Shinsuke Kobayashi

Safe clinical use of carbon nanotubes as innovative biomaterials.

Carbon nanotubes (CNTs) are structurally described as sheets of six-membered carbon atom rings (i.e., graphene) rolled up into cylinders. CNTs with only one layer are known as single-walled CNTs (SWCNTs), and those with two or more layers are known as multiwalled CNTs (MWCNTs). Cup-stacked carbon nanotubes and carbon nanohorns are also sometimes called CNTs.1−3 Currently, these very attractive carbon materials and nanomaterials are a subject of vigorous product development in a broad range of fields.4−11 The reasons are that CNTs have useful electrical, thermal, and mechanical characteristics, and their base material performance can be improved by combination with other materials.12−23 A recent industrial application of CNTs as an electrode additive to lithium-ion batteries is based on their excellent electrical characteristics. Addition of CNTs prevents battery deterioration and substantially lengthens time to recharging. It is doubtless that the demand for high-performance batteries will grow increasingly with multifunctionalization of personal computers and mobile phones, development of new mobile terminals, spread of electric vehicles, and other factors.24−30 Composite materials with the excellent mechanical characteristics of CNTs have already been used in sporting goods such as golf clubs, tennis rackets, and bicycles. CNTs are also expected to have applications that reduce the weight of aircraft and automobiles.10,14,31−35 A wide variety of advantages are gained from the use CNTs in precision parts as well. CNTs are also used in transistors and memory devices, and enhance their efficiency. The use of CNTs in various displays and TV screens continues to increase in rate. CNTs are also widely used in products designed to prevent static electricity, to shield electromagnetic waves, to store electricity, and for other purposes.36−45 Furthermore, Japan is now facing nuclear energy issues stemming from the accident at Tokyo Electric Power Company’s Fukushima No. 1 nuclear power plant. As a result, CNTs are expected to play a major role in developing new energy sources such as solar photovoltaic power generation and wind power generation.46−52 In the medical field, extensive research activities are underway to develop new CNTs biomaterials for use in the treatment and diagnosis of disease. For example, application of CNTs to cancer treatment and diagnosis, such as in drug delivery systems (DDSs) for treatment of cancer, hyperthermia, and in vivo imaging, has been investigated.53−57 In a study that aimed at applying CNTs to regenerative medicine, CNTs were found to work excellently as scaffold materials for nerve and bone tissue regeneration.58−63 Furthermore, R&D activities are underway to improve the mechanical strength and durability of implants by combining CNTs with existing biomaterials.64−67 Besides, numerous ideas have been put forth about how CNTs can be used in the treatment of a variety of diseases. Figure ​Figure11 shows the trend in the number of articles found in the PubMed database (http://www.ncbi.nlm.nih.gov/pubmed/) (accessed 20 March 2014) by searches using “carbon nanotubes” and “biomaterials” as keywords. The number has been soaring since 2005, suggesting that CNTs research has become a highly competitive field worldwide over the past few years. Of course, numerous articles on the biological applications of CNTs do exist that cannot be captured with these two simple keywords, and the graphic representation of this trend is no more than an indicator of the increase in this research over time. Figure 1 Time trends for the number of articles found in the PubMed database (http://www.ncbi.nlm.nih.gov/pubmed/) (accessed 20 March 2014) by search using “carbon nanotubes” and “biomaterials” as keywords. Recent years have seen ...

