Different morphologies of human embryonic kidney 293T cells in various types of culture dishes.

Human embryonic kidney 293T (HEK293T) cells are used in various biological experiments and researches. In this study, we investigated the effect of cell culture environments on morphological and functional properties of HEK293T cells. We used several kinds of dishes made of polystyrene or glass for cell culture, including three types of polystyrene dishes provided from different manufacturers for suspension and adherent cell culture. In addition, we also investigated the effect of culturing on gelatin-coated surfaces on the cell morphology. We found that HEK293T cells aggregated and formed into three-dimensional (3-D) multicellular spheroids (MCS) when non-coated polystyrene dishes were used for suspension culture. In particular, the non-coated polystyrene dish from Sumitomo bakelite is the most remarkable characteristic for 3-D MCS among the polystyrene dishes. On the other hand, HEK293T cells hardly aggregated and formed 3-D MCS on gelatin-coated polystyrene dishes for suspension culture. HEK293T cells adhered on the non- or gelatin-coated polystyrene dish for adherent culture, but they did not form 3-D MCS. HEK293T cells also adhered to non- or gelatin-coated glass dishes and did not form 3-D MCS in serum-free medium. These results suggest that HEK293T cells cultured on non-coated polystyrene dish may be useful for the tool to analyze the characteristics of 3D-MCS.

Investigation of a sterilization system using active oxygen species generated by ultraviolet irradiation.

We have been investigating an advanced sterilization system that employs active oxygen species (AOS). We designed the sterilization equipment, including an evacuation system, which generates AOS from pure oxygen gas using ultraviolet irradiation, in order to study the conditions necessary for sterilization in the system's chamber. Using Geobachillus stearothermophilus spores (10(6) CFU) in a sterile bag as a biological indicator (BI) in the chamber of the AOS sterilization apparatus, we examined the viability of the BI as a function of exposure time, assessing the role of the decompression level in the sterilization performance. We found that the survival curves showed exponential reduction, and that the decompression level did not exert a significant influence on the survival curve. Subsequently, we investigated the sterilization effect as influenced by the spatial and environmental temperature variation throughout the chamber, and found that the sterilization effect varied with position, due to the varying environmental temperature in the respective areas. We confirmed that temperature is one of the most important factors influencing sterilization in the chamber, and estimated the temperature effect on the distribution of atomic oxygen concentration, using the quartz crystal microbalance (QCM) method with fluorocarbon thin film prepared by radio frequency sputtering.

Behaviors of MC3T3-E1 cells on carbonated apatite films, with a characteristic network structure, fabricated on a titanium plate by aqueous spray coating.

Four carbonated apatite films having average thicknesses of 1.3-0.11µm, proportions of network sizes above 10µm of 41-68%, and average border heights of the characteristic network structure of 0.98-0.29µm were fabricated on a titanium plate by aqueous spray coating. These carbonated apatite films after heat treatment showed good mineralization ability in Hanks' balanced salt solution. Assessment of initial cell attachment and calcination on these films and on the Ti plate using osteoblastic MC3T3-E1 indicated that the carbonated apatite film heat treated at 600°C, whose film thickness, proportion of network sizes above 10µm, and border height were 0.11µm, 61%, and 0.31µm, respectively, was most preferred by osteoblastic cells. Field emission scanning electron microscopic observation of the cells attached to the films showed that the wide network and low border height of the network structure on the carbonated apatite film play an important role in the development of the filopodia of the osteoblastic cells.

Synthesis of novel oxide layers on titanium by combination of sputter deposition and micro-arc oxidation techniques.

A novel coating technique of thin ceramic layer resembling teeth color on titanium (Ti) surface was developed by combination of sputter deposition of metal zirconium (Zr) and subsequent micro-arc oxidation (MAO) treatment. The oxide layer grown by MAO treatment had a porous structure with a thickness of approximately 6 micrometers and was mainly composed of zirconium dioxide (ZrO(2)) with both tetragonal and monoclinic crystal structures. The surface of the specimen was hardened by this technique, and the hardness of the specimen was significantly larger than that of untreated Ti and MAO-treated Ti without Zr layer. The bonding strength test revealed that fracture occurred inside the oxide layer, indicating that adhesion between the oxide layer and the Ti substrate was sufficiently strong. From these results, this technique has an advantage for the development of novel dental materials with excellent mechanical and aesthetic properties.

