Papers by Author: Ayako Oyane

Abstract: FGF-2-apatite and FGF-2-zinc-apatite composite layers were formed on commercially available anodically oxidized Ti external fixation rods using FGF-2-and ZnCl₂-containing supersaturated calcium phosphate solutions. The FGF-2-zinc-apatite composite layers precipitated on the Ti external fixation rods significantly enhanced proliferation of fibroblastic NIH3T3 and osteoblastic MC3T3-E1 cells in vitro.

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.

Abstract: The present authors recently developed a new calcium phosphate (CaP) coating technique on an ethylene-vinyl alcohol copolymer substrate utilizing a laser-assisted biomimetic (LAB) process. In the present study, the LAB process was applied to a sintered hydroxyapatite (sHA) substrate for CaP coating. The LAB process was carried out by irradiating the sHA substrate immersed in a supersaturated CaP solution with a low-energy Nd-YAG pulsed laser. Within 30 min of irradiation, contiuous CaP layers with different morphologies were successfully formed on the laser-irradiated sHA surface. A submicron cavernous structure of the CaP layer was developed into a micron flake-like structure as the laser power increased from 1 to 3 W. This result suggests that the secondary nucleation and growth of CaP crystals were accelerated by laser irradiation in a power-dependent manner. Laser absorption by the sHA substrate and the resulting increase in ambient temperature locally near the surface should be responsible for the accelerated CaP nucleation and growth. The present CaP coating technique using the LAB process is simple and quick, hence it would be useful in orthopedic and dental applications as an on-demand surface-functionalization method for biomaterials consisting of sHA.

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.

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.

Abstract: A composite layer of fibroblast growth factor-2 (FGF-2) and low-crystalline apatite was formed on an ethylene-vinyl alcohol copolymer specimen using two types of aqueous calcium phosphate solutions supplemented with 10 !g·mL-1 FGF-2; one is a CP solution that is prepared by dissolving chemical reagents into ultrapure water and the other is an RKB solution that can be prepared by mixing clinically approved infusion fluids. In both solutions, a sufficient amount of FGF-2 for new skin tissue formation (1 !g·cm-2) was immobilized on the specimen surface.

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.

Abstract: A FGF-2-apatite composite layer (FGF-AP layer) was formed on the surface of Ti screws in a supersaturated calcium phosphate solution supplemented with FGF-2. By an in vitro study using fibroblastic NIH3T3 cells, it was confirmed that FGF-2 was immobilized in the layer without complete denaturation although the composite layer was formed at 37°C. When Ti screws with the FGF-AP layer were percutaneously implanted in the proximal tibial metaphysis of 16 rabbits, no osteomyelitis was observed in any rabbits although a FGF-2-free AP layer allowed osteomyelitis in some cases in our previous study. These results suggest that a FGF-AP layer formed on Ti screws is useful for resisting bacterial infection during external fixations.

Abstract: An ethylene-vinyl alcohol copolymer (EVOH) film with a laminin–apatite composite layer on its surface showed improved adhesion and compatibility to living epithelial tissue compared to untreated EVOH film. This result can be attributed to the good biocompatibility of apatite and the cell-adhesion activity of the laminin on the EVOH surface. This composite material, consisting of laminin, apatite, and EVOH, is considered a promising material for skin terminals to prevent epidermal downgrowth.