Trabecular minimodeling in human iliac bone

In adult human beings, remodeling creates nearly all of new bone tissue. However, Frost hypothesized that modeling can go on in trabeculae throughout life. As this hypothesis has not been verified, we looked for histologic evidence of trabecular modeling (minimodeling) during bone histomorphometry of transiliac bone biopsy specimens obtained from 34 patients (age range, 38-81 years; mean age, 58.4 years; female, 31/34) at the time of total hip arthroplasty. Before the bone biopsy study, we performed quantitative bone scintigraphy of bilateral hip joints and bilateral iliac crests in 10 other patients with unilateral hip disease and confirmed that the bone biopsy site was not affected by ipsilateral hip joint disease. Patients who had metabolic bone diseases or who had taken medications known to affect bone metabolism were excluded from the study. During modeling where bone formation and bone resorption are not coupled, bone formation can occur on quiescent bone surfaces without preceding bone resorption and create smooth cement lines. Therefore, the combination of fluorochrome labeling and a smooth cement line without interruption of surrounding collagen fibers was regarded as evidence of minimodeling. Histologic evidence of minimodeling was detected in 21 of the entire 34 specimens (62%) and 17 of 27 specimens obtained from postmenopausal patients (63%). Bone volume of minimodeling sites was less than 1% of the trabecular bone volume, and these sites accounted for less than 2% of the entire bone surface on average. However, osteoid volume of minimodeling sites comprised approximately one-tenth of the entire osteoid volume, and their labeled surface constituted one-fourth to half of the entire labeled surface on average. Therefore, when performing bone histomorphometry of adult cancellous bone, minimodeling should be taken into account when dealing with parameters related to osteoid volume and mineralization. A comparison of specimens with and without minimodeling demonstrated that the presence of minimodeling was correlated with smaller physique of patients, accelerated mineralization (as indicated by the higher mean MS/BS and MAR values and the shorter mean Omt), and higher metabolic turn-over of bone (as indicated by the higher mean BFR/BV value). Although the findings still need to be verified in a larger number of normal subjects without hip joint disease, they support Frosts hypothesis that minimodeling can continue throughout human life.

Culture medium type affects endocytosis of multi-walled carbon nanotubes in BEAS-2B cells and subsequent biological response.

We examined the cytotoxicity of multi-walled carbon nanotubes (MWCNTs) and the resulting cytokine secretion in BEAS-2B cells or normal human bronchial epithelial cells (HBEpCs) in two types of culture media (Hams F12 containing 10% FBS [Hams F12] and serum-free growth medium [SFGM]). Cellular uptake of MWCNT was observed by fluorescent microscopy and analyzed using flow cytometry. Moreover, we evaluated whether MWCNT uptake was suppressed by 2 types of endocytosis inhibitors. We found that BEAS-2B cells cultured in Hams F12 and HBEpCs cultured in SFGM showed similar biological responses, but BEAS-2B cells cultured in SFGM did not internalize MWCNTs, and the 50% inhibitory concentration value, i.e., the cytotoxicity, was increased by more than 10-fold. MWCNT uptake was suppressed by a clathrin-mediated endocytosis inhibitor and a caveolae-mediated endocytosis inhibitor in BEAS-2B cells cultured in Hams F12 and HBEpCs cultured in SFGM. In conclusion, we suggest that BEAS-2B cells cultured in a medium containing serum should be used for the safety evaluation of nanomaterials as a model of normal human bronchial epithelial cells. However, the culture medium composition may affect the proteins that are expressed on the cytoplasmic membrane, which may influence the biological response to MWCNTs.

Carcinogenicity evaluation for the application of carbon nanotubes as biomaterials in rasH2 mice

The application of carbon nanotubes (CNTs) as biomaterials is of wide interest, and studies examining their application in medicine have had considerable significance. Biological safety is the most important factor when considering the clinical application of CNTs as biomaterials, and various toxicity evaluations are required. Among these evaluations, carcinogenicity should be examined with the highest priority; however, no report using transgenic mice to evaluate the carcinogenicity of CNTs has been published to date. Here, we performed a carcinogenicity test by implanting multi-walled CNTs (MWCNTs) into the subcutaneous tissue of rasH2 mice, using the carbon black present in black tattoo ink as a reference material for safety. The rasH2 mice did not develop neoplasms after being injected with MWCNTs; instead, MWCNTs showed lower carcinogenicity than carbon black. Such evaluations should facilitate the clinical application and development of CNTs for use in important medical fields.

