Takashi Ikeo

A torque removal study on the primary stability of orthodontic titanium screw mini-implants in the cortical bone of dog femurs.

The aim of this study was to biomechanically evaluate the primary stability of pure titanium orthodontic mini-implants, inserted into pre-drilled cavities of differing diameters. Mini-implants (1.2 mm diameter) were placed into 1.0 mm and 1.2 mm diameter cavities prepared in the mid-region of the bilateral hind leg femurs of anesthetized beagles. Removal torque strengths were measured immediately, 1, 3, 6, 9 and 12 weeks post-insertion of the implant. For mini-implants placed into 1-mm cavities, removal torque values decrease over the first 6 weeks (p<0.01), after which values remained static. Average values obtained immediately, 1, 3 and 6 weeks post-insertion were 10.98, 8.83, 7.20 and 5.12 Ncm, respectively . Immediately post-insertion, removal torque values of mini-implants placed in a 1.2-mm cavity, were 11-fold lower than those placed in 1.0-mm cavities, which then demonstrated a significant increase in strength from 3 weeks (1.35 Ncm) to 6 weeks (5.17 Ncm) post-insertion (p<0.01). Measurements 6, 9 and 12 weeks post-insertion were similar to those in the 1.0-mm cavity. Initial stability of titanium mini-implants is considered necessary for immediate and early use in orthodontics, and an implant without this initial stability should be replaced or isolated until it develops the appropriate stability supported by osseointegration.

Suppression of alveolar bone resorption by etidronate treatment for periodontal disease: 4- to 5-year follow-up of four patients

Four women with periodontitis received intermittent cyclical etidronate (etidronate administered orally at a dose of 200 mg/day for 2 weeks, at intervals of 10-12 weeks or 6 months) for 4-5 years in addition to ordinary dental therapy. Alveolar bone density was measured using a new method comparing the percentage increase in density. Mean alveolar bone density increased significantly during intermittent cyclical etidronate treatment. Significant reductions were observed in the mobility of the teeth and the depth of periodontal pockets. There were significant correlations between alveolar bone density and both mobility of the teeth and the depth of the alveolar pockets. It is concluded that increases in alveolar bone density are associated with the clinical benefits of etidronate in the treatment of periodontitis.

Correlation of P-cadherin and β-catenin expression and phosphorylation with carcinogenesis in rat tongue cancer induced with 4-nitroquinoline 1-oxide

Using biochemical and immunohistochemical techniques, we have investigated P-cadherin, beta-catenin, c-src and c-met protein expression, and phosphorylation of beta-catenin in a rat model of tongue cancer induced with 4-nitroquinoline 1-oxide. Six-week-old male Sprague-Dawley rats were given either normal drinking water (controls) or 50 ppm 4NQO solution as drinking water for 16 and 20 weeks. This treatment produced dysplasia and well-differentiated squamous cell cancer in rat tongues after 16 and 20 weeks, respectively. In controls, P-cadherin and beta-catenin were expressed only in cell membranes of tongue suprabasal epithelial cells, whereas strong reaction to P-cadherin antibody was observed during carcinogenesis, especially in nests of cancer cells. However, dysplastic and cancer cells expressed beta-catenin not only in cell membranes but also in the nuclear and cytoplasmic compartments. During carcinogenesis, immunohistochemical reaction to phosphotyrosine increased gradually. Reaction to the c-src product was strongest at the dysplastic stage and, to the c-met product, at the cancer stage. In addition, western blotting analysis showed a marked increase in the expression of beta-catenin and phosphotyrosine in dysplastic and cancer cells compared with the controls. Using immunoprecipitation and western blotting techniques, we found that phosphorylated beta-catenin gradually increased during carcinogenesis. These experiments demonstrate that cell-cell adhesion in epithelial cells was reduced by phosphorylation of beta-catenin and that beta-catenin overexpression in nuclear and cytoplasmic compartments during carcinogenesis and the production of the c-met product that is associated with the phosphorylation of beta-catenin in tongue cancer.

