Analgesics effect of local diclofenac in third molar surgery: a randomized, controlled trial.

OBJECTIVES: This study aimed to evaluate the effect of locally diclofenac application on postoperative pain, sequalae, and adverse effects following mandibular third molar (MTM) surgery. METHODS: A randomized, crossover, double-blind, controlled trial was conducted in 20 patients who required surgical removal of bilateral symmetrical impacted MTM at two separate appointments. The 40 MTMs were randomly allocated to two groups. One side was assigned 0.1% w/v diclofenac sodium as the diclofenac group. The contralateral side was assigned phosphate-buffered saline (PBS) as the control group. Postoperative pain intensity was measured by visual analogue scale (VAS), where the time when the first pain emerged, the time to first rescue medication, pain at 6 and 24 h after surgery, and the total number of analgesics consumed were recorded. Postoperative swelling and trismus were assessed on postoperative days 2 and 7. The differences of continuous outcomes between two groups were analyzed by paired t-test or Wilcoxon signed-rank test. RESULTS: VAS scores were significantly lower when the first pain emerged and 6 h after surgery in diclofenac group (p < 0.05). The onset of pain in the diclofenac group was significantly longer than in the control group (p < 0.05). Two patients reported mild nausea and dizziness in the diclofenac group. CONCLUSION: This study demonstrates the analgesic effectiveness of 0.1% local application of diclofenac within 6 h postoperative with few side effects. CLINICAL RELEVANCE: Locally diclofenac application is an alternative of postoperative analgesic in MTM surgery which provides pain-free periods within 6 h.

Relationship Between O-GlcNAcase Expression and Prognosis of Patients With Osteosarcoma.

Several studies have demonstrated a role of O-GlcNAcylation (O-GlcNAc) in tumorigenesis of various carcinomas by modification of tumor-associated proteins. However, its implication in the pathogenesis of osteosarcoma remains unclear. This study aimed to investigate the levels of O-GlcNAc and the expressions of O-linked N-acetylglucosamine transferase (OGT) and O-GlcNAcase (OGA) in human osteosarcoma tissues, by using immunohistochemistry; and to find correlations between the levels or expressions and several clinicopathologic parameters. There were 109 first diagnosed osteosarcoma patients, including Enneking stage IIB (n=70) and III (n=39). Correlations between the immunoreactive score (IRS) and clinicopathologic parameters, overall survival, and metastasis-free survival were evaluated. A positive correlation was found between the IRS of OGA and the percentage of postchemotherapeutic tumor necrosis (r=0.308; P=0.017). Univariate analysis revealed significantly lower OGA IRS in metastatic patients (P=0.020) and poor chemotherapeutic-responder patients (P=0.001). By multivariate analysis, presence of tumor metastasis (P=0.002) and lower OGA IRS (P=0.004) was significantly associated with shorter overall survival. Subgroup analysis in stage IIB osteosarcoma (n=70) demonstrated that male sex (P=0.019), presence of tumor recurrence (P=0.026), poor chemotherapeutic responder (P=0.022), and lower OGA IRS (P=0.019) were significantly correlated with short metastasis-free survival. But, lower OGA IRS was the only independent predictor for short metastasis-free survival (P=0.006). Our findings suggested that O-GlcNAc pathway, especially OGA, may involve in pathogenesis and aggressiveness of osteosarcoma. Low level of OGA expression may be used as a poor prognostic indicator.

Involvement of the A disintegrin and metalloproteinase 9 in oral cancer cell invasion.

The aims of this study were to determine the functional roles of the transmembrane glycoprotein, Disintegrin and metalloproteinase domain-containing protein 9 (ADAM 9), in the phosphorylation of epidermal growth factor receptor (EGFR) and AKT and in the aggressiveness of oral cancer cells. Immunohistochemistry and immunoblotting were conducted to determine expression of ADAM 9 and the levels of EGFR phosphorylated at the tyrosine 1173 residue (p-EGFRtyr1173 ) and AKT phosphorylated at the serine 473 residue (p-AKTser473 ) in oral cancer tissues and in the oral cancer cell lines HN5, HN6, HN15, and HN008. Small interfering RNA (siRNA) was used to inhibit expression of ADAM9 mRNA, and thus production of ADAM9 protein, in oral cancer cells. ADAM9-knockdown cells were examined for p-EGFRtyr1173 and p-AKTser473 levels and used for cell proliferation and invasion assays. A positive correlation among overexpression of ADAM 9, p-EGFRtyr1173 , and p-AKTser473 was found in oral cancer tissues. These biomolecules were also overexpressed in HN6 and HN15 cell lines. Expression of ADAM9 in HN6 and HN15 cells was statistically significantly inhibited by siRNA against ADAM9 mRNA (siADAM9) compared with the negative-control siRNA (scramble). The levels of p-AKTser473 , but not those of p-EGFRtyr1173 , were statistically significantly blocked by siADAM9. Although the proliferation rates of ADAM9 knocked-down HN6 and HN15 cells did not differ from those of cells exposed to scramble, a statistically significant decrease in cell invasion was found in these ADAM9-silenced cells. These results suggest a functional role of the ADAM 9/AKT signaling pathway in oral cancer cell invasion, which may be beneficial as a therapeutic target of oral cancer.

