Mesenchymal stem cells (MSCs) are highly proliferative and can differentiate into multiple lineages. It has been demonstrated that S100A4, a S100 calcium-binding protein, can act as an inhibitor of mineralization. Therefore, inhibition of S100A4 might increase osteogenetic differentiation of MSCs. We investigated the effect of S100A4 inhibition by short interfering RNA (siRNA), which is a modified siRNA with improved silencing longevity. We also studied the specific effect of siRNA on the expression of many genes related to osteogenic differentiation. Inhibition of S100A4 by siRNA resulted in increased expression of many genes related to osteogenic differentiation. This study provided a tool for better understanding of osteogenesis differentiation in MSCs.
The experimental protocol of embryonic stem cell test (EST) using ES-D3 cells and 3T3 cells as an in vitro embryotoxicity test for biomaterials and medicine was established by H. Spielmann of Germany in 1997. In 1988, Imai proposed the cell recovery test as a variation of the cytotoxicity test. By exposing the material to cells, the transitory influence on recovery of the cell proliferation level is assessed. The cell recovery test was also developed similarly allowing the examination of not only recovery of cell proliferation but also recovery from chemical disruption of cell differentiation. After exposing both cells to NaF and SnF2, used to prevent tooth decay, we compared the results of each culture in fresh medium with the findings obtained by the EST. For "5-day recovery" and "7-day recovery", each value increased slightly. NaF and SnF2 both showed "non-embryotoxicity" under all conditions. Based on these findings, no influence of cellular recovery was seem with demonstrated in NaF and SnF2. These chemicals are both fluorine compounds, and the embryotoxicity level of SnF2 is presumed to be slightly stronger than that of NaF. No influence of cellular recovery was demonstrated with NaF and SnF2. Stronger embryotoxicity is a concern when the recovery culture shows that the toxicity level of a chemical remains constant; therefore, it is necessary to consider the utility of examining recovery from embryotoxicity.
Adiponectin, a relatively abundant plasma protein secreted by adipocytes, exhibits various biological functions. Low plasma adiponectin levels are known to cause insulin resistance and metabolic syndrome. We previously demonstrated the expression of adiponectin receptors (AdipoR1 and AdipoR2) in osteoblasts, indicating that adiponectin may affect bone metabolism. In this study, we investigated gene expression in osteoblasts which showed suppressed receptor expression due to small interfering RNA (siRNA), to elucidate the influences of the failure of adiponectin action on bone metabolism. Two days after transfecting siRNA against adiponectin receptors (siRNA-AdipoR), murine pro-osteoblastic cells were stimulated with ascorbic acid and β-glycerophosphate to induce osteoblast differentiation. The mRNA expression level was measured using real-time quantitative RT-PCR two days after stimulation. Ascorbic acid and β-glycerophosphate-dependent induction of BMP-2 and osteocalcin were significantly suppressed by siRNA-AdipoR2 compared with non-silencing siRNA controls. Both mRNA expression levels of BMP-2 and osteocalcin were correlated with that of AdipoR2, while there was no correlation of osteoblast differentiation markers with AdipoR1; therefore, the failure of adiponectin action may suppress the progression of osteoblast differentiation and bone formation.
The purpose of this study was to examine the biodegradation and bone replacement of resorbable hydroxyapatite derived from red algae (RHGA) and to investigate the effect of particle size and surface properties on osteogenesis. RHGA particles (300-1000 micrometers), which had a smooth surface, and crushed small RHGA particles (100-300 micrometers), which had a rough surface with open pores, were used. Calvarial bone defects were created in 30 rats. The defects were filled with blood clot (Group A), original particles (Group B), or small particles (Group C). The animals were sacrificed 4 (n=15) or 8 (n=15) weeks later. The bone was examined histologically and histomorphometrically. In Group A at 8 weeks, new bone extended towards the defect's center, but the initial thickness was not restored. In Group B, connective tissue encapsulated most particles at 4 weeks; at 8 weeks, some particles collapsed from the originally concave surface, and immature bone was observed around this area. In Group C, new bone and particles occupied most of the defect at 8 weeks. The particles' pore system lost its septal structure and was invaded by mature bone. The present results indicate that structural architecture, such as a pore system, appears to be crucial for particle resorption and new bone formation and that small RHGA having rough surface with open pore is potential as bone substitute.
We had previously developed hydroxyapatite fiber material (HF) consisting of 100% hydroxyapatite fibers of 5-15µm in diameter. The purpose of the present study was to examine whether HF supports proliferation and differentiation of osteogenic cells as scaffold and whether it is effective as bone substitute. Firstly, rat bone marrow cells were prepared and cultured. Then, these cells were incorporated into HF and cultured in osteogenic medium. After 7, 10 and 14 days of culture, DNA content, alkalinephosphatase (ALP) activity and gene expression of osteogenic genes (osteocalcin and type I collagen) were examined. Secondary, the bone defects of 3mm in diameter were prepared in tibias of nine Japanese rabbits. The defects of the right tibias were filled with HF while the defects of the other side were untreated. The animals were sacrificed at 1, 2 and 3 months. The defects were analyzed with soft X-ray and micro CT and histologically. In the culture experiment increase in DNA content and ALP activity and osteogenic gene expression was evident suggesting that bone marrow cells can grow and differentiate to osteoblastic cells in HF. Radiological and histological analyses in animal experiments demonstrated disappearance of HF from bone marrow without eliciting inflammatory response to the surrounding tissue and complete regeneration of the bone defects at 3 months. These results indicate that HF could be favorable scaffold for bone regeneration and effective as resorbable bone substitute.
