To examine the effect of fluoride on the survival of osteoblasts, media containing various concentrations of fluoride, 0〜5.0×10-2 mol/L, were prepared and mouse osteoblast MC3T3-E1 cells were cultured. At the same time, media containing 0〜5.0×10-2 mol/L of chloride as an indicator of pH were also adjusted. Although osteoblasts grew well at fluoride levels below 1.0×10-3 mol/L, osteoblast survival was dramatically inhibited over 5.0×10-3 mol/L of fluoride despite the pH of the medium scarcely decreasing when compared with the control pH 7.81, and osteoblasts showing favorable survival with 2.0×10-2 mol/L of chloride even at pH 6.81. Fluorospectrophotometric observation showed quite different features between control cells and at higher concentrations of fluoride. These results suggested that fluoride dramatically inhibits osteoblast growth at concentrations above 5.0×10-3 mol/L in the medium solution.
Chemokines regulate the homeostatic trafficking of lymphocytes and lymphocyte influx into sites of injury and inflammation. Chemokines can induce rapid changes in integrin-dependent adhesion. Although activation of phosphatidy-linositol 3-kinase (PI 3-K) is critical to integrin activation induced by Ig superfamily members such as CD2, CD3 and CD28, the role of PI 3-K in chemokine-induced integrin activation is unclear. We examined the role of PI 3-K in several functional responses induced by ligation of the CXCR4 chemokine receptor expressed on Jurkat T cells with the CXC chemokine stromal cell-derived factor-1α (CXCL12). Enhanced Jurkat cell adhesion induced by CXCL12 was inhibited by pertussis toxin and by the PI 3-K inhibitors, wortmannin and LY294002. CXCL12 also induced F-actin polymerization on Jurkat cells that was only partially inhibited by PI 3-K inhibitors. In contrast, CXCL12 -mediated phosphorylation of the PI 3-K-dependent enzyme AKT was markedly prolonged, with AKT activation being detectable as late as 60 min.
Adipocytokines are a group of adipocyte-derived biologically active molecules, including adiponectin, leptin, tumor necrosis factor-α (TNF-α), resistin. A proteolytic cleavage product of adiponectin/Acrp30, gAcrp30, is found to circulate in plasma and to have a three-dimensional structure similar to that of TNF-α. In this study, we demonstrated the expression of adiponectin receptors in cultured pro-osteoblastic cells, and investigated the influences of gAcrp30 and TNF-α on osteoblast differentiation. RT-PCR analysis demonstrated the expression of transcripts for both adiponectin receptors, AdipoR1 and AdipoR2, in murine pro-osteoblastic cell line, MC3T3-E1. Both gAcrp30 and TNF-α suppressed mRNA levels of type I collagen and RANKL compared with time-matched controls. Interestingly, gAcrp30 had no significant influence on the promotion of osteocalcin expression induced by the stimulation of ascorbic acid and β-glycerophosphate, although TNF-α inhibited the increase of osteocalcin expression. These results indicated that adiponectin may not affect osteoblast differentiation but may suppress osteoclastogenesis in an endocrine manner, while TNF-α may inhibit the differentiation of pro-osteoblastic cells.
We prepared the DNA/cationic polyamino acid complex and examined their cell viability and histopathological responses. As cationic polyamino acids, poly-L-arginine (Poly-arg), poly-L-histidine (Poly-his) and poly-L-lysine (Poly-lys) were used. A water-insoluble white powder (DNA/poly-arg complex, DNA/poly-his complex and DNA/poly-lys complex) was obtained from the reaction of each cationic polyamino acid with DNA. The yields were approximately 37-45%. SEM observation revealed that prepared complexes had a porous structure. The porous disks could be easily prepared. In cell culture tests using L-929 mouse fibroblast cells, DNA/poly-arg and DNA/lys complexes showed slight cytotoxicity and DNA/poly-his complex showed moderate cytotoxicity. However, in vivo test, all complexes showed a mild tissue response after the implantation into the back of skin of rats. These results suggested that these complexes could be useful materials for biomaterials with functional properties such as pH responsibility and cell adhesion to be used as a scaffold.
