Nobutaka Hanagata

Hokkaido University, Sapporo, Hokkaidō, Japan

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Publications (154)555.05 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Gene delivery is often accomplished by the forward or reverse transfection protocol. In either protocol, a transfection reagent (usually cationic) is added to increase the delivery efficiency. In this study, we employed a series of nanosheet networks to facilitate the delivery of naked plasmid DNA into human mesenchymal stem cells (hMSCs). By adding different chemicals into the reaction mixture for etching the silica glass, we were able to fabricate inorganic/organic hybrid nanosheet networks with different physico-chemical characteristics. We then analyzed the transfection efficiency on different nanosheets and the possible dependence of the transfection efficiency on the physico-chemical parameters of nanosheets. The results showed that all nanosheet networks were noncytotoxic and demonstrated a high cell survival rate (∼90%) after transfection. The transfection efficiency was critically determined by the aspect ratio (height/thickness of the wall) of the nanosheets. The effects of chemistry or other surface properties were not significant. Moreover, the transfection efficiency may be successfully predicted by the initial cell migration rate and the activation of integrin β3 on the nanosheets. Compared to the conventional method, transfection using concurrent cell/plasmid seeding on the nanosheets is not only more effective but also much safer. Future efforts may focus on combining the inorganic/organic hybrid nanosheets with soft substrates for in situ transfection.
    Physical Chemistry Chemical Physics 09/2015; DOI:10.1039/c5cp03483c · 4.49 Impact Factor
  • Nobutaka Hanagata · Hiromi Morita
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    ABSTRACT: Contradictory results have been reported for in vitro evaluations of whether zinc oxide nanoparticles (ZnO NPs) are cytotoxic. Though there have been reports of ZnO NPs cytotoxicity due to Zn ions released from the nanoparticles, there have also been reports concluding that Zn ions are not cytotoxic. This inconsistency is mostly attributed to the types of cells used. In this research, we investigated the difference in the level of ZnO NPs cytotoxicity due to culturing conditions. The sensitivity of human lung epithelial cells to ZnO NPs cytotoxicity differed depending on the dispersing medium, physiological state of the cells resulting from their growth stage, and composition of the medium. Further, with regard to the toxicity of ZnO NPs, NPs internalized into cells had a greater cytotoxic effect than Zn ions released from ZnO NPs. Instead of inducing cell death, ZnO NPs internalized into cells slowed the rate of cell proliferation. Furthermore, the cytotoxicity of ZnO NPs depended greatly on the concentration of calcium ions (Ca(2+)) in the medium. When the concentration of Ca(2+) was low, the cytotoxicity of ZnO NPs increased markedly. However, the toxicity of ZnO NPs was mitigated by the addition of CaCl2 to the medium. Global gene expression analysis revealed that Ca(2+)-induced upregulation of cell cycle functions could be attributable to the mitigation of ZnO NP toxicity by Ca(2+).
    The Journal of Toxicological Sciences 09/2015; 40(5):625-35. DOI:10.2131/jts.40.625 · 1.29 Impact Factor
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    ABSTRACT: Toll-like receptor 9 recognizes CpG oligodeoxynucleotides (ODNs) and induces immune-mediator cytokines. Natural phosphodiester class B CpG ODNs induce interleukin-6 (IL-6), but not interferon-α (IFN-α). We prepared silicon nanoparticles (Si NPs) with different positive surface charge density and bound negatively charged class B CpG ODN molecules electrostatically. No significant differences in the amount of class B CpG ODN molecules or negative surface charge after binding of the molecules onto naked NPs was observed. However, the profile of cytokine induction from peripheral blood mononuclear cells was correlated with a positive surface charge density of naked NPs prior to binding of CpG ODN molecules. The level of IL-6 induction slightly decreased as the positive surface charge density was increased, while the IFN-α induction significantly increased as the positive surface charge density was increased. This observation demonstrates that the bifurcated cytokine induction can be regulated by the surface charge of naked NPs.
