periods. The expression of ICAM-1, IL-1RI, and IL-1RII messenger RNA (mRNA) was evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). ICAM-1 protein expression was examined by western blotting. Soluble ICAM-1 (sICAM-1) level in the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). Activation of Akt and ERK by IL-1ß was measured by Pathscan p-Akt ELISA or western blot. Viable cell number was evaluated by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. RESULTS: Dental pulp cells expressed IL-1RI, but little IL-1RII. IL-1ß stimulated COX-2 and ICAM-1 mRNA and protein expression as well as sICAM-1 production of pulp cells. Aspirin and eugenol enhanced the IL-1ß-induced sICAM-1 production and ICAM-1 expression. IL-1ß rapidly activated Akt and ERK. LY294002 and U0126 attenuated IL-1ß-induced ICAM-1 expression and sICAM-1 production. CONCLUSIONS: These results reveal that IL-1ß may be involved in the pulpal inflammatory processes by stimulating ICAM-1 expression and secretion. These events are associated with IL-1RI expression and differential activation of PI3K/Akt and MEK/ERK and COX. CLINICAL RELEVANCE: Pharmacological inhibition of IL-1ß, IL-1RI, COX-2, ICAM-1, and related signaling pathways (MEK/ERK and PI3K/Akt) may be useful for the control of pulpal inflammation.