Enamel Matrix Derivative Protein Stimulated Wound Healing via Phosphoinositide 3-Kinase

BACKGROUND Enamel matrix derivative (EMD) protein has been clinically used for periodontal regeneration, but the molecular mechanisms are not clear. Previous studies suggested that the activation of phosphoinositide 3-kinase (PI 3-kinase) plays a key role in facilitating cell migration. Given that the migration of osteoblasts is one of the key steps in the wound-healing processes, we hypothesized that EMD protein would stimulate osteoblast migration by activating PI 3-kinase. In this study, we tested this hypothesis using MG-63 cells as model systems to evaluate mechanisms of migration by stimulation with EMD protein. METHODS Confluent MG-63 cells were mechanically scratched using a sterilized 1-mm pipette tip that removed the cells within a circular area. The wells were incubated for 24 hours in various stimulation conditions (25, 50, or 100 microg/ml EMD protein) with or without the PI 3-kinase inhibitor wortmannin (1, 10, and 100 nM) or LY294002 (1, 10, and 100 microM). Migrated cells in the wound section were counted by randomly selecting three areas from one well. The activation of PI 3-kinase by EMD protein was evaluated by the phosphorylation of Akt using Western blot analysis. RESULTS Although EMD protein did not affect proliferation, it enhanced migration into wounds on MG-63 cells. We showed that EMD protein enhanced the phosphorylation of Akt in a dose-dependent manner. We demonstrated that the PI 3-kinase inhibitors wortmannin and LY294002 blocked migration into wounds and the phosphorylation of Akt enhanced by EMD protein in MG-63 cells. CONCLUSION These results demonstrated that the activation of PI 3-kinase plays a key role in the EMD protein-stimulated migration of osteoblasts.

RNA interference-mediated knockdown of Smad1 inhibits receptor activator of nuclear factor κB ligand expression induced by BMP-2 in primary osteoblasts

OBJECTIVE BMP-2 induces osteoblast differentiation and activates osteoclast formation. Here, we investigated the role of Smad1, a molecule that signals downstream of BMP-2, in mediating the effects of BMP-2 on osteoclast differentiation induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in osteoblasts. DESIGN The effects of 1,25(OH)2D3 and BMP-2 in osteoclasts were examined using polymerase chain reaction and Western blotting to measure changes in target gene and protein expression. Immunostaining was carried out to investigate the localization of the vitamin D receptor (VDR) in the nucleus in response to BMP-2. RESULTS Stimulation with both 1,25(OH)2D3 and BMP-2 resulted in significantly greater osteoclast formation and receptor activator of nuclear factor κB ligand (RANKL) mRNA expression compared to stimulation with 1,25(OH)2D3 alone. In addition, expression of the VDR protein was increased, enhancing the activity of 1,25(OH)2D3. Interestingly, knockdown of Smad1 resulted in reduced osteoclast formation, RANKL mRNA expression, and VDR protein expression compared with control cells. Costimulation with 1,25(OH)2D3 and BMP-2 enhanced VDR localization in the nucleus. CONCLUSIONS We found that BMP-2 induced Smad1 activation, thereby influencing the localization of VDR in the nucleus in the presence of 1,25(OH)2D3 and resulting in increased RANKL mRNA expression. These effects ultimately resulted in enhanced osteoclast differentiation.

Expression patterns of CD66a and CD117 in the mouse submandibular gland

The epithelial tissue of the salivary gland consists of the acinar and ductal parts, the latter of which is further divided into the intercalated, striated and excretory duct segments and is the residential site for salivary stem/progenitor cells. In the present study, the expression patterns of two cell surface molecules, CD66a and CD117, were investigated in the adult mouse submandibular glands (SMG) by immunofluorescence microscopy. Combinations of the two molecules differentially marked several types of SMG epithelial cells, including acinar cells (CD66a-intense, CD117-negative), intercalated duct cells (CD66a-intense, CD117-positive), a subset of the striated and excretory duct cells (CD66a-weak, CD117-positive). Most of the CD117-positive ductal cells were negative for cytokeratin 5 and overlapped with the NKCC1-expressing cells. The CD117- and keratin 5-positive cells resided only in the excretory duct were suggested to correspond to the recently identified salivary stem cells. CD66a and CD117 may be useful markers to isolate several cell types consisting of SMG epithelium and to analyze their molecular and cellular nature. Our data also suggest that CD117-expressing epithelial cells of the gland include at least two distinct populations of the stem/progenitor cells.