    Journal of Drug Delivery Science and Technology 09/2015; 29:251-260. DOI:10.1016/j.jddst.2015.08.008 · 0.48 Impact Factor
  • International Journal of Nanomedicine 09/2015; DOI:10.2147/IJN.S85265 · 4.38 Impact Factor
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    Huijie Zhang · Shini Feng · Ting Yan · Chunyi Zhi · Xiao-Dong Gao · Nobutaka Hanagata
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    ABSTRACT: CpG oligodeoxynucleotides (ODNs) stimulate innate and adaptive immune responses. Thus, these molecules are promising therapeutic agents and vaccine adjuvants against various diseases. In this study, we developed a novel CpG ODNs delivery system based on polyeth-yleneimine (PEI)-functionalized boron nitride nanospheres (BNNS). PEI was coated on the surface of BNNS via electrostatic interactions. The prepared BNNS–PEI complexes had positive zeta potential and exhibited enhanced dispersity and stability in aqueous solution. In vitro cytotoxicity assays revealed that the BNNS–PEI complexes with concentrations up to 100 μg/mL exhibited no obvious cytotoxicity. Furthermore, the positively charged surface of the BNNS– PEI complexes greatly improved the loading capacity and cellular uptake efficiency of CpG ODNs. Class B CpG ODNs loaded on the BNNS–PEI complexes enhanced the production of interleukin-6 and tumor necrosis factor-α from peripheral blood mononuclear cells compared with CpG ODNs directly loaded on BNNS. Contrary to the free CpG ODNs or CpG ODNs directly loaded on BNNS, class B CpG ODNs loaded on the BNNS–PEI complexes induced interferon-α simultaneously. PEI coating may have changed the physical form of class B CpG ODNs on BNNS, which further affected their interaction with Toll-like receptor 9 and induced interferon-α. Therefore, BNNS–PEI complexes can be used to enhance the immunostimulatory effect and therapeutic activity of CpG ODNs and the treatment of diseases requiring interleukin-6, tumor necrosis factor-α, and interferon-α.
    International Journal of Nanomedicine 08/2015; 2015:10-5343. DOI:10.2147/IJN.S88774 · 4.38 Impact Factor
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    Yi Xu · Peter Claiden · Yufang Zhu · Hiromi Morita · Nobutaka Hanagata
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    ABSTRACT: Abstract In this study, we proposed to modify mesoporous silica nanoparticles (MSNs) with 3-aminopropyltriethoxysilane (NH2-TES), aminoethylaminopropyltriethoxysilane (2NH2-TES) and 3-[2-(2-aminoethylamino)ethylamino] propyl-trimethoxysilane (3NH2-TES) for binding of cytosine-phosphate-guanosine oligodexynucleotides (CpG ODN), and investigated the effect of different amino groups of MSNs on the CpG ODN delivery. Serum stability, in vitro cytotoxicity, and cytokine interleukin-6 (IL-6) induction by MSN-NH2/CpG, MSN-2NH2/CpG and MSN-3NH2/CpG complexes were investigated in detail. The results showed that three kinds of aminated-MSN-based CpG ODN delivery systems had no cytotoxicity to RAW264.7 cells, and binding of CpG ODN to MSN-NH2, MSN-2NH2 and MSN-3NH2 nanoparticles enhanced the serum stability of CpG ODN due to protection by the nanoparticles. However, three aminated MSN-based CpG ODN delivery systems exhibited different CpG ODN delivery efficiency, and MSN-NH2/CpG complexes had the highest ability to induce IL-6 secretion.
