Honeycomb collagen (HC) with its unique geometric structure has been used in cell culture experiments to study the three-dimensional (3D) effects of the artificial matrix. When implanted in animal tissue however, HC was easily distorted and its geometric structure crushed. In order to elucidate the effect of 3D geometry of this scaffold in both in vitro and in vivo, we attempted to give it rigidity by calcifying and coating HC with hydroxyapatite. By using a calcifying solution with a high calcium (15 mM) and phosphate (9 mM) concentration, hydroxyapatite was evenly precipitated on the surface of HC, obtaining 10 times higher weight than the original HC. A mechanical strength test of the calcified HC showed a 4 times higher compression modulus than the original HC. Implantation of calcified HC into rats subcutaneously with BMP effectively induced bone, comparable with the HC and another conventional scaffold. Moreover, implantation of calcified HC without BMP induced vasculature along individual tunnels of the calcified HC, while the implantation of HC alone without BMP led to the degradation of the scaffold at 4-8 weeks.

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Carbon nanotubes (CNTs) are one of the most representative nanomaterials that have newly appeared and drawn much attention for various applications. To examine the effect of CNTs on biological organisms, the reaction behavior of human hepatocytes, Hc cells, to CNTs was investigated in comparison with phagocytes. After hepatocytes were incubated in the culture medium with the addition of CNTs, CNTs were found in cytoplasms by OM (Optical Microscopy), SEM (Scanning Electron Microscopy), TEM (Transmission Electron Microscopy). In the successive observation using the time-lapse OM system, hepatocytes showed the phagocytosis-like behavior to CNTs by changing their shapes and forming the long, straight pseudopods. The quantitative analysis showed that cell activities to scavenge CNT debris were constant during the observation for more than 22 hrs. Since these behaviors of hepatocytes apparently resemble those of phagocytes, the cell functional analysis was done and compared by measuring the expression of cytokines and superoxide dismutase (SOD) activity. When incubated with CNTs, THP-1 monocytes released TNF-α in a dose dependent manner to CNT content, while the amount released from hepatocytes were very low and there was almost no difference from control. These results indicate that hepatocytes have the phagocytosis-like ability to uptake foreign objects. However, their mechanism is completely different from that of phagocytotic cells in the point that they have no functions to lead to inflammation through cytokine cascade as monocytes exhibit. Throughout the study, hepatocytes could work without the acute toxicity such as cell death by CNTs. In situ observation could image the dynamic behavior of hepatocytes to perform the functions to treat the foreign objects. The results confirm that liver is an important organ to deal with them under noninflammatory circumstances.

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Imogolite is a naturally occurring aluminosilicate clay mineral with a nanotube structure of 2 nm in diameter and 60 nm-2 μm in length. In this study, synthetic imogolite nanotubes were used to fabricate a scaffold for cell culture and the properties of the resulting scaffold were compared with those of a conventional culture dish and a carbon nanotube (CNT) scaffold. The surface characteristics of the imogolite scaffold were drastically changed depending on the amount of imogolite on the dish. With the increase of imogolite concentration, the surface morphology of scaffolds changed from an island-like shape in random orientation to a self-organized fiber texture aligned in the same direction, and finally full coverage in a random orientation with plural layers. Osteoblast-like cells (Saos-2) cultured on imogolite showed a flat-form morphology developed in all directions in contrast to a spindle-shaped morphology developed in one direction on cell culture dishes (Cntl) and carbon nanotube scaffolds (CNT). The large growth of the cells proliferated on imogolite limited the smaller number of cells than the Cntl and CNT. However, the amount of normalized protein per cell on imogolite showed the value more than twice of that on Cntl. Imogolite contributed more to the enlargement of cell volume than proliferation. The cells cultured on the imogolite scaffold become more strongly bound to the substrate and showed high osteoblastic mineralization than those cultured on Cntl and CNT. The results indicate the good biocompatibility between cells and the imogolite scaffold, and suggest that imogolite could be useful for various bioapplications, such as for the fabrication of scaffolds for cell cultures, some useful proteins production (i.e. antibody, enzyme) and surface modified implants.