Differences in the bone differentiation properties of MC3T3-E1 cells on polished bulk and sputter-deposited titanium specimens.

The roughness and cleanness of a titanium surface must be controlled in order to investigate the expression mechanism of hard tissue compatibility on titanium. In this study, osteogenic MC3T3-E1 cells were cultured and differentiation-induced on bulk and sputter-deposited titanium specimens, and the osteogenesis were investigated. For the preparation of bulk specimens, titanium discs were mirror-polished. On the other hand, titanium was sputter-deposited on smooth and clean cover glasses as sputter-deposited specimens. As a result, no significant difference was observed in the cell morphology and attached number. On the other hand, the time showing maximum activity in the alkaline phosphatase and gene expressions, which are related to bone differentiation on the bulk titanium, were superior to those on the sputter-deposited titanium. From the surface observation of the specimens with a scanning electron microscope and a scanning probe microscope, the surface on the sputter-deposited titanium was more uniform and cleaner than that on the bulk titanium. According to X-ray photoelectron spectroscopy, the thickness of surface oxide film on the sputter-deposited titanium was smaller than that on the bulk titanium. In addition, the proportions of TiO and Ti(2)O(3) in the surface oxide film on the sputter-deposited titanium were larger than those on the bulk titanium. These differences might influence the differentiation of osteoblastic cells.

Microstructure and magnetic susceptibility of as-cast Zr-Mo alloys.

The microstructures and magnetic susceptibilities of Zr-Mo alloys were investigated to develop a Zr alloy with a low magnetic susceptibility for magnetic resonance imaging (MRI). The microstructure was evaluated with an X-ray diffractometer (XRD), an optical microscope (OM) and a transmission electron microscope (TEM), and the magnetic susceptibility was measured with a magnetic susceptibility balance. The alpha' phase with acicular structure was dominant in Zr-1Mo alloys, while the omega and beta phases with the equiaxed and relatively flat (no acicular) microstructure was dominant in Zr-3Mo. The mixed microstructural features of Zr-1Mo and Zr-3Mo were observed in Zr-2Mo, which consists of the alpha', omega and beta phases. The beta phase is stabilized when the Mo content exceeds over 3 mass% Mo. As-cast Zr-Mo alloys showed a minimum value of magnetic susceptibility at 3 mass% Mo, and the value abruptly increased up to 10% Mo before remaining stable up to 15 mass% Mo. XRD, OM and TEM revealed that the minimum value of the susceptibility was closely related to the appearance of the athermal omega phase in the beta phase. As the Mo content decreases from 3 mass%, the alpha' phase appears with the omega and beta phases. On the other hand, as the Mo content increases from 3 mass%, the beta phase increases and the omega phase decreases. Thus the appearance of the alpha' and beta phase leads to an increase in magnetic susceptibility. The magnetic susceptibility of as-cast Zr-3Mo alloy was almost one-third that of Ti-6Al-4V, which is commonly used for medical implant devices. Zr-Mo alloys are useful for medical devices used under MRI.

Calcification by MC3T3-E1 cells on RGD peptide immobilized on titanium through electrodeposited PEG.

The effect of a cell-adhesive peptide containing Arg-Gly-Asp (RGD) immobilized through poly(ethylene glycol) (PEG) on titanium (Ti) on calcification by MC3T3-E1 cells was investigated to develop a new surface modification technique using biofunctional molecules. RGD was immobilized on Ti through PEG, both terminals of which were terminated with -NH(2) and -COOH to combine with the Ti surface and RGD. PEG was immobilized on Ti with electrodeposition, and RGD, with immersion. For comparison, glycine was employed because it is the simplest molecule containing both -NH(2) and -COOH at its terminals. MC3T3-E1 cells were cultured and differentiation-induced on each specimen, and the cell calcification properties were investigated. As a result, there was no significant difference in the morphology and extension of MC3T3-E1 cells cultured on each specimen, while the number of cells cultured on RGD/PEG/Ti was the largest. After differentiation-induction, there was no significant difference in the ALP activity among all specimens. On the other hand, the level of cell calcification on RGD/PEG/Ti was the highest. Therefore, the hard tissue compatibility of Ti is improved by immobilizing RGD through functional molecules which have a long molecular chain.