    Science and Technology of Advanced Materials 08/2015; 16(045006):11. DOI:10.1088/1468-6996/16/4/045006 · 3.51 Impact Factor
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    ABSTRACT: In the present study, the interaction of 5-Fluorouracil with herring sperm DNA is reported using spectroscopic and molecular modeling techniques. This binding study of 5-FU with hs-DNA is of paramount importance in understanding chemico-biological interactions for drug design, pharmacy and biochemistry without altering the original structure. The challenge of the study was to find the exact binding mode of the drug 5-Fluorouracil with hs-DNA. From the absorption studies, a hyperchromic effect was observed for the herring sperm DNA in the presence of 5-Fluorouracil and a binding constant of 6.153 × 103M-1 for 5-Fluorouracil reveals the existence of weak interaction between the 5-Fluorouracil and herring sperm DNA. Ethidium bromide loaded herring sperm DNA showed a quenching in the fluorescence intensity after the addition of 5-Fluorouracil. The binding constants for 5-Fluorouracil stranded DNA and competitive bindings of 5-FU interacting with DNA-EB systems were examined by fluorescence spectra. The Stern-Volmer plots and fluorescence lifetime results confirm the static quenching nature of the drug-DNA complex. The binding constant Kb was 2.5 × 104 L mol-1 and the number of binding sites are 1.17. The 5-FU on DNA system was calculated using double logarithmic plot. From the Forster nonradiative energy transfer study it has been found that the distance of 5-FU from DNA was 4.24 nm. In addition to the spectroscopic results, the molecular modeling studies also revealed the major groove binding as well as the partial intercalation mode of binding between the 5-Fluorouracil and herring sperm DNA. The binding energy and major groove binding as -6.04 kcal mol-1 and -6.31 kcal mol-1 were calculated from the modeling studies. All the testimonies manifested that binding modes between 5-Fluouracil and DNA were evidenced to be groove binding and in partial intercalative mode.
    Biophysical Reviews and Letters 07/2015; 10(2):115-133. DOI:10.1142/S1793048015500034
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    ABSTRACT: A versatile method for the rapid fabrication of aligned fullerene C60 nanowhiskers (C60NWs) at the air-water interface is presented. This method is based on the vortex motion of a sub-phase (water), which directs floating C60 nanowhiskers (C60NWs) to align on the water surface according to the direction of rotational flow. Aligned C60NWs could be transferred onto many different flat substrates and, in this case, aligned C60NWs on glass substrates were employed as a scaffold for cell culture. Bone forming human osteoblast MG63 cells adhered well to the C60NWs and their growth was found to be oriented with the axis of the aligned C60NWs. Cells grown on aligned C60NWs were more highly oriented with the axis of alignment than when grown on randomly oriented nanowhiskers. A study of cell proliferation on the C60NWs implied their non-toxicity indicating their potential for use in biomedical applications.
    ACS Applied Materials & Interfaces 06/2015; 7(28). DOI:10.1021/acsami.5b04811 · 6.72 Impact Factor
  • Nobutaka Hanagata
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    ABSTRACT: Interferon-induced transmembrane protein 5 (IFITM5) is an osteoblast-specific membrane protein that has been shown to be a positive regulatory factor for mineralization in vitro. However, Ifitm5 knockout mice do not exhibit serious bone abnormalities, and thus the function of IFITM5 in vivo remains unclear. Recently, a single point mutation (c.-14C>T) in the 5′ untranslated region of IFITM5 was identified in patients with osteogenesis imperfecta type V (OI-V). Furthermore, a single point mutation (c.119C>T) in the coding region of IFITM5 was identified in OI patients with more severe symptoms than patients with OI-V. Although IFITM5 is not directly involved in the formation of bone in vivo, the reason why IFITM5 mutations cause OI remains a major mystery. In this review, the current state of knowledge of OI pathological mechanisms due to IFITM5 mutations will be reviewed.
    Journal of Bone and Mineral Metabolism 06/2015; DOI:10.1007/s00774-015-0667-1 · 2.46 Impact Factor
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    ABSTRACT: Background Extrahepatic cholangiocarcinoma is very difficult to diagnose at an early stage, and has a poor prognosis. Novel markers for diagnosis and optimal treatment selection are needed. However, there has been very limited data on the proteome profile of extrahepatic cholangiocarcinoma. This study was designed to unravel the proteome profile of this disease and to identify overexpressed proteins using mass spectrometry-based proteomic approaches.Methods We analyzed a discovery set of formalin-fixed paraffin-embedded tissues of 14 extrahepatic cholangiocarcinomas employing shotgun mass spectrometry, and compared proteome profiles with those of 7 controls. Then, selected candidates were verified by quantitative analysis using scheduled selected reaction monitoring-based mass spectrometry. Furthermore, immunohistochemical staining employed a validation set of 165 cases.ResultsIn total, 1,992 proteins were identified and 136 proteins were overexpressed. Verification of 58 selected proteins by quantitative analysis revealed 11 overexpressed proteins. Immunohistochemical validation for 10 proteins showed positive rates of S100P (84%), CEAM5 (75%), MUC5A (62%), OLFM4 (60%), OAT (42%), CAD17 (41%), FABPL (38%), AOFA (30%), K1C20 (25%) and CPSM (22%) in extrahepatic cholangiocarcinomas, which were rarely positive in controls.Conclusions We identified 10 proteins associated with extrahepatic cholangiocarcinoma using proteomic approaches. These proteins are potential targets for future diagnostic biomarkers and therapy.
