Tsukasa Akasaka

Effect of carbon nanotubes on cellular functions in vitro

Carbon nanotubes (CNTs) have been shown to affect cell behavior. But how and why the CNTs affect potential differentiation of the attached cells has not been largely known. In this study, multiwalled carbon nanotubes (MWNTs) and graphite (GP) were pressed as compacts. Higher ability of CNTs to adsorb proteins, compared with GP, was shown. Myoblastic mouse cells (C2C12) were cultured and the cell responses to the two kinds of compacts were compared in vitro. Meanwhile, we used cell culture on the culture plate as a control. During the conventional culture, significantly better cell attachment, proliferation, and differentiation of cells on the MWNTs were found. To confirm the hypothesis that the larger amount of protein adsorbed on the CNTs was crucial for this, we made the compacts adsorb more proteins in culture medium with 50% fetal bovine serum (FBS) before cell culture. With the adsorption of the proteins in advance, the increments of the total-protein/DNA and alkaline phosphatase (ALP)/DNA for the MWNTs was respectively as about 11 times and 18 times as the increments of those for GP and the control at both day 4 and day 7. Therefore, the CNTs might induce cellular functions by adsorbing more proteins, which indicated that the CNTs might be a candidate for scaffold material for tissue engineering.

Material nanosizing effect on living organisms: non-specific, biointeractive, physical size effects

Nanosizing effects of materials on biological organisms was investigated by biochemical cell functional tests, cell proliferation and animal implantation testing. The increase in specific surface area causes the enhancement of ionic dissolution and serious toxicity for soluble, stimulative materials. This effect originates solely from materials and enhances the same functions as those in a macroscopic size as a catalyst. There are other effects that become prominent, especially for non-soluble, biocompatible materials such as Ti. Particle size dependence showed the critical size for the transition of behaviour is at approximately 100 μm, 10 μm and 200 nm. This effect has its origin in the biological interaction process between both particles and cells/tissue. Expression of superoxide anions, cytokines tumour necrosis factor-α and interleukin-1β from neutrophils was increased with the decrease in particle size and especially pronounced below 10 μm, inducing phagocytosis to cells and inflammation of tissue, although inductively coupled plasma chemical analysis showed no dissolution from Ti particles. Below 200 nm, stimulus decreases, then particles invade into the internal body through the respiratory or digestive systems and diffuse inside the body. Although macroscopic hydroxyapatite, which exhibits excellent osteoconductivity, is not replaced with natural bone, nanoapatite composites induce both phagocytosis of composites by osteoclasts and new bone formation by osteoblasts when implanted in bone defects. The progress of this bioreaction results in the conversion of functions to bone substitution. Although macroscopic graphite is non-cell adhesive, carbon nanotubes (CNTs) are cell adhesive. The adsorption of proteins and nano-meshwork structure contribute to the excellent cell adhesion and growth on CNTs. Non-actuation of the immune system except for a few innate immunity processes gives the non-specific nature to the particle bioreaction and restricts reaction to the size-sensitive phagocytosis. Materials larger than cell size, approximately 10 μm, behave inertly, but those smaller become biointeractive and induce the intrinsic functions of living organisms. This bioreaction process causes the conversion of functions such as from biocompatibility to stimulus in Ti-abraded particles, from non-bone substitutional to bone substitutional in nanoapatite and from non-cell adhesive to cell adhesive CNTs. The insensitive nature permits nanoparticles that are less than 200 nm to slip through body defence systems and invade directly into the internal body.

Capture of bacteria by flexible carbon nanotubes

Capture of bacteria with flexible carbon nanotubes (CNTs) was done in vitro. Bundles of single-walled carbon nanotubes (SWCNTs) or multi-walled carbon nanotubes (MWCNTs) was mixed with Streptococcus mutans. Precipitation assays and colony-forming unit formation assays showed free S. mutans in the solution was significantly decreased by the addition of the CNTs. Observation of the precipitate by scanning electron microscopy showed bacterial adhesion to CNTs. It has been shown that CNTs of different diameters have significantly different effects on the precipitation efficiency, and the manners in which they capture the cells are different. We found that MWCNTs (diameter of approximately 30 nm) had the highest precipitation efficiency, which was attributable to both their adequate dispersibility and aggregation activity. From observations by scanning electron microscopy, bundles of SWCNTs and thin MWCNTs (diameter of approximately 30 nm), which were moderately flexible, were easily wound around the curved surface of S. mutans. Bare CNTs having high adhesive ability could be useful as biomaterials, e.g., as tools for the elimination of oral pathogens at the nano-level.