Effects of low-dose irradiation on human osteoblasts and periodontal ligament cells.

OBJECTIVE: To investigate the effects of dental x-ray on proliferation and mineralization in human primary osteoblasts as well as on proliferation and apoptotic potential in human periodontal ligament (PDL) cells. DESIGN: Primary osteoblasts and PDL cells were irradiated with various doses of periapical radiography by repeated exposures and further incubated for 1, 3 or 7 days. Cell proliferation was assayed by BrdU incorporation. The effect of dental x-ray on mineralization in osteoblasts either before or after x-ray exposures was determined by Alizarin red staining. Both mRNA and protein expressions of BCL-2, an anti-apoptotic gene, and BAX, a pro-apoptotic gene, in PDL cells were analyzed by RT-qPCR and immunoblotting analysis, respectively. RESULTS: Neither the proliferative nor the mineralization ability of irradiated osteoblasts was different from that of non-irradiated osteoblasts at any doses or time points. By contrast, there was a significant decrease in the proliferation of PDL cells on day 3 after repeated exposures to dental x-ray for 20 times (P < 0.05), whereas the ratio of BCL-2 to BAX mRNA and protein expressions in these irradiated PDL cells was significantly increased (P < 0.05). CONCLUSIONS: Upon multiple exposures to dental x-ray used in intraoral radiography up to 20 times, there is no effect on the proliferation or the mineralization of osteoblasts, whereas the proliferative and apoptotic potentials of PDL cells are transiently decreased.

O-GlcNAcylation in oral squamous cell carcinoma.

BACKGROUND: Two post-translational mechanisms commonly demonstrated in various cancers are protein phosphorylation and glycosylation by O-linked ß-N-acetylglucosamine (O-GlcNAc). However, only phosphorylation of the epidermal growth factor receptor (EGFR)/Akt pathway has been reported in oral squamous cell carcinoma (OSCC). Therefore, we aimed to determine both post-translational modifications in OSCC tissues and in oral cancer cells compared to normal tissues and oral keratinocytes and to find correlations of these modifications with histological grading. METHODS: Thirty-two OSCC and ten normal formalin-fixed and paraffin-embedded sections were probed with the anti-O-GlcNAc, anti-O-GlcNAc transferase (OGT), anti-phosphorylated-EGFRtyr1173 , and anti-phosphorylated-Aktser473 antibodies following standard immunohistochemistry. The immunohistochemical (IHC) score was determined using the Fromowitz standard. Whole cell lysates of oral cancer cells and normal oral keratinocytes were immunoblotted with the anti-O-GlcNAc antibody. RESULTS: The median IHC scores of O-GlcNAc or OGT between OSCC and normal tissues were not different, whereas those of phosphorylated-EGFRtyr1173 and phosphorylated-Aktser473 were significantly higher in OSCC than normal tissues (P < .001 and P < .01, respectively). Similarly, expression of O-GlcNAcylated proteins in oral cancer cells and normal oral keratinocytes did not differ. In the OSCC group, the median IHC scores of O-GlcNAc and OGT were significantly lower than those of phosphorylated-EGFRtyr1173 and phosphorylated-Aktser473 (P < .01 and P < .001, respectively). The IHC scores of O-GlcNAc or OGT were not determined to correlate with histological grading. CONCLUSION: Unlike other types of cancers, our findings demonstrate that the levels of O-GlcNAcylation are not significantly increased in OSCC tissues or in oral cancer cells and are not associated with the histological grading of OSCC.

The antimicrobial peptide, human ß-defensin-1, potentiates in vitro osteoclastogenesis via activation of the p44/42 mitogen-activated protein kinases.