Basic potential of carbon nanotubes in tissue engineering applications

Carbon nanotubes (CNTs) are attracting interest in various fields of science because they possess a high surface area-to-volume ratio and excellent electronic, mechanical, and thermal properties. Various medical applications of CNTs are expected, and the properties of CNTs have been greatly improved for use in biomaterials. However, the safety of CNTs remains unclear, which impedes their medical application. Our group is evaluating the biological responses of multiwall CNTs (MWCNTs) in vivo and in vitro for the promotion of tissue regeneration as safe scaffold materials. We recently showed that intracellular accumulation is important for the cytotoxicity of CNTs, and we reported the active physiological functions CNTs in cells. In this review, we describe the effects of CNTs in vivo and in vitro observed by our group from the standpoint of tissue engineering, and we introduce the findings of other research groups.

Endocytosis of Multiwalled Carbon Nanotubes in Bronchial Epithelial and Mesothelial Cells

Bronchial epithelial cells and mesothelial cells are crucial targets for the safety assessment of inhalation of carbon nanotubes (CNTs), which resemble asbestos particles in shape. Intrinsic properties of multiwalled CNTs (MWCNTs) are known to cause potentially hazardous effects on intracellular and extracellular pathways. These interactions alter cellular signaling and affect major cell functions, resulting in cell death, lysosome injury, reactive oxygen species production, apoptosis, and cytokine release. Furthermore, CNTs are emerging as a novel class of autophagy inducers. Thus, in this study, we focused on the mechanisms of MWCNT uptake into the human bronchial epithelial cells (HBECs) and human mesothelial cells (HMCs). We verified that MWCNTs are actively internalized into HBECs and HMCs and were accumulated in the lysosomes of the cells after 24-hour treatment. Next, we determined which endocytosis pathways (clathrin-mediated, caveolae-mediated, and macropinocytosis) were associated with MWCNT internalization by using corresponding endocytosis inhibitors, in two nonphagocytic cell lines derived from bronchial epithelial cells and mesothelioma cells. Clathrin-mediated endocytosis inhibitors significantly suppressed MWCNT uptake, whereas caveolae-mediated endocytosis and macropinocytosis were also found to be involved in MWCNT uptake. Thus, MWCNTs were positively taken up by nonphagocytic cells, and their cytotoxicity was closely related to these three endocytosis pathways.

Biological responses according to the shape and size of carbon nanotubes in BEAS-2B and MESO-1 cells

This study aimed to investigate the influence of the shape and size of multi-walled carbon nanotubes (MWCNTs) and cup-stacked carbon nanotubes (CSCNTs) on biological responses in vitro. Three types of MWCNTs – VGCF®-X, VGCF®-S, and VGCF® (vapor grown carbon fibers; with diameters of 15, 80, and 150 nm, respectively) – and three CSCNTs of different lengths (CS-L, 20–80 μm; CS-S, 0.5–20 μm; and CS-M, of intermediate length) were tested. Human bronchial epithelial (BEAS-2B) and malignant pleural mesothelioma cells were exposed to the CNTs (1–50 μg/mL), and cell viability, permeability, uptake, total reactive oxygen species/superoxide production, and intracellular acidity were measured. CSCNTs were less toxic than MWCNTs in both cell types over a 24-hour exposure period. The cytotoxicity of endocytosed MWCNTs varied according to cell type/size, while that of CSCNTs depended on tube length irrespective of cell type. CNT diameter and length influenced cell aggregation and injury extent. Intracellular acidity increased independently of lysosomal activity along with the number of vacuoles in BEAS-2B cells exposed for 24 hours to either CNT (concentration, 10 μg/mL). However, total reactive oxygen species/superoxide generation did not contribute to cytotoxicity. The results demonstrate that CSCNTs could be suitable for biological applications and that CNT shape and size can have differential effects depending on cell type, which can be exploited in the development of highly specialized, biocompatible CNTs.

