Tetsushi Taguchi

Apatite coating on hydrophilic polymer-grafted poly(ethylene) films using an alternate soaking process

Previously, we developed a novel alternate soaking process and clarified that bone-like apatite was formed on/in organic polymer hydrogel matrices using this process. The present study focused on the apatite coating on hydrophilic polymer grafted poly(ethylene) (PE) films with various grafting densities and commonly used hydrophilic polymers, poly(acryl amide) (PAAm) and poly(acrylic acid) (PAAc) were employed. From X-ray diffraction analysis, hydroxyapatite was coated on PAAm- or PAAc- grafted PE films. The amount of apatite coated on PAAm-grafted PE (PAAm-g-PE) films increased with an increase in the reaction cycles and the grafting density of PAAm. Similar to PAAm-g-PE, the amount of apatite coated on PAAc-grafted PE (PAAc-g-PE) films increased linearly with an increase in the grafting density of the PAAc up to around 30 microg/cm2. While, no significant increase in the apatite coating on the PAAc-g-PE films was observed even after 50 reaction cycles when the grafting densities of PAAc were over 30 microg/cm2. Apatite coating was not observed on original PE films. Scanning electron microscopic images reveal that the aggregation of apatite crystals on all PAAm-g-PE films and PAAc-g-PE films with grafting density from 10 to 30 microg/cm2. On the other hand, a dense apatite layer with some cracks was coated when the grafting density of the PAAc chains was over 30 microg/cm2. These results indicated that it was possible to coat apatite on hydrophilic polymer grafted PE films by an alternate soaking process and that the apatite crystal morphology could be controlled as a function of polymer type and density.

Apatite formation on/in hydrogel matrices using an alternate soaking process: II. Effect of swelling ratios of poly(vinyl alcohol) hydrogel matrices on apatite formation

In our previous study, we reported a novel method of apatite formation on/in a three-dimensional hydrogel matrix. Using this method, bone-like apatite could be formed on/in the hydrogel matrix under normal conditions in vitro. A poly(vinyl alcohol) (PVA) gel was used as a model matrix. The method consists of two steps: first, water is transformed in a PVA gel with a CaCl2/Tris-HCl aqueous solution (pH 7.4) and second, the gel is soaked in a Na2HPO4 aqueous solution. In the present study, we report a detailed study of the effects of the swelling ratios of PVA gels on apatite formation. Cross-sectional observations and gravimetric measurements of PVA gels with various swelling ratios were done. The amount of apatite formed on/in PVA gels increased almost linearly with an increase in the reaction cycles. The rates of apatite formation on/in PVA gels largely depended on the swelling ratios, which were approximately 0.48, 0.61, 1.28, and 1.55 mg per cycle for swelling ratios of 4.1, 10.4, 16.8, and 30.1, respectively. The apatite content in PVA-apatite composites that was obtained by this method also increased with an increase of the reaction cycles. After six reaction cycles, a PVA gel with a high swelling ratio contains approximately 70 wt% of formed apatite in the composite. On the other hand, a gel with a low swelling ratio contains about 15 wt% of formed apatite in the composite. Cross-sectional views of the PVA gels after each cycle showed that apatite crystals were formed, not only on the surface of the gel but also within it after fifteen reaction cycles. The hydrogel-apatite composites that were obtained using an alternative soaking process will be useful as not only bone substitute materials but also as soft tissue adhesive materials.

Preparation and characterization of apatite deposited on silk fabric using an alternate soaking process

Apatite-deposited silk fabric composite materials were developed using a new alternate soaking process. The characteristics of deposited apatite were studied using scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectrophotometry (FTIR), and X-ray photoelectron spectroscopy (XPS). Apatite weight increased with alternating soaking in a calcium solution [200 mM aqueous calcium chloride solution buffered with tris(hydroxymethyl) aminomethane and HCl (pH 7.4)] and a phosphate solution (120 mM aqueous disodium hydrogenphosphate) changed every hour. SEM showed that apatite deposited after 21 or more repeated soakings was over 20 microm thick. XRD showed that with alternate soakings, the apatite crystals deposited on silk fabric elongated along the c axis. FTIR and XPS indicated the existence of carbonate, HPO(4)(2-), and Na(+) ions in addition to constituent ions of hydroxyapatite. A loss of HPO(4)(2-) and Na(+) ions in the deposit upon further soaking might be associated with an increasing apatite crystallinity. Apatite deposited on silk by the alternate soaking process was a deficient apatite containing carbonate, HPO(4)(2-), and Na(+) ions as in a natural bone tissue. Thus, this apatite-silk composite material might be potentially bioactive.