The hydrogel formation of bisphosphonate was investigated after the immersion of apatite coated titanium web (TW) in bisphosphonate solution. Apatite thin film was coated onto TW (porosity of 85 %, pore size of 200-300 µm) using molecular precursor method. Molecular precursor solution was obtained by adding dibutylammonium diphosphate salt to Ca-EDTA/amine ethanol solution by adjusting Ca/P=1.67. Sintered cylindrical TW (2.8mm diameter x 6 mm length) was immersed into molecular precursor solution and then heated at 600 °C for 2 hr. The immersion and heating process was repeated three times. Uncoated and apatite coated TW were immersed into the bisphosphonate solution (pamidronate disodium, 1x10-2M) for 24 hr at 37 °C. The viscosity of bisphosphonate solution in apatite coated TW was increased. The formation of hydrogel was clearly observed around the apatite coated TW after taking out it from the bisphosphonate solution. It is expected that apatite coated TW will contribute the hydrogel formation of molecules with phosphate groups.
The most important and useful property of stem cells is that of self-renewal. Through this property, striking parallels can be found between stem cells and cancer cells. Natural killer (NK) cells play a key role in inflammation and tumor regression through their ability to cytotoxicity. The infiltrating NK cells are thought to kill cancer cells independently of major histocompatibility complex (MHC). Generally, NK cells are unable to exhibit their cytotoxicity unless adhering to target cells via an adhesion molecule. In order to clarify part of the mechanism for the killing of cancer cells by NK cells, we investigated the adhesion molecule between two cell types, i.e., NK92 MI cells and WM35 cells, and evaluated the effect of the cell adhesion on the manifestation of the cytotoxicity of NK cells. We used NK92 MI cells, which were obtained by non-viral transfection of NK92 cells with the cDNA for human IL-2. It was confirmed that NK92 MI cells kill WM35 cells. We proceeded to investigate the effect of the adhesion molecule CD54 on the killing of WM35 cells by IL-2 activated NK92 MI cells. After a reaction with anti-CD54 antibody, WM35 cells were subjected to a cytotoxicity experiment with NK92 MI cells. The anti-CD54 antibody significantly suppressed the killing of WM35 cells by IL-2 activated NK92 MI cells. These results suggest that the killing of CD54 expressed melanoma cells by IL-2 activated CD56 positive NK cells might be CD54-dependent.
Thin carbonate-containing apatite (CA) coatings were deposited onto titanium webs (TW) using a molecular precursor method. The volumetric porosity and fiber diameter of the TW were 87 % and 50 mm, respectively. A molecular precursor solution was obtained by adding dibutylammonium diphosphate salt to Ca-EDTA/amine ethanol solution by adjusting the Ca/P ratio to 1.67. Two molecular precursor solutions with different calcium ion concentrations, 0.157 mmol/g (0.157-precursor) and 0.470 mmol/g (0.470-precursor), were prepared. Each sintered TW was immersed in the molecular precursor solution and then heated at 600°C for 2 hr under oxygen gas introduction. Without oxygen gas, the CA coated TW appeared darker and blacker. Oxygen gas introduction produced a whiter TW appearance after CA coating. Three-times coating of 0.157-precursor produced a CA coating that covered the surface of the TW as well as its center. By using the 0.470-presursor, a single application of CA covered the inside as well as the surface of the TW. Increasing the calcium ion concentration of the molecular precursor solution enabled one-time application for effective CA coating not only of the surface of the TW but also its center. In conclusion, one-time application of 0.470-precursor solution during oxygen gas introduction produces a sufficiently CA coated TW.
Development of reliable new cures is expected in clinical dentistry, although there are still very few examples of the regenerative dentistry. In order to achieve breakthrough in these areas, new approache is proposed. Root canal therapy and filling methods using an optical irradiation lamp for composite resin polymerization. A transparent point for root canal restoration will be used and weak phototoxicity will facilitate apical tissue regeneration. Phototoxicity will also be used also for the medical treatment of root canal infection by optical irradiation only, without requiring extraction. And, crown removal and root filling points can be judged using light irradiation by observing the progress of symptoms.
Worldwide, animal use figures for scientific purposes have been conservatively estimated as approximately 127 million in 2005. Over 11 million living non-human vertebrates were used within Japan in 2004. This was second only to the USA, which used approximately 17 million animals in 2005. The scientific and regulatory limitations inherent within the use of animal models during human clinical and toxicological investigations are substantial, and increasingly recognized. A previous trend of decreasing animal use is reversing, largely due to increased use of genetically-modified animals, and the implementation of large-scale chemical testing programs. These developments demonstrate the need for considerably greater awareness and implementation of the 3Rs -the replacement, reduction and refinement of laboratory animal use -within governmental, academic and commercial sectors. These principles are widely recognized as essential to good laboratory animal practice. They may increase research quality and the robustness of procedures; result in reduced timeframes and resource consumption; and provide more reliable human clinical and toxicological outcomes. An overview of 3Rs principles, and of strategies likely to increase their implementation, is therefore provided. Combinations of such strategies may have synergistic effects, improving scientific outcomes whilst decreasing animal use.