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In order to improve bio compatibility and biological function of carbon nanotubes (CNTs), the surface of CNTs was modified by precipitating calcium phosphate compounds in a calcifying solution. To accelerate calcification process and efficacy of modification, a solution containing 6-times higher concentration of calcium and phosphate ions than that of physiological body fluid was used (the 6-times solution). Precipitation of calcium phosphate (Ca-P) upon CNTs was investigated, concerning the incubation time, influence of concentration of CNTs, elemental analyses of products and protein adsorption on the chromatographic column. Time-dependent observation of the reaction products by SEM indicates that the moss-like precipitation of Ca-P at first, then the granular bodies appeared at the beginning of incubation. These moss-like bodies seemed to be replaced gradually by granular bodies, and later, cotton fiber-shaped structures appeared. After 24 hr of incubation, the aggregation of spheroid bodies, composed of hydroxyapatite and CNTs, became the dominant constitute of the products. Ca and P were detected by EDS even when there were no precipitations in SEM image, giving the evidence that the surface CNTs was slightly covered by calcium and phosphate compounds from very early stage. This was further strengthened by a chromatographic result that the chromatography of slightly modified CNTs clearly showed the greater affinity for an acidic protein (albumin) than the unmodified CNT column. Combining with the evidence by EDS for Ca and P contents, this chromatographic result clearly indicates that the presence of calcium phosphates on the surface of CNTs, even though the observation by SEM could not detect it. The improvement of biocompatibility and the functionalization of CNTs could be done using calcification solution and the possible applications were suggested for the adsorption and release of various growth factors and medicines.

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The elemental concentration, shape and chemical state of debris contained in the articular capsules of metal-on-metal hip joints were examined. Co derived from Co-Cr alloy was detected with high concentration. By TEM observation, some larger debris in several hundred nanometers and a lot of smaller debris smaller than several ten nanometers were observed. The debris could be observed in the severe metallosis region. Most of Co contained in the articular capsules could be estimated as the metallic state by fluorescence XAFS analysis, which provides high sensitivity for chemical state analysis. Therefore, the metallosis of the articular capsule could be assumed as mainly caused by the metallic debris.

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Multi-walled carbon nanotubes (MWNTs) and the hydroxyapatite (HA) precursor of CaHPO₄·H₂O and Ca(OH)₂ were mixed in water slurry and sintered at 1200^oC at 120 MPa by spark plasma sintering (SPS). MWNTs were dispersed in the composite as agglomerates less than 50 mm in length and 5 mm in width, consisting of MWNTs and alumina nanocrystals smaller than 100 nm. The bulk density and Vickers hardness were decreased with increasing content of MWNTs. The strength of the composite was improved by the addition of MWNTs. Pull-out MWNTs were observed on the fracture surface of the agglomerate and contributed to strengthen the composite.

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In this study, we have succeeded in synthesizing and characterizing a water-soluble multi-walled carbon nanotube. Carbon nanotubes have been functionalized with many hydrophilic carboxylic groups on the surface (3-10 wt%) by a poly-carboxylation reaction and the obtained products show stable aqueous dispersion. To examine the nanotube's preliminary biocompatibility, we injected the functionalized carbon nanotube solution into mice through the tail vein. The time-profile of the subjects' body weight had a similar tendency to that of mice in a control group during 4-week post-injection examination. The injected carbon nanotube derivatives were observed in specimens of the lung and liver using a transmission electron microscope. These results suggest that the injected carbon nanotubes reach some organs through blood circulation after injection but they did not cause serious acute toxicity in mice.

