Aiko Kamada

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.

Disaccharide analysis of chondroitin sulphate in human gingival crevicular fluid using high-performance liquid chromatography.

Gingival crevicular fluid (GCF) was collected into capillary tubes from healthy gingiva and sites of advanced periodontitis. Following digestion with Pronase E, the glycosaminoglycans were isolated by successive precipitation into 5% cetylpyridinium chloride and 95% ethanol. Unsaturated disaccharide isomers of chondroitin sulphate, obtained following chondroitinase ACII digestion, were analysed by high-performance liquid chromatography. Chondroitin sulphate was found in all GCF samples, with greater amounts in patients with periodontal disease than at control sites with a relatively healthy periodontium. The predominant isomer in the periodontal diseased group was delta Di-4S, while that in the control group and serum samples was delta Di-0S. Comparison of the relative proportions of the unsaturated disaccharides in GCF with previously reported values for alveolar bone, cementum, gingiva and periodontal ligament, as well as for serum, indicates that the chondroitin sulphate present in GCF of patients with periodontal disease originated from the mineralized connective tissues of the periodontium, notably alveolar bone, possibly with some contributions from soft connective tissues of gingiva and periodontal ligament and from serum.

Adsorption of glycosaminoglycans onto hydroxyapatite using chromatography

Proteoglycans are known to play an important role in the mineralization process, acting either as promoters or inhibitors. In this study the binding affinity of a variety of constituent glycosaminoglycan to hydroxyapatite was studied. Glycosaminoglycans (10-1000 microg ml(-1) in 0.02 M sodium acetate (pH 6.8) were constantly circulated through a hydroxyapatite column for 1 h. The total amount of glycosaminoglycan bound was determined by dimethylmethylene blue assay. The relative affinities of the different glycosaminoglycans remaining bound to hydroxyapatite was investigated by examining their release in a 0-1 M sodium phosphate gradient. Differences were noted between the desorption profiles of dermatan sulfate with two elution peaks and chondroitin 4-sulfate and chondroitin 6-sulfate each with a single peak. Dermatan sulfate and chondroitin 6-sulfate had a higher affinity for hydroxyapatite than chondroitin 4-sulfate possibly due to the presence of differing di-sulfated disaccharide ratios in the glycosaminoglycan chains. These findings suggest the presence of a variety of binding forms of each glycosaminoglycan or the differing orientation of these forms to yield different complexes with hydroxyapatite. The Ca2+ co-ordinates of the glycosaminoglycans are known to vary and may in part explain these findings.

CHARACTERISTICS AND LOCALIZATION OF RAT SUBMANDIBULAR GLAND PROTEOGLYCANS

The submandibular gland proteoglycans were investigated biochemically and immunohistochemically in male Sprague-Dawley rats. Proteoglycans were extracted with 4 M guanidine-HCl, followed by ultracentrifugation in a CsCl density gradient, and fractionated by ion-exchange chromatography and gel filtration. The molecular weight of PGs was estimated by SDS-PAGE and immunoblot analysis with monoclonal antibodies (HepSS-1 or 6-B-6). The glycosaminoglycan side-chains in the proteoglycan fractions were identified by electrophoresis on cellulose acetate membrane. Three proteoglycan fractions were obtained. One was a heparan sulphate proteoglycan that migrated as a diffuse band of about 210 kDa. The other two fractions contained at least two dermatan sulphate proteoglycans of 70-85 kDa and 40-50 kDa. Digestion of these two proteoglycans with chondroitinase ABC, but not heparitinase, produced two bands of 50 and 21 kDa, which were core proteins. The smaller dermatan sulphate proteoglycan may be a portion of the other, as the core protein of both bound to 6-B-6 antibody, and sugar chains of both were the same (20-30 kDa). Heparan sulphates recognized by antibody HepSS-1 were observed widely in the basement membrane, fibrous connective tissue, and striated and excretory ductal cells, while dermatan sulphate proteoglycans recognized by antibody 6-B-6 were located in the connective tissue surrounding striated and excretory ducts.

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 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.