Biomineralisation occurs in nature in many forms, some of which are beneficial to humans and some detrimental. Biomineralisation leads to the formation of the skeleton but also to pathogenic calcification in arteries, on heart valves, kidney stones and medical devices such as urinary catheters. Several pathogenic calcifications are associated with bacterial activity. Can we exploit this capacity for our benefit? Serratia sp. NCIMB 40259 is a non-pathogenic Gram-negative bacterium which is capable of growing as a biofilm on almost all surfaces. A bacterial cell-wall located acid phosphatase enzyme liberates phosphate ions from organic phosphates and these combine with Ca2+ ions to form hydroxyapatite (HA) crystals. We discuss the potential use of Serratia HA for medical and other applications.
Cell proliferations on carbon nanotube scaffolds were investigated for the embryonic stem cells (ES cells) of mouse and the bone marrow-derived mesenchymal stem cells (MSC) of rat. Although ES cells were well attached and proliferated on gelatin-coated plastic dishes, they did not attach on the CNT-coated scaffold, and formed the spheroids growing in the culture medium. For MSC, they attached on the CNT-coated scaffold and grew to form mineralized particles as on the plastic dishes of control. The growth rate of the MSC was lower than control but showed the higher expression of alkaline phosphatase activity per cell in 2 weeks and degree of mineralization per cell for 3 and 4 weeks.
This study evaluated the influence of fibronectin (FN) immobilization on poly-L-lactic acid (PLA) films on attachment and proliferation of MC3T3-E1 osteoblast-like cells. FN immobilized PLA (FN-PLA) was prepared through a condensation reaction between the carboxylic acid groups on the hydrolyzed PLA surface and the amino groups of FN using water soluble carbodiimide. The number of attached cells onto FN-PLA films was significantly greater than on PLA films. At 10 days, the greatest number of attached cells was observed on FN-PLA surface. After 7 days of culture, the morphological differences of attached osteoblast-like cells were observed between PLA films and FN-PLA films. The cell attached on FN-PLA films appeared more flat. On the other hand, cell attached on PLA films were more rounded. The present study revealed the effectiveness of fibronectin immobilization of PLA for cell attachment and proliferation. Immobilization of cell adhesive protein will provide a wide variety of useful application of PLA in dental clinics.
The elements in discolored nails were assessed by X-ray fluorescence analysis (XRF), and the chemical state of Zn in the nail samples was assessed by X-ray absorption fine structure (XAFS) analysis. Compared to the normal nail, a part of the yellow nail contained a considerably high amount of Ca, whereas the black nail contained higher amounts of Ca and Zn. The chemical state of Zn in the yellow nail was similar to that in the normal nail. The Zn in the black nail was in a slightly different chemical state, suggesting the existence of different chemical species of Zn in the black nail.
Previously we have shown that honeycomb-shaped hydroxyapatite ceramics (HC-HAP) of different tunnel sizes control the formation bone and cartilage when implanted into rat skin with bone morphogenetic protein (BMP). The HC-HAP of larger tunnel size (300-400 μm) induced bone directly, while the smaller size (90-110 μm) induced endochondral ossification. Concerning this interesting tendency of bone-cartilage biphasic phenomena that was observed in hydroxyapatite ceramics, we wanted to know whether the same phenomena occur in β-tri-calcium phosphate (β-TCP) ceramics. We prepared honeycomb-shaped β-TCP ceramics (HC-β-TCP) of disk type with three-different tunnel sizes, 300, 75 and 50 μm, having tunnel numbers of 37, 568 and 907 per ceramic, respectively within a cross-section of 3.1 mm disk diameter. When implanted into rats subcutaneously with BMP-2, HC-β-TCP of three different tunnel diameters all induced active bone tissue formation, with the highest ALP value in HC-β-TCP with tunnel diameter of 75 μm. Interestingly, an appreciable amount of cartilage formation was observed only in the early stage of implantation of the HC-β-TCP with a tunnel size of 50 μm, but not with ones of 75 μm, and as expected, of 300 μm. The results were essentially similar to ones obtained using hydroxyapatite with tunnel sizes of 90-110 μm, in which clear endochondral processes were observed. We hypothesized that the slightly different tendency may be attributed mainly to the chemical properties of the two ceramics: hydroxyapatite is non-biodegradable, while β-TCP is a biodegradable ceramic.