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Transparent PMMA/layered silicate nanocomposites were fabricated by a solution intercalation method. Montmorillonite (MMT), organically modified with alkylammonium ions, was selected as the filler for reinforcement. Platelets of organically modified MMT were well-dispersed macro-scopically in dissolved PMMA. X-ray diffraction patterns showed that a peak regarded as a basal spacing was shifted toward the lower 2θ values, indicating a diffusion of PMMA polymer chains into interlayer regions of MMT. TEM micrographs revealed well-intercalated and partially exfoliated structures of organically modified MMT. Board-shaped specimens used for flexural tests were fabricated by compression molding at 230°C. The specimens were transparent enough for their use as esthetic orthodontic wires. The flexural modulus of the nanocomposites increased marginally, relative to that of PMMA, with the increasing amount of clay.

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The effects of calcium and phosphate contents on bone formation activity by BMP-2 with nano-hydroxyapatite-collagen composites (nHACs) were evaluated. Nanosize crystallites with different concentrations of hydroxyapatite were successfully fabricated by changing the mixing ratio of calcium solution and phosphate-containing neutralization buffer. Composites were implanted into the subcutaneous tissue in the Wistar male rats with BMP-2 for 2 and 4 weeks. nHAC was degraded and inflammation in the surrounding area decreased with time.
Ectopic bone formation was observed around nHACs with higher contents of nano-hydroxyapatite in BMP-2 application at 2 weeks, but was not observed with lower contents at 4 weeks. These results suggest that nHAC with a higher content of nano-hydroxyapatite is suitable as a carrier for BMP-2.

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This study design is experimental study using a syringomyelia model. Fibroblast growth factor (FGF) 2 is an endogenous neurotrophic growth factor in the central nervous system (CNS). The aim of this study was to examine how the spatial and temporal expression of FGF2 in the rat spinal cord changes over the 20-week period following the induction of experimental syringomyelia in rats. Rats were subjected to intracisternal injection of kaolin, which causes syringomyelia. The rats were sacrificed at 0, 3, 7, 10, and 12 weeks, and the spinal cord was histologically examined. The localization of FGF-2 and glial fibrillary acidic protein (GFAP) was also examined by immunohistochemistry. In the normal, slight FGF2 immunoreactivity was observed in a few axons and myelin sheaths in the white matter of the spinal cord. Three weeks after injection, no syrinx formation and little vacuolar degeneration were seen in the white matter, while a mild increase in FGF2 immunoreactivity were in some axons and myelin sheaths. At 7, 10, and 20 weeks, the central canal and the syrinx formation were enlarged. These changes were followed by demyelination. The number of FGF2-positive axons and myelin sheaths increased over time, and this seemed to correlate positively with the progress of the vacuolar degenerative changes. These results showed a detailed expression pattern of FGF2 during the establishment process of experimental syringomyelia, and suggest that a role played by FGF2 in the pathophysiology of this disease.

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There are many research reports about the safety of the nanomaterials inside of the human body. However, the embryotoxicity risk of nano material is still not clear. Since numerous nanomaterials are currently being developed, a method for testing embryotoxicity in vitro in a speedy, effective, and precise manner is needed. In 1997. Spielmann et al. developed the embryonic stem cell test (EST), which is an in vitro embryotoxicity test method that can be used to estimate the risk of embryotoxicity of chemical substances relatively quickly compared to the conventional methods that involve animal experiments. The EST has been evaluated in a formal validation study funded by the ECVAM (European Centre for the Validation of Alternative Methods) in which two other in vitro embryotoxicity tests (micromass test, whole embryo culture test) were validated against a set of test chemicals characterized by high levels of in vivo embryotoxicity data in laboratory animals and humans. In a validation study in Europe, the EST was found to be reproducible, demonstrating an overall accuracy of 80% and 100% correct prediction of strong embryotoxicity for chemicals studied under blind conditions. However, use of EST is impossible for the nanomaterial which is not dissolved in a culture medium. Therefore, some improvement of EST protocol is proposed.

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