Previous studies have demonstrated increased expression and raised levels of human ß-defensin (hBD)-1 in gingival tissue and crevicular fluid of patients with chronic periodontitis and peri-implantitis, oral bone-resorbing diseases caused by enhanced osteoclastogenesis. Therefore, we aimed to investigate the effect of hBD-1 on osteoclast formation and function and to elucidate the involved signaling pathway in vitro. Human peripheral blood mononuclear cells (PBMCs) were first incubated with various doses of hBD-1 and cell viability was assayed by MTT. PBMCs were treated with macrophage-colony stimulating factor and receptor activator of nuclear factor kappa-B ligand (RANKL) in the presence or absence of non-toxic doses of hBD-1. In vitro osteoclastogenesis was analyzed by tartrate-resistant acid phosphatase (TRAP) staining, osteoclast-specific gene expression, and a resorption pit assay. Involvement of mitogen-activated protein kinases (MAPKs) was studied by immunoblotting and specific MAPK inhibitors. HBD-1 potentiated induction of in vitro osteoclastogenesis by RANKL, as shown by significantly increased number of TRAP-positive multinuclear cells and resorption areas on the dentin slices, and further up-regulated expressions of osteoclast-specific genes compared to those by RANKL treatment (p <0.05). However, hBD-1 treatment without RANKL failed to induce formation of osteoclast-like cells. A significant and further increase in transient phosphorylation of the p44/42 MAPKs was demonstrated by hBD-1 co-treatment (p<0.05), consistent with the inhibitory effect by pretreatment with U0126 or PD98059 on hBD-1-enhanced osteoclastogenesis. Collectively, hBD-1 potentiates the induction of in vitro osteoclastogenesis by RANKL via enhanced phosphorylation of the p44/42 MAPKs.

In vitro oral epithelium cytotoxicity and in vivo inflammatory inducing effects of anesthetic rice gel.

In vitro cytotoxicity of lidocaine hydrochloride (LH) and prilocaine hydrochloride (PH) to oral epithelial cells, isolated from tissue specimens of healthy volunteers, were evaluated. Cell vitality after treating with 1-20% anesthetic solutions for 5 and 30 min was investigated using F-actin and 4',6-diamidino-2-phenylindole staining technique and observed by fluorescence microscopy. Vitality rate of more than 90% was found in all anesthetic groups at both durations whereas no survived cell was found in a positive control group (sodium dodecyl sulfate). Lactate dehydrogenase (LDH) assay was performed to confirm the safety of both anesthetic solutions. Cell culture medium after treating with LH or PH for 5 and 30 min were collected and analyzed using commercial kits. The results showed no significant difference between the test groups and negative control group (untreated culture) with low LDH levels. In vivo inflammatory inducing effect of 5, 10, 20% LH or PH loaded rice gels was investigated in healthy volunteers. Tumor necrosis factor alpha (TNF-α) in gingival cervicular fluid was determined by ELISA technique. It was found that the expression of TNF-α was not different from the baseline. The expression of this inflammatory mediator caused by the commercial gel was higher than those of both anesthetic rice gels. It might be due to the effects of other excipients in the formulation of the commercial product. It is concluded that LH or PH possess no cytotoxicity to oral epithelium and the developed rice gel base and LH and PH rice gels do not induce inflammatory effect to oral tissues.

Akt2 and p-Akt overexpression in oral cancer cells is due to a reduced rate of protein degradation.

OBJECTIVE: To quantitatively measure the increased expression of Akt2 and its phosphorylated form (p-Akt) in oral cancer cell lines and investigate the post-translational mechanism for Akt2 and p-Akt overexpression. METHODS: Three oral cancer cell lines and three cell lines of primary human oral keratinocytes (HOKs) were cultured and the degrees of Akt2 and p-Akt expression was evaluated by immunoblot analysis and flow cytometry. Each cell line was incubated with cycloheximide, an inhibitor of new protein synthesis, for various times to quantitatively determine the remaining expression levels of Akt2 and p-Akt by flow cytometry. The localization of Akt2 and p-Akt was assessed by immunofluorescence. RESULTS: The levels of Akt2 and p-Akt proteins were significantly higher in cancer cell lines than those in HOKs (P < 0.05). When the new protein synthesis was blocked by cycloheximide treatment, the degradation rate of Akt2 and p-Akt in oral cancer cells was significantly lower than that in HOKs (P < 0.05). Both Akt2 and p-Akt were more intensely stained in the cytoplasm of cancer cells, whereas HOKs expressed Akt2 and p-Akt only minimally. CONCLUSION: Both Akt2 and p-Akt were overexpressed in oral cancer cells, which may be partly explained by a reduced rate of protein degradation in order to maintain high cytosolic levels.

CLCN7 and TCIRG1 mutations differentially affect bone matrix mineralization in osteopetrotic individuals.

