The early growth response (EGR) gene family is nuclear transcription factor and is implicated in regulating cell proliferation. In this study, we investigated the effect of insulin-like growth factor (IGF) -I, which relates to oral tissue reconstruction, on the expression and localization of EGR-1 and EGR-2 in cultured human periodontal ligament fibroblasts (PDLFs) using molecular biological and immunocytochemical techniques. Three PDLF cell lines were first cultured for two days in serum-free Dulbecco's modified Eagle medium (DMEM) and further stimulated with various concentrations of IGF-I (1-100 ng/mL in DMEM). Stimulation of the cells with IGF-I increased, in a dose-dependent manner, the proliferation index of the cells as measured by 5-bromo 2'-deoxyuridine incorporation. The mRNA levels of EGR-1 peaked after 45 min of incubation of the cells with IGF-I at a concentration of 10 ng/mL. Further, the expression of EGR-2 mRNA was observed subsequent to incubation with IGF-I at a concentration of 1ng/mL. These changes correlated well with those seen at the protein levels. Immunocytochemical analyses showed strong immunostaining in or around the nucleus for EGR-1 or EGR-2 in IGF-I-treated cells. These results suggest that the EGR genes induced in IGF-I-stimulated human PDLF and may involve a potential important role in PDLF growth during tissue regeneration and healing in vivo.
It has recently been reported that statins, the cholesterol-lowering drug, activate the promoter of the bone morphogenic protein-2 (BMP-2) gene, and stimulate bone forma-tion. However, the mechanism of the stimulation of bone metabolism by statins is not precisely clarified. In this study, we investigated whether statins effect the expression of osteogenic master transcription factors, Runx2/Cbfa1 and Dlx5, as a downstream target of BMP-2. The human osteoblastic osteosarcoma cell line, SaOS-2, was used for this experiment. RT-PCR analysis demonstrated continuous overexpression of Runx2 in SaOS-2, though the expression was hardly observed in normal human osteoblasts. Therefore we considered SaOS-2 a model for the overexpression of the transcription factor. When the osteosarcoma cells were incubated with compactin, there was a significant decrease in mRNA level of Runx2 compared with controls (p<0.01). There appeared to be no overall changes in Dlx5. These results demonstrate that statins suppress the overexpression of Runx2 mRNA, and it hardly effects the expression of Dlx5. It is known that Runx2 is essential for the initial osteoblastic differentiation, but it inhibits the final differentiation. Hence, it is indicated that statins promote not only the initial osteoblastic differentiation but also the final differentiation through regulation of transcription factors.
The aim of this study was to investigate the effect of bioabsorbable material on the direct pulp capping to know whether this material has a function in dentin regeneration by bioabsorption. Class V cavities with the exposed pulp area were prepared on the buccal surface of first molars in young beagle dogs. All cavities were conditioned with a self-etching primer (Clearfil SE Primer). In the cavities of right side (experimental group), the exposed pulp was covered with a bioabsorbable sheet (Kurabio AG), and then the cavities were coated with an adhesive agent (Clearfil SE Bond) and resorted with a resin composite. The left side cavities (control group) were applied the adhesive agent without the bioabsorbable sheet and restored with a resin composite. On three and six months after surgery, the teeth were extracted, immersion-fixed and decalcified. Afterward, the teeth were frozen-sectioned, stained with hematoxylin and eosin and observed using a light microscopy. In the experimental group, a large amount of the tertiary dentin was formed into the prepared cavity. On the other hand, the tertiary dentin was almost not formed into the cavity in the control group. The significantly tertiary dentin formation was recognized into the prepared cavity when the exposed pulp area had been covered by a bioabsorbable material. This result suggested that the space making by a bioabsorbable material could produce the derivation of pulp tissue and then dentin regeneration into the pre-pared cavity.
Spheroids were formed for human fetal hepatocytes on poly-L-glutamic acid-coated polystyrene dishes that had a negatively charged surface at neutral pH. The optimum concentration of poly-L-glutamic acid solution for coating the surface of the dish was 1mg/ml. The optimum cell density of inoculation for the spheroid formation was 3.4×104 cells/cm2 (4.0×105 cells/35mm-dish). The cytoskeletal actin filaments of the cells were reorganized from stress fiber in the monolayer to cortical actin in the spheroid. The spheroid formation was inhibited by cytochalasin D, which is an inhibitor of actin polymerization. Furthermore, the activity (6.6 pmol/106nuclei/min) of cytochrome P450 1A1/2 of cells in the spheroid was almost threefold higher than that of those in the monolayer, and was comparable with that of human mature hepatocytes of primary culture. These results suggest that the reorganization of actin filaments in hepatocytes would be essential for the spheroid formation, and the spheroid culture seems to be suitable for the expression of differentiated functions of human hepatocytes.