Maturation of osteoblast-like SaoS2 induced by carbon nanotubes

Osteogenic maturation of the osteoblast is crucial for bone formation. In this study, multi-walled carbon nanotubes (MWCNTs) and graphite (GP) were pressed as compacts. The greater ability of carbon nanotubes to adsorb proteins, compared with graphite, was shown. Human osteoblast-like SaoS2 cells were cultured and the cell response to the two kinds of compacts was compared in vitro. Meanwhile, we used cell culture on the culture plate as a control. Assays for osteonectin, osteopontin and osteocalcin gene expression, total protein (TP) amount, alkaline phosphatase activity (ALP) and DNA of cells cultured on the samples were done. During the conventional culture, significantly higher osteonectin, osteopontin and osteocalcin gene expression level, ALP/DNA and TP/DNA on carbon nanotubes were found. To confirm the hypothesis that the larger amount of specific proteins adsorbed on the carbon nanotubes was crucial for this, the compacts were pre-soaked in culture medium having additional recombinant human bone morphogenetic protein-2 (rhBMP-2) before cell culture. Compared with GP, osteonectin, osteopontin and osteocalcin gene expression level, ALP/DNA and TP/DNA of the cells tested increased more on the MWCNTs after the compacts were pre-soaked in the culture medium with rhBMP-2. The results indicated that the carbon nanotubes might induce osteogenic maturation of the osteoblast by adsorbing more specific proteins.

Development of a 3D Collagen Scaffold Coated With Multiwalled Carbon Nanotubes

Carbon nanotubes (CNTs) have attractive biochemical properties such as strong cell adhesion and protein absorption, which are very useful for a cell cultivation scaffold. In this study, we prepared a multiwalled carbon nanotube-coated collagen sponge (MWCNT-coated sponge) to improve the surface properties of the collagen sponge, and its cell culturing properties were examined. The suface of the collagen sponge was homogeneously coated with MWCNTs by dispersion. MC3T3-E1 cells were cultured on and inside the MWCNT-coated sponge. The DNA content on the MWCNT-coated sponge after 1 week of culture was significantly higher than on an uncoated collagen sponge (p < 0.05). There was no significant difference between the estimated ALP activity normalized by DNA quantity on the MWCNT-coated sponge and that on the uncoated collagen sponge which is well known as one of the best scaffolds for cell cultivation. In addition, the MWCNT-coated surface shows strong cell adhesion. Therefore, the MWCNT-coated collagen sponge is expected to be a useful 3D scaffold for cell cultivation. (c) 2009 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2009.

In Vitro Evaluation of Porous Poly(L-Lactic Acid) Scaffold Reinforced by Chitin Fibers

In this study, the previously reported porous three-dimensional poly(L-lactic acid) (PLLA) scaffolds reinforced by the chitin fibers (PLLA/CF) with and without the link were evaluated in vitro. Firstly, pH value of the phosphate buffered saline lixiviums of the PLLA/CF with different content of the chitin fibers was measured to get an appropriate content of the chitin fibers in the PLLA/CF. Then, the cell functions (attachment, proliferation, alkaline phosphatase per unit cell, total protein per unit cell, and osteonectin, osteopontin, and osteocalcin gene expression) of human osteoblast-like cells (SaOS2) cultured on the PLLA/CF with the link, PLLA/CF without the link and PLLA scaffold were compared. The results showed that the link treatment did not significantly influence the pH value of the lixiviums of the scaffolds, 30% volume content might be an appropriate content of the chitin fibers in PLLA/CF scaffold to keep the pH value of the lixiviums of the scaffolds between 7.0 and 7.2 during the lixiviation time of 16 weeks, the PLLA/CF scaffold was significantly better for the attachment, proliferation, differentiation, and mineralization of the osteoblast than PLLA, the link treatment did not significantly influence these cells activities, which further suggested that PLLA/CF with the link treatment might be an appropriate scaffold for tissue engineering.

Synthesis of polymethacrylate derivatives having sulfated maltoheptaose side chains with anti‐HIV activities

Anti-HIV (human immunodeficiency virus) active polymethacrylates having pendant sulfated oligosaccharides were synthesized, and the relationship between structures and biological activities of the polymethacrylates was examined. Acetylated 1-O-methacryloyl maltoheptaoside (MA-AcM7) was polymerized with AIBN as an initiator to give polymethacrylates having a pendant acetylated maltoheptaose in every repeating unit, poly(MA-AcM7)s. After hydroxyl groups were recovered by deacetylation, the polymethacrylates having maltoheptaose units, poly(MA-M7)s, were sulfated to give polymethacrylates having sulfated maltoheptaose side-chains, poly(MA-SM7)s, with degrees of sulfation of 1.1 to 2.7 (maximum, 3.0). These polymethacrylates including sulfated oligosaccharides exhibited low anti-HIV activities represented by the 50% protecting concentration (EC 50 ) in the range of 15-62 μg/mL and low blood anticoagulant activities around 10 unit/mg (standard dextran sulfate, 22.7 unit/mg). The anti-HIV activity increased with increasing degree of sulfation to reach EC 50 of 15-16 μg/mL. In addition, copolymerization of MA-AcM7 with methyl methacrylate (MMA) and subsequent sulfation gave polymethacrylates consisting of various proportions of highly sulfated maltoheptaose and MMA units. It was revealed that the anti-HIV activity increased with decreasing proportion of the sulfated oligosaccharide moiety and that a copolymethacrylate having 22 mol % of sulfated maltoheptaose units (DS = 3.0) had a high anti-HIV activity in the EC 50 of 0.3 μg/mL. The blood anticoagulant activity increased slightly from 9 to 18 unit/mg with decreasing proportion of the sulfated maltoheptaose units. These results suggested that the biological activities were influenced strongly by the spatial distance between sulfated oligosaccharide substituents in the polymethacrylate main chain. Distinction and conformation of the oligosaccharide side chains also played an important role.