Cytokine-mediated activation of human ex vivo-expanded Vγ9Vδ2 T cells

Vγ9Vδ2 T cells, the major subset of the human peripheral blood γδ T-cell, respond to microbial infection and stressed cells through the recognition of phosphoantigens. In contrast to the growing knowledge of antigen-mediated activation mechanisms, the antigen-independent and cytokine-mediated activation mechanisms of Vγ9Vδ2 T cells are poorly understood. Here, we show that interleukin (IL) -12 and IL-18 synergize to activate human ex vivo-expanded Vγ9Vδ2 T cells. Vγ9Vδ2 T cells treated with IL-12 and IL-18 enhanced effector functions, including the expression of IFN-γ and granzyme B, and cytotoxicity. These enhanced effector responses following IL-12 and IL-18 treatment were associated with homotypic aggregation, enhanced expression of ICAM-1 and decreased expression of the B- and T-lymphocyte attenuator (BTLA), a co-inhibitory receptor. IL-12 and IL-18 also induced the antigen-independent proliferation of Vγ9Vδ2 T cells. Increased expression of IκBζ, IL-12Rβ2 and IL-18Rα following IL-12 and IL-18 stimulation resulted in sustained activation of STAT4 and NF-κB. The enhanced production of IFN-γ and cytotoxic activity are critical for cancer immunotherapy using Vγ9Vδ2 T cells. Thus, the combined treatment of ex vivo-expanded Vγ9Vδ2 T cells with IL-12 and IL-18 may serve as a new strategy for the therapeutic activation of these cells.

Expression of Plectin-1 and Trichohyalin in Human Tongue Cancer Cells

In basal squamous cells, plectin-1 interacts with intermediate filaments, whereas trichohyalin, which is distributed primarily in the medulla and inner root sheath cells of human hair follicles, plays a role in strengthening cells during keratinization. Although both cytoskeletal proteins occur in trace amounts in human tongue epithelial cells, there are minimal data on their expression in human tongue primary cancer cells. We therefore investigated the expression of plectin-1 and trichohyalin in human tongue epithelial cell line (DOK) and tongue cancer cell line (BICR31) using western blotting and FITC-labeled immunocytochemistry techniques. DOK and BICR31 cells were cultivated to subconfluence in Dulbecco’s Modified Eagle’s Medium containing 0.4 μg/ml of hydrocortisone and 10% fetal bovine serum, and the levels of trichohyalin and plectin-1 were determined by western blot analysis and immunocytochemical staining. Trichohyalin expression was clearly observed, with no differences between DOK and BICR31 cells. Although DOK cells expressed trace levels of plectin-1, obvious plectin-1 bands were detected in western blot analyses of BICR31 cells. Immunocytochemical staining revealed that trichohyalin and plectin-1 localize in the cytoplasm. Trichohyalin was diffusely distributed in both cell lines, and colocalization of trichohyalin and cytokeratin 1/10 was observed in almost all BICR31 cells. There were no correlations between western blot and immunocytochemical data for trichohyalin. Conversely, correlations in immunochemical reactions for plectin-1 were observed. Most DOK cells showed no localization of plectin-1, but strong reactions were detected in the cytoplasm of BICR31 cells. These results indicate that trichohyalin is expressed by cancerous tongue epithelial cells during various stages of malignancy and that plectin-1 provides an index of malignancy.

Enamel matrix derivative protein enhances production of matrixmetalloproteinase-2 by osteoblasts

BackgroundMatrix metalloproteinases (MMPs) degrade the extracellular matrix (ECM) and regulate remodeling and regeneration of bone. Enamel matrix derivative (EMD) protein has been used clinically for periodontal regeneration, although its molecular mechanisms are not clear. We evaluated the role of matrix metalloproteinases (MMPs) in regulating EMD-dependent degradation of gelatin on oeoblast-like cell line MG63.MethodsMG-63 cells (osteoblast cell line) were incubated with 100 μg/ml EMD protein in the presence or absence of MMP-2 tissue inhibitor for 20 h followed by incubation on DQ-gelatin-coated plates for 4 h. MG-63 cells (1 × 106) were preincubated with SB203580 for 30 min at 37°C and were then placed in 100 μg/ml EMD protein for 24 h. Conditioned media were collected and detected by Western blot analysis.ResultsEMD protein enhanced cell-mediated degradation of gelatin, which was inhibited by the MMP inhibitor TIMP-2. Furthermore, MMP-2 was produced by MG63 cells in response to EMD protein in a P38 MAPK-dependent manner. In addition, blocking of p38 MAPK activation by SB203580 significantly inhibited generation of the active form of MMP-2.ConclusionP38 MAPK pathway promotes expression MMP-2 in EMD activated osteoblasts, which in turn stimulates periodontal regeneration by degrading matrix proteins in periodontal connective tissue.