To observe the dynamic response behavior of cells under exposure to nano / micro particles, time-lapse optical microscopic observation method was applied. Though the conventional SEM observation after chemical fixation indicated only static morphological information, this method allows the observation of dynamic cell morphology and functional activity. The dynamic behavior of cells observed by time-lapse method showed qualitatively a similar tendency to the results obtained by the conventional cytotoxicity test. TiO2 and ITO nanoparticles showed little effect on cell behavior. Cells in contact with CuO particles showed a decrease in activity. However, cells without contact were as active as control. The activity decrease was observed more rapidly under nanoparticle exposure due to the higher probability of nanoparticles to contact to cells, compared with microparticles in the same concentration. The present experimental method can allow to evaluate the influence of materials on cell function in a dynamical activity state.
Carbon nanotubes (CNTs) have the unique characteristics such as cell proliferation, extension of filopodium and enhancement of alkaline phosphatase activity to osteoblast-like cells. Proteins play important roles for cell proliferation and differentiation. In this study, adsorption of proteins was investigated by liquid chromatography using CNTs with different purification treatments to clarify the relation of proteins and CNTs. Two different sorts of CNTs were used, bold straight shape with high purity, and fine curled shape with low purity. The former CNTs showed rather weak adsorption. With processing of purification treatments adsorption ability was increased. Electrophoresis, protein sequence and mass spectrum indicated that proteins with a lower molecular weight had the tendency to be more strongly adsorbed. On the other hand, the latter CNTs showed strong adsorption regardless pretreatments.
The use of dental implants currently entails problems associated with lack of adhesion to the oral mucosa at the point of abutment, which differs from that of natural teeth. Usually, the experimental evaluation of adhesion between test implant materials and soft tissue requires animal experiments. In this study, to facilitate easier evaluation, adhesion experiments were performed using a three-dimensional (3D) in vitro epidermal cell culture model. The implant materials examined were nano hydroxyapatite (nHAp) coated polyethylene terephthalate (PET), uncoated PET, zirconia, and titanium. The nHAp particles exhibited high cell adhesion, and nHAp coated PET exhibited high tissue-adhesion in the 3D epidermal cell culture in vitro. Zirconia exhibited substantial tissue adhesion to the 3D epithelial tissue, but titanium exhibited little adhesion. This 3D in vitro epidermal cell culture model could be used instead of animal experiments to facilitate easier evaluation of adhesion between tissue and materials, and it could be useful in screening tests for the development of tissue adhesive materials and processing conditions.
Why do mesenchymal stem cells (MSCs) regenerate good cartilage tissue in vitro under condition of microgravity generated by a rotating vessel? To answer this question, we developed a PIV measuring apparatus equipped with a RWV (rotating wall vessel) bioreactor, which enabled visualization of the three dimensional medium flow applied to the aggregated cartilaginous cells. The spacial distribution of cartilage marker genes (sox-9, aggrecan, and collagen type II) was measured using a microdisection system and discussed in association with the PIV results.
To identify the respective contributions of released zinc and solid particles on the cytotoxicity of zinc oxide nanoparticles (ZnO-NPs), we exposed A549 cells to ZnO-NP suspensions, their extractions collected after centrifugation, and medium containing zinc chloride (ZnCl2). We then assayed the cytotoxicity of these samples using water-soluble tetrazolium salts (WSTs) and intracellular reactive oxygen species (ROS) with 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) as outputs. Only the ZnO-NP suspension caused cytotoxicity and increased intracellular ROS; the extractions and ZnCl2 did not cause cytotoxicity or oxidative stress. Global gene expression analysis revealed that both ZnCl2-containing medium and the ZnO-NP suspension caused upregulation of the "cadmium binding" gene functional category. This category consisted of metallothioneins (MTs), important zinc-homeostasis proteins. To further investigate the role of MTs in ZnO-NP-dependent cytotoxicity, we inhibited the overexpression of MTs with corresponding siRNA and found that released zinc contributed to ZnO-NP-dependent cytotoxicity. We conclude that both solid particles and released zinc contributed to ZnO-NP-dependent cytotoxicity. Additionally, we propose a synergic relationship between ZnO-NPs and MTs.