    Journal of Hepato-Biliary-Pancreatic Sciences 04/2015; 22(9). DOI:10.1002/jhbp.262 · 2.99 Impact Factor
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    Shanmugavel Chinnathambi · Nobutaka Hanagata
    Sweden - Japan Seminar on Nanomaterials and Nanotechnology; 03/2015
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    ABSTRACT: The interaction of antimetabolite 5-fluorouracil (5FU) with bovine serum albumin (BSA) under UVC (253.7 nm) irradiation was investigated in the present study using UV-Vis spectroscopy, steady state/time resolved fluorescence spectroscopic techniques. The stability of protein was found to be very strong when BSA gets bind to 5FU and moreover it is compared with the free BSA under UVC irradiation. From the fluorescence spectroscopic study, the stability of the complex was found to acquire 2-fold stronger than free protein. From the molecular modelling studies, we came to know the hydrogen bonds between BSA and antimetabolite 5FU are strong, up to 70.4 J/m 2 under UVC irradiation.
    International Journal of Spectroscopy 01/2015; 2015:1-12. DOI:10.1155/2015/315764
  • Song Chen · Qiqing Zhang · Lan Jia · Xinxin Du · Nobutaka Hanagata
    Journal of Materials Chemistry B 01/2015; DOI:10.1039/C5TB01270H · 4.73 Impact Factor
  • Cuilian Tao · Yufang Zhu · Xianglan Li · Nobutaka Hanagata
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    ABSTRACT: We developed a potential cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) delivery system by binding of CpG ODN onto aminated mesoporous silica nanoparticles (MSNs) to form CpG/MSN-NH2 complexes for Toll-like receptor 9 (TLR9)-mediated induction of cytokines. Serum stability, in vitro cytotoxicity, cellular uptake, and interleukin-6 (IL-6) induction of CpG/MSN-NH2 complexes were investigated. The results showed that MSN-NH2 nanoparticles had no cytotoxicity to Raw 264.7 cells, and binding of CpG ODN to MSN-NH2 nanoparticles enhanced serum stability of CpG ODN due to the protection by nanoparticles. Furthermore, CpG/MSN-NH2 complexes could be efficiently taken up by cells due to small particle size and good dispersity. Most importantly, CpG/MSN-NH2 complexes significantly enhanced the level of IL-6 induction, stimulated by interaction between CpG ODN and TLR9 in endolysosomes. Therefore, MSNs would be a promising carrier for enhancing the delivery efficiency of CpG ODN.
    Microporous and Mesoporous Materials 11/2014; 204. DOI:10.1016/j.micromeso.2014.11.007 · 3.45 Impact Factor
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    ABSTRACT: Theranostic nanoparticles currently have been regarded as an emerging concept of 'personalized medicine' with diagnostic and therapeutic dual-functions. Eu3+ doped hydroxyapatite (HAp) has been regarded as a promising fluorescent probe for in vivo imaging applications. Additionally, substitution of Ca2+ with Fe3+ in HAp crystal may endow the capability of producing heat upon exposure to a magnetic field. Here we report a preliminary study of doping mechanism and photoluminescence of Eu3+ and Fe3+ doped HAp nanoparticles (Eu/Fe:HAp). HAp with varied concentration of Eu3+ and Fe3+ doping are presented as Eu(10 mol%):HAp, Eu(7 mol%)-Fe(3 mol%):HAp, Eu(5 mol%)-Fe(5 mol%):HAp, Eu(3 mol%)-Fe(7 mol%):HAp, and Fe(10 mol%):HAp in the study. The results showed that the HAp particles, in nano-size with rod-like morphology, were successfully doped with Eu3+ and Fe3+, and the particles can be well suspended in cell culture medium. Photoluminescence analysis revealed that particles have prominent emissions at 536 nm, 590 nm, 615 nm, 650 nm and 695 nm upon excitation at a wavelength of 397 nm. Moreover, these Eu/Fe:HAp nanoparticles belonged to B-type carbonated HAp, which has been considered an effective biodegradable and biocompatible drug/gene carrier in biological applications.