We fabricated quartz crystal microbalance sensors coated with titanium, stainless steel (SS), alumina (Al2O3), and yttria tetragonal zirconia polycrystal (Y-TZP), which were prepared by DC magnetron sputtering. The crystal resonance frequency shift was measured immediately after injection of bovine salivary proteins, namely, bovine serum albumin (BSA) and bovine submaxillary gland mucin (BSM), in phosphate-buffered saline solution. The crystal resonance frequency shift changed depending on the amount of adsorbed protein. Differences in surface topography and roughness were observed among the four sensor types by scanning probe microscopy. X-ray photoelectron spectroscopy indicated that the outer surface of each sensor was a metal oxide. Of the four sensor types, sensors with Al2O3 and Y-TZP coatings exhibited the lowest and highest surface roughness, respectively. Adsorption of BSA and BSM were significantly the lowest and highest on the SS sensor, respectively. This study has revealed the technical basis for distinguishing protein-adsorbing materials of various orthodontic appliances and may contribute to the development of oral rinses that prevent biofilm formation.
To examine the effects of C60 fullerene on angiogenesis, we investigated the effects of C60 fullerene on capillary regeneration from cells in the early stage of tubule-like structures using a human angiogenesis kit (Kurabo, Tokyo). In addition, using 8-Hydroxydeoxyguanosine (8-OHdG), we investigated its effects as an antioxidant to inhibit oxidative stress. The area ration and length of new blood vessels in the 5mg/mL group were 57.4 and 54.3%, respectively, compared with the control group. At 2.5 and 5 mg/mL, no significant difference was noted compared with the control group. The amounts of 8-OHdG in the 2.5 and 5 mg/mL groups were 89.4 and 87.5%, respectively, on comparison with the control group. Thus, no significant difference was noted. C60 fullerene was not sufficiently dispersed in the medium. Thus, C60 fullerene particles should be homogeneously dispersed in media. In the future, our results should be compared with those from dissolution using Polyvinylpyrrolidone (PVP) to clarify biological evaluation of new capillary formation.
It is important to know quantitatively the dilute concentration of carbon nanotubes (CNTs) for the view point of their risk assessment and biomedical applications. However, the measurement of less than 100 ppm is difficult for the conventional weight measuring method. In this study, the optical absorptiometric method was newly proposed and its validity was investigated. The method showed the linear relationships between absorption and CNT concentrations in the range from 3 ppm to 100 ppm for various solutions, with or without proteins and phenol-red in the medium at different pH, and for various wave lengths. The higher sensitivity than the conventional weight measuring method and the validity in the various conditions suggested that the newly developed absorptiometric measuring method is efficient for the quantitative analysis of CNTs in the low concentration range.
We have developed a specific procedure for cryopreservation of Paramecium caudatum that does not require either a controlled-rate deep freezer or liquid nitrogen. Optimum conditions for cryoprotectants, freezing rates, freezing temperatures, and thawing conditions have been determined by monitoring three cellular characteristics, namely, cellular morphology, ciliary movement and contractile vacuole contraction. Cells at the exponential phase in test tube culture were pre-incubated in the cryoprotectant (5% dimethyl sulfoxide, DMSO) for several hours and then frozen in a glass capillary. Thawing was performed at 35 oC as quickly as possible, then recovered cells were suspended in a post-thawing recovery solution of cell free culture medium (CFCM) containing bovine serum albumin (BSA). About 80% of the recovered cells grew and formed clones in fresh culture medium. Our method gives about 4% of normal viable cells after one week in a freezer at -84 oC. The clones derived from the recovered cells showed normal characteristics with regard to growth, swimming behavior and sexual activity. Our method offers a relatively simple method for routine cryopreservation of Paramecium caudatum.