The effect of hypothyroidism induced by thiamazole on the toxicity of fluconazole was studied in chick embryos. To our knowledge, no information is now available on the effects of thyroid gland function on the regeneration and differentiation of tissue. Fertilized eggs of White Leghorns were incubated and investigated. 1.2 mg/0.2 mL/egg of thiamazole was injected into the albumen of fertilized eggs on the 9th day of incubation. Fluconazole at 0.4 mg/egg was injected into the air sac of fertilized eggs on the 16th day of incubation. Electrocardiograms were recorded 0 to 60 min after the injection. After the injection of fluconazole into the thiamazole-treated eggs, the heart rate was significantly decreased compared with the untreated eggs. These findings indicate that hypothyroidism induced by thiamazole has a marked influence on the toxicity of fluconazole in chick embryos.
This study was designed to examine the effects of bone formation induced by recombinant human BMP-2 (rhBMP-2), which was implanted together with a poly-lactate-polyglycolate-copolymer/gelatine sponge (PGS) at palatal sites in 30-week- old male Wister rats. The rats were divided into four groups according to the dose of rhBMP-2: 0µg, 4µg, 8µg and 16µg. New bone formation was observed in every group, and new bone was continuous with the original bone at 6 weeks after implantation. Thickness of new bone (TNB) increased as the dose increased from 0µg to 8µ, reached the maximal level at 8µg. In contrast, TNB decreased significantly as the dose increased from 8µg to 16µg. The results of this study indicate that new bone formation varies according to the dose of rhBMP-2 at palatal sites in adult rats.
The present study was designed to examine the crystal nature of dentin, and the relationship between the crystal orientation and the mechanism of tooth formation among the monophyodont teeth, diphyodont teeth and polyphyodont teeth. The apatite crystal of dentin was studied using the curve IP x-ray Laue system and the laser Raman microprobe spectrometry. The crystal orientation was very poorly orientated in the enamel-covered crown dentin of the diphyodont. In the enamel-covered dentin, calcospherites were present. In the presence of calco-spherites, the crystal orientation was arranged radially. The crystals were orientated indistinctly in the cementum-covered root dentin of the diphyodont. In the cementum-covered dentin, the axis of crystal orientation was almost parallel to the tooth axis and to the direction of the collagen fibers. In the enamel-covered crown dentin from the monophyodont and polyphyodont, the crystal orientation was recognized as similar to the cementum-covered root dentin. In the Raman spectrum of dentin, the peak of 1074cm-1 (CO32-) in the monophyodont teeth was lower than that of diphyodont teeth. The results suggest that there is a relationship between the mechanism of the tooth formation and the characteristic of dentin crystal. It is considered that the analysis of structural mechanism of the dentin gives much information to the regeneration research.
The information about periodontal conditions is necessary for regeneration of periodontal tissues. The measurement of sulfated glycosaminoglycans (S-GAGs) in gingival crevicular fluid (GCF) is important in the search for disease biomarkers. To analyze the amount of S-GAGs in GCF quickly and accurately, we have modified a microplate method previously used for urinary S-GAGs to estimate the content of S-GAGs in GCF without interference of other GCF components. GCF samples were collected from six maxillary anterior teeth and diluted GCF samples were divided into two groups (20 µ:l each), with or without chondroitinase ABC treatment, mixed with a dimethylmethylene blue (DMB) dye solution (180 µl) to detect S-GAG. Absorbance at 540 nm was measured using a microplate reader and the amount of S-GAG was calculated from calibration curves for standard S-GAGs where a linear relationship existed in the concentration range (0-3 µg/100 µl). Other GCF components such as glycoprotein and serum constituents did not influence the measurement of S-GAGs. Although trace amounts of S-GAGs were found in controls with a healthy periodontium (0.05 µg/100 µl ± 0.04, mean±S.E., n=5), considerable amounts of S-GAGs were found in patients with advanced periodontal disease (0.42 ± 0.11, n=6, p<0.05). The accuracy of this simple and rapid method of measuring S-GAGs in GCF makes it useful for screening periodontal conditions.