    Science and Technology of Advanced Materials 09/2014; 15(5):055005. DOI:10.1088/1468-6996/15/5/055005 · 3.51 Impact Factor
  • Cuilian Tao · Yufang Zhu · Xianglan Li · Nobutaka Hanagata
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    ABSTRACT: We developed a potential cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) delivery system based on magnetic mesoporous silica (MMS) nanoparticles by binding of CpG ODN onto aminated MMS (MMS-NH2) nanoparticles to form CpG/MMS-NH2 complexes for Toll-like receptor 9 (TLR9)-mediated induction of cytokines. Magnetization, serum stability, in vitro cytotoxicity, cellular uptake, and interleukin-6 (IL-6) induction of CpG/MMS-NH2 complexes were evaluated. The results showed that MMS nanoparticles exhibited superparamagnetic behavior with a saturation magnetization of 6.5 emu/g. Also, MMS-NH2 nanoparticles had no cytotoxicity to Raw 264.7 cells, and CpG/MMS-NH2 complexes enhanced serum stability of CpG ODN and could be localized in the endolysosomes after endocytosis by cells. Importantly, CpG/MMS-NH2 complexes significantly enhanced the TLR9-mediated IL-6 induction compared to free CpG ODN. Therefore, CpG/MMS-NH2 complexes could be magnetic targeted delivery and significantly enhance the TLR9-mediated cytokine induction for stimulating immune responses.
    RSC Advances 09/2014; 4(86). DOI:10.1039/C4RA08003C · 3.84 Impact Factor
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    ABSTRACT: T cell receptor (TCR) phosphorylation requires the kinase Lck and the phosphatase CD45. CD45 activates Lck by dephosphorylating an inhibitory tyrosine of Lck to relieve autoinhibition. However, CD45 also dephosphorylates the TCR, and the spatial exclusion of CD45 from TCR clustering in the plasma membrane appears to attenuate this negative effect of CD45. To further investigate the role of CD45 in signal initiation, we reconstituted membrane TCR clusters in vitro on supported lipid bilayers. Fluorescence microscopy of single clusters showed that incorporation of CD45 enhanced phosphorylation of TCR clusters, but only when Lck co-clustered with TCR. We found that clustered Lck autophosphorylated the inhibitory tyrosine and thus could be activated by CD45, whereas diffusive Lck molecules did not. In the TCR-Lck clusters and at low CD45 density, we speculate that the effect of Lck activation may overcome dephosphorylation of TCR, resulting in a net positive regulation. The CD45 density in physiological TCR clusters is also low owing to the exclusion of CD45. Thus, we propose that the spatial organization of TCR/Lck/CD45 in T cell membranes is important not only for modulating the negative role of CD45 but also for creating conditions in which CD45 has a positive role in signal initiation.
    Journal of Biological Chemistry 08/2014; 289(41). DOI:10.1074/jbc.M114.574319 · 4.57 Impact Factor
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    ABSTRACT: To evaluate the in vivo foreign body reaction to bio-inert 2-methacryloyloxyethyl phosphorylcholine (MPC) polymers, MPC polymer-coated porous substrates, with large surface area, were implanted subcutaneously in mice for 7 and 28 days, and the surrounding tissue response and cells infiltrating into the porous structure were evaluated. The MPC polymer surface induced low angiogenesis and thin encapsulation around the porous substrate, and slightly suppressed cell infiltration into the porous substrate. M1-type macrophage specific gene (CCR7) expression was suppressed by the MPC polymer surface after 7 days, resulting in the suppression of inflammatory cytokine/chemokine gene expression. However, the expression of these genes on the MPC polymer surface was higher than on the non-coated surface after 28 days. These findings suggest that MPC polymer surfaces successfully inhibit inflammatory responses during the early stage of tissue response, and seem to retard its occurrence over time.