Osteopetrosis is an inherited disorder of impaired bone resorption, with the most commonly affected genes being CLCN7 and TCIRG1, encoding the Cl(-) /H(+) exchanger CLC-7 and the a3 subunit of the vacuolar H(+) -ATPase, respectively. We and others have previously shown that the disease is frequently accompanied by osteomalacia, and that this additional pathology is also found in Tcirg1-deficient oc/oc mice. The remaining question was whether osteoid enrichment is specifically associated with TCIRG1 inactivation, or whether CLCN7 mutations would also cause skeletal mineralization defects. Here we describe a complete osteologic assessment of one family carrying a novel mutation in CLCN7 (D145G), which impairs the activation and relaxation kinetics of the CLC-7 ion transporter. The two siblings carrying the mutation in the homozygous state displayed high bone mass, increased serum levels of bone formation markers, but no impairment of calcium homeostasis when compared to the other family members. Most importantly, however, undecalcified processing of an iliac crest biopsy from one of the affected children clearly demonstrated a pathological increase of trabecular bone mass, but no signs of osteomalacia. Given the potential relevance of these findings we additionally performed undecalcified histology of iliac crest biopsies from seven additional cases with osteopetrosis caused by a mutation in TNFRSF11A (n=1), CLCN7 (n=3), or TCIRG1 (n=3). Here we observed that all cases with TCIRG1-dependent osteopetrosis displayed severe osteoid accumulation and decreased calcium content within the mineralized matrix. In contrast, there was no detectable bone mineralization defect in the cases with TNFRSF11A-dependent or CLCN7-dependent osteopetrosis. Taken together, our analysis demonstrates that CLCN7 and TCIRG1 mutations differentially affect bone matrix mineralization, and that there is a need to modify the current classification of osteopetrosis.

Long-term survival in infantile malignant autosomal recessive osteopetrosis secondary to homozygous p.Arg526Gln mutation in CLCN7.

Infantile malignant autosomal recessive osteopetrosis (ARO; OMIM 259700) has been reported to be associated with mutations in TCIRG1, CLCN7, or OSTM1. ARO caused by homozygous (or compound heterozygous) mutations in CLCN7, as described here, is usually diagnosed at birth or early in infancy due to generalized osteosclerosis and severe hematologic deficits. The maximal life expectancy of patients with ARO in the absence of bone marrow transplantation is thought to be 10 years. We report on a 25-year-old Thai man who is affected with ARO. Clinical features include proportionate short stature, vision impairment, esotropia, exophthalmos, mild hearing loss, and hepatosplenomegaly. Pancytopenia was present and the patient had frequent illnesses. Radiographs showed generalized osteosclerosis with almost no visible of bone marrow spaces. Dense maxilla and mandible with impacted and malformed teeth were observed. Multiple fractures were reported. He developed osteomyelitis of the mandible on four separate occasions, and partial mandibulectomy was performed. Molecular studies showed that there were no pathogenic mutations in TCIRG1. However, mutation analysis of CLCN7 revealed a homozygous missense mutation (p.Arg526Gln). This patient is, it appears, the longest lived individual with ARO ever reported. Evaluation of osteoclastogenesis in our patient demonstrated very large immature osteoclasts with a high number of nuclei.

Traditional chewing and smoking habits from the point of view of Northern Thai betel quid vendors.

PURPOSE: To evaluate knowledge of betel quid (BQ) vendors in relation to traditional chewing and smoking habits in Northern Thailand. MATERIALS AND METHODS: Interviews of vendors selling BQ and other traditional chewing and smoking items were conducted. Questions related to side effects of BQC were included, as well as questions focusing on why traditional chewing and smoking habits were on the decline. RESULTS: Nineteen stalls in 10 markets were visited and 18 vendors were interviewed (16 women, 2 men, average age 55.0 years, range 28-75 years). Vendors had been present for an average of 21.8 years (range 2-60 years). The number of customers buying BQ regularly was 2-3 per day. More elderly women than men bought BQ. Side effects of BQ on the oral mucosa were largely unknown to vendors. Most respondants thought BQ to be good for teeth. Reasons why young people have given up the BQ habit were black teeth. Miang (fermented tea leaves) and khi yo (traditional cigar) were rarely sold and were considered vanishing habits. CONCLUSIONS: BQ vendors had poor knowledge of the side effects of BQC. BQ vendors unanimously considered traditional habits such as chewing of BQ, miang and smoking of traditional cigars to be on the decline. Nowadays, most of these items are bought to be offered during ceremonies. Generally, traditional habits seem to be replaced by 'modern' lifestyle habits such as cigarette smoking and alcohol consumption. With these changes, general and oral disease patterns will eventually occur.