Reaction conditions for sidewall carboxylation of multi-walled carbon nanotubes (MWCNTs) by radical addition reaction using acid peroxide as the radical source were investigated with the goal of high-yield carboxylation. In particular, the parameters of reaction time and reactant (succinic acid peroxide) concentration were examined. The carboxylation ratio is defined as the ratio of the mole of carboxylic group to the molar amount of carbon in the MWCNT. It was concluded that the carboxylation ratio depends more on reactant concentration rather than on reaction time. This tendency is reasonably explained by assuming that the carboxylation will be almost complete within a short period, and the yield of carboxylation will ultimately reach a constant value. The final value of the carboxylation ratio is hence determined by the reactant concentration. The results were explained by the kinetics of pseudo-first-order reaction.
We compared cytotoxicities of metal oxide nanomaterials, including different sizes (20 and 60 nm) of ZnO nanoparticles (NPs), Al-doped ZnO-NPs, Al2O3-NPs, Al2O3 nanowhiskers (NWs) and SiO2-NPs, on the basis of viabilities of human lung epithelial A549 cells exposed to these nanomaterials. Al2O3-NPs, Al2O3-NWs and SiO2-NPs showed no significant cytotoxic effects on viability. On the other hand, ZnO-NPs, which included ZnO-NPs (20 nm), ZnO-NPs (60 nm), and Al-doped ZnO-NPs, all showed toxic effects; however, no significant differences in cytotoxicity were observed among these ZnO-NPs, suggesting that nanoparticle size and Al-doping do not contribute to cytotoxicity. Global gene expression analysis using a DNA microarray revealed that 22 and 8 genes were consistently up- and down-regulated, respectively, in cells exposed to the 3 ZnO-NPs. Our results suggest that up-regulation of genes by ZnO-NPs leads to maintenance of zinc homeostasis and protection against oxidative stress, while down-regulation of genes causes disruption of zinc homeostasis, DNA damage, and apoptosis. The balance between protective mechanisms induced by up-regulated genes and cellular damages induced by down-regulated genes may determine the fate of cells.
The residual metals were partially eliminated by purifying the pristine multi-walled carbon nanotubes (MWCNTs) with hydrochloric acid. The purified MWCNTs showed toxicities which were almost identical in magnitudes to that observed for the pristine MWCNTs. The experiments were conducted by cultivation of Arabidopsis T87 suspension cells in media containing MWCNTs. Contents for reactive oxygen species (ROS), catalase and glutathione were quantitatively analyzed. ROS and catalase increased and glutathione decreased were observed for the cells after the exposure to MWCNTs. The MWCNTs exposure also caused cell membrane disintegration and cellular organs structure changes. Our experimental data gave evidences to conclude that MWCNTs are toxic to the Arabidopsis T87 suspension cells and the overproduction of ROS is responsible for causing the toxicity.
In many cases, nanoparticles have higher physical and chemical activities than fine particles. These activities are beneficial for industrial use; however, they may have biological effects resulting from properties such as high toxicity. Rutile titanium dioxide (TiO2) is one of the most important metal oxide nanoparticles. It has been used not only for industrial purposes but also in cosmetics such as sunscreen. Compared to anatase TiO2, cellular influences of rutile TiO2 are not well understood. In the present study, we examined cells for examining the effects of cosmetic-grade Al(OH)3-treated rutile TiO2 nanoparticles. It has been suggested that metal ion release and the subsequent oxidative stress play an important role in the cytotoxicity of metal oxide nanoparticles. Therefore, we focused on the effect of released soluble metal and the induction of oxidative stress due to rutile TiO2 nanoparticles. First, the properties of TiO2 medium dispersion were detected. Rutile TiO2 nanoparticles had strong protein adsorption ability. Titanium ion release from TiO2 nanoparticles in the medium dispersion was not detected, and aluminum ions were found to be slightly released. Next, two kinds of cultured cells-human lung carcinoma A549 cells and human keratinocyte HaCaT cells-were exposed to stable TiO2 medium dispersion. Cellular influences (oxidative stress, apoptosis, and colony formation) of the TiO2 nanoparticles were small. These results suggest that metal ion release is the most important cytotoxic factor influencing the toxic activity of metal oxide nanoparticles. On the other hand, the cellular effect of adsorbed serum proteins is small. Therefore, cellular influences of Al(OH)3-treated rutile TiO2 nanoparticles that did not release metal ions are limited. The detection of metal ion release is essential for the evaluation of the cellular influence of metal oxide nanoparticles.