    Journal of Biomaterials Science Polymer Edition 07/2014; 25(14-15):1-15. DOI:10.1080/09205063.2014.939917 · 1.65 Impact Factor
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    ABSTRACT: Graphene-like two-dimensional materials (2DMats) show application potential in optoelectronics and biomedicine due to their unique properties. However, environmental and biological influences of these 2DMats remain to be unveiled. Here we reported the antibacterial activity of two-dimensional (2D) chemically exfoliated MoS2 (ce-MoS2) sheets. We found that the antibacterial activity of ce-MoS2 sheets was much more potent than that of the raw MoS2 powders used for the synthesis of ce-MoS2 sheets possibly due to the 2D planar structure (high specific surface area) and higher conductivity of the ce-MoS2. We investigated the antibacterial mechanisms of the ce-MoS2 sheets and proposed their antibacterial pathways. We found that the ce-MoS2 sheets could produce reactive oxygen species (ROS), different from a previous report on graphene-based materials. Particularly, the oxidation capacity of the ce-MoS2 sheets toward glutathione oxidation showed a time and concentration dependent trend, which is fully consistent with the antibacterial behaviour of the ce-MoS2 sheets. The results suggest that antimicrobial behaviors were attributable to both membrane and oxidation stress. The antibacterial pathways include MoS2-bacteria contact induced membrane stress, superoxide anion (O2˙(-)) induced ROS production by the ce-MoS2, and the ensuing superoxide anion-independent oxidation. Our study thus indicates that the tailoring of the dimension of nanomaterials and their electronic properties would manipulate antibacterial activity.
    Nanoscale 07/2014; 6(17). DOI:10.1039/c4nr01965b · 7.39 Impact Factor
  • Song Chen · Xuetao Shi · Akiyoshi Osaka · Hong Gao · Nobutaka Hanagata
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    ABSTRACT: Novel silica hollow flowers (1–5 μm) were synthesized using globular apatite flowers as sacrificed template via a sol–gel route and then employed as biocompatible carrier of bone morphogenetic protein-2 (BMP-2) to stimulate osteoblast differentiation. Apatite was bio-mimetically synthesized from a well-known Kokubo’s simulated body fluid (SBF), then coated with silica in a Stöber-type silica sol–gel system, and finally dissolved in an acetic solution to yield silica hollow flowers. Analyses of SEM and TEM images show that the resultant silica flowers had a porous and hollow structure due to removal of apatite template by acetic treatment and their shell was constructed by numerous silica nanosheets (∼10 nm in silica shell). A larger specific surface of 890 m2/g was obtained for silica hollow flowers compared to silica-coated apatite due to the presence of porous and hollow structure. Silica hollow flowers had no significant toxicity after incubation with osteoblast MC3T3-E1 cells, indicating a good biocompatibility. They favored adsorption and supported a sustained release behavior of BMP-2. The released BMP-2 was biological active and enhanced osteoblast differentiation with higher ALP activity and larger amount of osteocalcin. The present silica hollow flowers are thus applicable to delivery system in tissue generation.
    Chemical Engineering Journal 06/2014; 246:1–9. DOI:10.1016/j.cej.2014.02.053 · 4.32 Impact Factor

Publication Stats

2k Citations
555.05 Total Impact Points


  • 2010–2015
    • Hokkaido University
      • Graduate School of Life Science
      Sapporo, Hokkaidō, Japan
  • 2006–2015
    • National Institute for Materials Science
      • • Nanotechnology Innovation Station
      • • International Center for Young Scientist (ICYS)
      Tsukuba, Ibaraki, Japan
  • 2014
    • Tokyo Institute of Technology
      • Department of Metallurgy and Ceramics Science
      Edo, Tōkyō, Japan
  • 2013
    • Anna University, Chennai
      • Department of Medical Physics
      Chennai, Tamil Nādu, India
  • 2003–2006
    • Tokyo University of Technology
      • School of Bionics
      Edo, Tōkyō, Japan
  • 1993–2006
    • The University of Tokyo
      • Research Center for Advanced Science and Technology
      Tokyo, Tokyo-to, Japan