Papers by Author: Hideo Tsurushima

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Authors: Subhadip Bodhak, Masanori Kikuchi, Ayako Oyane, Yu Sogo, Hideo Tsurushima, Atsuo Ito
Abstract: Calcium phosphate (CaP) coating is an effective technique for surface functionalization of biomaterials. The objective of our research is to prepare calcium phosphate (CaP) coatings on a hydroxyapatite/collagen (HAp/Col) nanocomposite and subsequently provide it with gene delivery function through the immobilization of DNA in the coating. We have specifically selected the HAp/Col nanocomposite since it has the high potential as bone substitutes due to its similar composition, nanostructure, and biological properties to those of human bone. CaP coatings consisting of different sized particles were prepared on the HAp/Col nanocomposite membrane by immersing it in supersaturaterd CaP solutions (so-called RKM solutions) with the varied Ca and P concentration levels. We immobilized DNA in the CaP coatings together with lipid and fibronectin by supplementing DNA, lipid, and fibronectin to the RKM solutions (DLF-RKM solutions). Gene transfer capability of the resulting HAp/Col nanocomposite membrane was improved with decreasing concentration level of the DLF-RKM solution. It was confirmed that the present CaP coating technique was effective in providing the HAp/Col nanocomposite membrane with gene transfer capability and that the Ca and P concentration level of the DLF-RKM solution was a controlling factor affecting the gene transfer efficiency.
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Authors: Ayako Oyane, Hiroko Araki, Yu Sogo, Atsuo Ito, Hideo Tsurushima
Abstract: A surface-mediated gene transfer system using DNA-calcium phosphate (CaP) composite layers (D-CaP layers) would be useful in tissue engineeing. In previous studies, D-CaP layers were fabricated in supersaturated CaP solutions prepared using chemical reagents. In this study, a so-called RKM solution prepared using clinically approved infusion fluids was employed as a supersaturated CaP solution. A D-CaP layer consisting of submicron spherical particles was successfully fabricated on a polystyrene substrate by immersing the substrate in the RKM solution for 24 h. When the immersion period was prolonged from 24 to 72 h, amount of CaP and DNA on the substrate increased. However, the gene transfer capability of the D-CaP layer for the CHO-K1 cells was kept unchanged irrespective of the immersion period. In the RKM solution process, immersion period of 24 h was found to be long enough for gene transfer application of the D-CaP layer. More importantly, the D-CaP layer fabricated by the RKM solution process exhibited a significantly higher gene transfer capability than our previous D-CaP layer fabricated in the conventional CaP solution with the same DNA concentration. The RKM solution process for the fabrication of D-CaP layers was found to be advantageous to the previous process in terms of not only safety but the layers gene transfer capability.
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Authors: Subhadip Bodhak, Masanori Kikuchi, Ayako Oyane, Yu Sogo, Hideo Tsurushima, Atsuo Ito
Abstract: Hydroxyapatite/collagen (HAp/Col) nanocomposites with bone-like self-organized nanostructure show excellent bioactivity in vivo. However, they show quite high absorbability for cationic ions and lower culture medium ionic concentrations which adversely affects bone cell proliferation and osteogenic differentiation in in vitro cell culture condition. To address this limitation, in this study we have supplemented Ca2+ and Mg2+ ions to the HAp/Col nanocomposite membrane sample prior to cell culture to improve it’s in vitro biological properties. The HAp/Col nanocomposite membrane samples were fabricated by the simultaneous titration method using Ca(OH)2, type-I atelocollagen and H3PO4 as starting precursor materials. Prior to in vitro cell culture experiments, the HAp/Col samples were pretreated with Ca2+ and/or Mg2+ ions by immersing in 10 ml of 20 mM CaCl2 solution, 20 mM MgCl2 solution, or a solution containing 20 mM CaCl2 and 20 mM MgCl2 for 7 days. In vitro bone cell-material interactions on the pretreated and untreated HAp/Col samples were studied by culturing MC3T3-E1 cells up to 7 days. Enhanced bone cell proliferation was found on all the pretreated HAp/col samples as confirmed by the CCK-8 assay. Interestingly, the HAp/Col samples pretreated with both Ca2+ and Mg2+ ions showed the maximum viable bone cell density.
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Authors: Ayako Oyane, Hideo Tsurushima, Atsuo Ito
Abstract: A laminin–DNA–apatite composite layer was successfully formed on the surface of an ethylene–vinyl alcohol copolymer. The immobilized DNA was transferred to the cells adhering onto the laminin–DNA–apatite composite layer more efficiently than those adhering onto a lamininfree DNA–apatite composite layer. It is considered that laminin immobilized in the surface layer enhances cell adhesion and spreading, and DNA locally released from the layer is effectively transferred into the adhering cells, taking advantage of the large contact area. The present gene transferring system, which shows high efficiency and safety, would be useful in gene therapy and tissue engineering.
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