Advantages of concurrent use of anabolic and antiresorptive agents over single use of these agents in increasing trabecular bone volume, connectivity, and biomechanical competence of rat vertebrae

The purpose of this study was to evaluate the usefulness of a combination regimen of anabolic and antiresorptive agents in increasing skeletal quantity and quality in comparison to a single-drug regimen with these agents. We examined histomorphometrically and biomechanically the effects of separate and combined administration of intermittent parathyroid hormone (PTH) and estrogen or bisphosphonate on both axial and appendicular skeletons of male Wistar rats, which were 4 months old and weighed approximately 300 g at the beginning of the treatment. The animals were untreated or injected with vehicle, recombinant human PTH(1-84) (PTH; 100 microg/kg daily), 17beta-estradiol (E2, 500 microg/kg every other day), incadronate disodium (YM175, 80 microg/kg every other day), combined PTH and E2 (PTH + E2), or a combination of PTH and YM175 (PTH + YM175). After 1 month of treatment, the three groups in which PTH was administered (PTH, PTH + E2, and PTH + YM175) had significantly higher trabecular bone volume and connectivity in the proximal tibial metaphyses (PTMs) compared with the untreated and vehicle-treated groups, whereas only combination groups (PTH + E2 and PTH + YM175) showed significant increases in these indices in the lumbar vertebrae. This site-related discrepancy was attributed to the fact that PTH significantly elevated bone resorption not in the PTMs but in the vertebrae. This increased bone resorption in the vertebrae was suppressed by the addition of an antiresorptive agent. As a result, trabecular bone mass, connectivity, and mechanical strength of the vertebrae were significantly increased from control levels only in the concurrent treatment groups (PTH + E2 and PTH + YM175). The superior skeletal effects of PTH cotherapy over PTH monotherapy were not seen with regard to bone mass, but with increased connectivity and mechanical strength. The concurrent use of PTH and an antiresorptive agent has been shown to be superior to the single use of PTH for enhancing these properties of rat vertebrae, which encourages future research, especially in larger animals.

Specific biological responses of the synovial membrane to carbon nanotubes.

Biological evaluation of carbon nanotubes (CNTs) is typically performed in the lung or abdominal cavity; however, biological reactions to CNTs are predicted to be markedly different in other tissues. In applications of CNTs as reinforcement for artificial joints and drug delivery systems, including their use in bone regeneration, the intra-articular synovial membrane makes contact with the CNTs. Herein, we analyzed the reaction of the synovial membrane with multiwalled CNTs (MWCNTs). Injection of MWCNTs into rat knee joints revealed their dose-dependent incorporation into deep synovial membranes and the formation of granulation tissue, without long-term inflammation. MWCNTs were incorporated into human fibroblast-like synoviocytes (HFLSs), with less cytotoxicity than that observed in macrophages (RAW264 cells). Moreover, MWCNTs inhibited the release of cytokines and chemokines from HFLSs. The reaction of the synovial membrane with MWCNTs differed from that observed in other tissues; thus, detailed biological evaluation at each target site is necessary for clinical applications.

Titanium Fiber Plates for Bone Tissue Repair

Titanium plates are widely used in clinical settings because of their high bone affinity. However, owing to their high elastic modulus, these plates are not suitable for bone repair since their proximity to the bone surface for prolonged periods can cause stress shielding, leading to bone embrittlement. In contrast, titanium fiber plates prepared by molding titanium fibers into plates by simultaneously applying compression and shear stress at normal room temperature can have an elastic modulus similar to that of bone cortex, and stress shielding will not occur even when the plate lies flush against the bones surface. Titanium fibers can form a porous structure suitable for cell adhesion and as a bone repair scaffold. A titanium fiber plate is combined with osteoblasts and shown that the titanium fiber plate is better able to facilitate bone tissue repair than the conventional titanium plate when implanted in rat bone defects. Capable of being used in close contact with bone for a long time, and even capable of promoting bone repair, titanium fiber plates have a wide range of applications, and are expected to make great contributions to clinical management of increasing bone diseases, including bone fracture repair and bone regenerative medicine.