Amide bond formation between carboxylated multi-walled carbon nanotubes (MWCNT-COOHs) and a glass surface with a water-soluble condensing agent in aqueous media was examined to make a transparent conductive material. The condensing agent used was DMT-MM (4-(4,6-dimethoxy- 1,3,5-triazin-2-yl)-4-methylmorpholinium chloride). The extent of immobilization depended on the DMT-MM content in solution; and an optimum concentration of DMT-MM was found to exist. Finally, a mechanism for the variation of the extent of immobilization as a function of DMT-MM content was proposed.
The in vivo reaction against nano-apatite is still not well understood. We self-prepared four samples using medical-grade collagen and commercial (40 nm) nano-apatite particles (n-HAp), namely (i) collagen sponge (sample C), (ii) n-HAp collagen composite sponge (sample AC), (iii) collagen sponge with basic fibroblast growth factor (b-FGF) (sample CF) and (iv) n-HAp collagen composite sponge with b-FGF (sample ACF); and implanted them in subcutaneous tissues of the back of mice for 1 and 2 weeks. Visual inspections and histological observations revealed that sample ACF caused most intensified angiogenesis with time, followed by sample CF and sample AC, while sample C caused little angiogenesis. More the angiogenetic, more the foreign body reactions. It was confirmed that n-HAp bound in collagen sponge was safe and bio-absorbable; and n-HAp had minor capability of angiogenesis, and if coupled with b-FGF, strong capability of angiogenesis at the implanted site in soft tissues. This biological property could be useful for re-generation of hard tissues as well as soft tissues in the future clinical applications.
Carbon nanotubes have various features, and many industries have an interest in carbon nanotubes. Recently, however, artificial nanoparticles have become a public health concern. Current experiments have indicated that the survival rate of cultured cells gradually declined by exposure to single-walled carbon nanotubes (SWCNTs) despite SWCNT purification. These results suggest that SWCNTs have not only the toxicity of catalyst metals but also their own toxicity. However, SWCNTs did not induce DNA mutagenesis, which means that SWCNTs do not transform cells directly from a normal to malignant state. In contrast, the uptake process of SWCNTs was clarified with a transmission electron microscope (TEM). SWCNTs contained other risk of toxicity because of the needlelike structure and persistence in biological systems. TEM observation revealed that SWCNTs brought into cells by clathrin-mediated endocytosis were transported subsequently to the lysosome. Such accumulated SWCNTs appeared to injure the cells in several ways that pierced the organelle and broke the membrane during cytokinesis or movement. SWCNTs that pierced the cell membrane were found frequently.
This study examined in vivo biocompatibility of poly-L-lactide (PLLA) scaffolds coated with hydroxyl apatite (HA) by pulsed laser deposition (PLD). A thin HA film was made on PLLA discs by an ArF PLD operating at a repletion rate of 10 Hz. Cranial bone defects were created in rats and filled with non-HA-coated PLLA scaffold (PLLA) or HA-coated PLLA scaffold (HAPLLA). All specimens were evaluated micro-radiographically and with histological analysis at 2, 4, and 6 weeks. The presence of calcium and phosphorous on the surface as well as in the interior of the PLLA was confirmed by energy dispersive analysis. In vitro cell-spread tests showed that the human osteoblasts spread more on HAPLLA than on PLLA. Upon transplanting calvarial defects, the in vivo hard tissue responses suggested earlier restoration of the defects with HAPLLA than with PLLA.
In this study, we investigated the biodistribution of water-soluble carbon nanotubes (CNTs) after administration through the tail vein of mice. The biodistribution was investigated using an optical and transmission electron measurement. Some of the CNTs remained in the liver and lung even 4 weeks post-injection. We also succeeded in direct observation of CNTs in the excited organs using Raman spectroscopy. In future, this method can allow to observation as a Raman mapping images of CNTs in body.