3D collagen scaffolds coated with multiwalled carbon nanotubes : Initial cell attachment to internal surface

The cell adhesion in a multiwalled carbon nanotube-coated collagen sponge (MWCNT-coated sponge) was investigated. Immediately after seeding, the cells adhered to the inner surface of the MWCNT-coated sponge and a significantly larger number of cells were observed there than for a pure collagen sponge used as control. On the MWCNT-coated sponge, the cells appeared favorable adhesion and spread in the early stages in the center part of the sponge which cells rarely attached without MWCNT-coating. It was suggested that the physical structure of MWCNTs was effective for initial adhesion of cells from the result of serum-free culture. MWCNT-coating makes the material a suitable three-dimensional scaffold for cell culturing, as opposed to other scaffold systems where such an effect is not seen.

Effects of Micro/Nano Particle Size on Cell Function and Morphology

The cytotoxicity of micro/nano particles in Ti, TiO and carbon nanotube was investigated by in vitro biochemical analyses using human neutrophils. The particles smaller and larger than the neutrophils were used to determine the relationship between cell and pa rticle size with respect to cytotoxicity. As the particle size decreased, the cell survival rate was decreased and, with the good corresponding relation to this, the value of lactate dehydrogenase (LDH ), which is the indication of cell disruption, was increased. The release of superoxide anion showed t he increasing tendency. Proinflammatory cytokines were detected distinctly for 3μm or sm aller particles and very little in more than 10μm, which is closely related to the phagocytosis by neutrophil s. ICP elemental analysis showed that the dissolution from Ti particles was below detection li mit. Micro and nano particles stimulated the cell reactions according to the results of the huma n neutrophil functional tests. As the particle size was smaller, the inflammation was pronounced. The fine particl es less than 3μm caused distinctly the inflammation in the surrounding tissue. All these results i ndicated that the cytotoxicity was induced due to the physical size effect of particles, which is different from the ionic dissolution effect. The clinical phenomenon confirmed the result obtained in vitro ce ll tests. The neutrophils stimulated by fine particles may cause the inflammatory cascade and harm the surrounding tissue.

Comparative study of bioactivity of collagen scaffolds coated with graphene oxide and reduced graphene oxide

Background Graphene oxide (GO) is a single layer carbon sheet with a thickness of less than 1 nm. GO has good dispersibility due to surface modifications with numerous functional groups. Reduced graphene oxide (RGO) is produced via the reduction of GO, and has lower dispersibility. We examined the bioactivity of GO and RGO films, and collagen scaffolds coated with GO and RGO. Methods GO and RGO films were fabricated on a culture dish. Some GO films were chemically reduced using either ascorbic acid or sodium hydrosulfite solution, resulting in preparation of RGO films. The biological properties of each film were evaluated by scanning electron microscopy (SEM), atomic force microscopy, calcium adsorption tests, and MC3T3-E1 cell seeding. Subsequently, GO- and RGO-coated collagen scaffolds were prepared and characterized by SEM and compression tests. Each scaffold was implanted into subcutaneous tissue on the backs of rats. Measurements of DNA content and cell ingrowth areas of implanted scaffolds were performed 10 days post-surgery. Results The results show that GO and RGO possess different biological properties. Calcium adsorption and alkaline phosphatase activity were strongly enhanced by RGO, suggesting that RGO is effective for osteogenic differentiation. SEM showed that RGO-modified collagen scaffolds have rough, irregular surfaces. The compressive strengths of GO- and RGO-coated scaffolds were approximately 1.7-fold and 2.7-fold greater, respectively, when compared with the non-coated scaffold. Tissue ingrowth rate was 39% in RGO-coated scaffolds, as compared to 20% in the GO-coated scaffold and 16% in the non-coated scaffold. Conclusion In summary, these results suggest that GO and RGO coatings provide different biological properties to collagen scaffolds, and that RGO-coated scaffolds are more bioactive than GO-coated scaffolds.