Magnetically activated cell sorting (MACS) system is one isolation technique of mesenchymal stem cells (MSC) from bone marrow cells. TEM/EDX observation revealed that its micro-beads had the size of about 0.5 to 1.0 µm and consisted of matrix and inorganic phases. The inorganic phase contained small particles of about 40 nm size, and consisted of Fe, Si, O, Cl (e.g., Fe3O4 + SiO2 + FeCl3). In MACS system, lineage (hematopoietic) cell depletion kit reduced the cell number of bone marrow cells to 1.2 %. Subsequent positive selection by CD117, anti-Sca-1, CD34 and CD44 micro-beads lead to the cell collection rates of 0.57 %, 0.10 %, 0.13 % and 0.14 %, respectively. A combination of three micro-beads for lineage cell depletion kit, CD117 and anti-Sca-1 micro-beads succeeded in reducing the cell collection rate down to 0.04 %, matching a normal existing rate of MSC in bone marrow cells (0.01 to 0.10 %).
We studied rat femur bone marrow stromal cells (BMSCs) and microenvironment turnover in capsaicin (CAP)-conditioned media in vitro. Proliferation, differentiation and TRPV1 expression at the mRNA level (RT-PCR) of conditioned BMSCs, and pH changes and calcium assay of the conditioned culture media were examined; the obtained data were statistically analyzed. Elapsed time-dependent cell proliferation of BMSCs peaking at day 10 was observed in alkaline phase and 37°C microenvironment. Although there were no significant differences of proliferation in different conditions on designated experimental days, day 10 was the turning point of proliferation phase where intermittent administration of 25 µM CAP resulted in the least amount of cell proliferation than other culture conditions on day 14. The present RT-PCR study revealed expression of TRPV1 mRNA during 3 to 4 days (either day 3 or day 7) after single or intermittent exposure under 25 µM CAP. This study elucidated that by intermittent addition of 25 µM CAP into the culture medium, the CAP-sensing TRPV1 of the rat BMSCs was activated to significantly mediate Ca2+ influx leading to cell death, and the TRPV1 was desensitized between day 7 and day 10 in vitro.
The positive dissolution of the trace element ion from the alloy for prosthetic dentistry is expected in Regenerative Dentistry. Vascularization, which carries oxygen and nutrition, is indispensable to tissue generation. We investigated the influence of the human skin model on cell viability test and the formation test of tubule-like structures is very low concentration copper, titanium and zinc ions. In both experiments, the influences of metal ions significantly differed. The cell viability test using a human skin model showed significant differences in the reaction to slight cell stimulate on. In testing the formation of tubule-like structures, the influence of the singularity of HUVEC should be considered. In the dentistry field especially, the dissolution of chemical substances from material, applications in reparative dentistry from prosthetic dentistry material to saliva is expected. The development of technology to control the dissolution of metal ions is also required. Basic experiments focusing on specific metal ions or changes in the exposure period need to be accumulated in future studies.
This study was designed to evaluate the effects of recombinant human BMP-2 (rhBMP-2) injected into the palatal periosteal sites in Wister rats on bone formation. The implant sites were separately divided into four groups according to the dosage of rhBMP-2 : 0 µg, 0.1 µg, 0.5 µg and 1.0 µg. The rats were sacrificed at 3 weeks after implantation, followed by histopathologic observation and histometric evaluation of new bone. Implants had disappeared at every group and new bone formation was observed at 0.1 µg, 0.5 µg and 1.0 µg. New bone was continuous with the original bone at 0.5 µg and 1.0 µg. Thickness of new bone increased as the dosage increased from 0 µg to 0.5 µg (P<0.05), and did not significantly change as the dosage increased from 0.5 µg to 1.0 µg. These results suggested that rhBMP-2 injected into the palatal periosteal sites could lead to induce new bone formation, although its effects varied according to the dosage of rhBMP-2.