Apatite formation on/in hydrogel matrices using an alternate soaking process (III) : Effect of physico-chemical factors on apatite formation on/in poly(vinyl alcohol) hydrogel matrices

The aim of this study is to clarify the physico-chemical factors which influence apatite formation on/in a hydrogel during a novel alternate soaking process. A poly(vinyl alcohol) (PVA) gel was used as a model matrix. The amount of apatite formed on/in PVA gels decreased with an increase in the reaction temperature during the same reaction cycles. This suggested that the equilibrium swelling ratios decreased with increasing reaction temperatures; that is, the diffusion of calcium and phosphate ions reduced at high reaction temperature. However, the crystallinity of apatite formed on/in PVA gels was greater at higher reaction temperatures. The amount of apatite formed on/in PVA gels increased with an increase in the calcium and phosphate solution concentrations, and increased by shaking at the first three reaction cycles. A few influences could be observed when the solution volume was changed, however, the soaking order was not effective in this study. These results indicate that the amount of apatite formation on/in PVA gels can be controlled by changing the reaction temperature and the Ca- and P-solution concentrations, and that the crystallinity of apatite can be also changed by controlling the reaction temperatures.

A study on hydroxyapatite formation on/in the hydroxyl groups-bearing nonionic hydrogels

Using the biomimetic method, we formed a hydroxyapatite (HAp) layer on/in certain types of nonionic hydrogels that contain hydroxyl groups. The hydrogels used were poly(vinyl alcohol) (PVA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(glucosyloxyethyl methacrylate) (PGEMA), and agarose. Under an optical microscope, we observed a thin, continuous HAp layer on the top surface of the PVA, PHEMA, and PGEMA gels. On the other hand, we only observed an intermittent HAp layer on the surface of the agarose gel. The swelling ratio and the bound water content of these hydrogels were measured as an essential character in HAp formation. There was some relation among the HAp formation, the swelling ratios, and the bound water content.

Ca-adsorption and apatite deposition on silk fabrics modified with phosphate polymer chains.

Silk fabric was modified with polymethacryloyloxyethylphosphate (pMOEP) by graft copolymerization. Ca-adsorption onto pMOEP-grafted silk fabric was significantly enhanced compared to that onto original silk fabric. SEM observation indicated that some crystallites were deposited on the pMOEP-grafted silk fabric after 1 week of immersion in simulated body fluid, whereas no change occurred on the surface of the original silk fabric. X-ray diffraction showed that this crystallite contained hydroxyapatite. These results indicate that pMOEP-grafted silk fabric induce hydroxyapatite formation more effectively than the original silk fabric.

Preparation of a novel functional hydrogel consisting of sulfated glucoside-bearing polymer: activation of basic fibroblast growth factor.

Sulfated poly(glucosyloxyethyl methacrylate) [poly(GEMA)-sulfate] and its hydrogel were synthesized and evaluated to develop novel tissue regeneration assist devices. The poly(GEMA)-sulfate hydrogel was obtained by gamma-ray irradiation. Basic fibroblast growth factor (bFGF) was expected to be subsequently incorporated with hydrogel matrices and activated or released from the matrices. A sustained release of bFGF from the poly(GEMA)-sulfate hydrogel was not realized; however, bFGF from poly(GEMA)-sulfate hydrogel was effectively activated. Poly(GEMA)-sulfate hydrogels are thought to be potential component materials for a tissue engineering matrix.

Immobilization of Human Vascular Endothelial Growth Factor (VEGF165) onto Biomaterials: An Evaluation of the Biological Activity of Immobilized VEGF165

Human vascular endothelial growth factors (VEGF) are angiogenic factors that induce specific endothelial cell proliferation. In this study, VEGF165, which contains 165 amino acids, was immobilized onto poly(acrylic acid) grafted polyethylene) films (PAAc-g-PE). VEGF165 was immobilized via a reaction between the amino group of VEGF165 and the carboxyl group of PAAc using water-soluble carbodiimide. Using cultured human umbilical vein endothelial cells (HUVEC), adhesion, proliferation, and migration of the cells were evaluated with three kinds of protein that were immobilized on the PE films. The proteins used were collagen (type IV), fibronectin (FN), and VEGF165. The adhesion of HUVEC was enhanced by the immobilization of collagen or FN and by the co-immobilization of VEGF with FN, but not VEGF alone. The VEGF FN-co-immobilized surface showed cell growth promotion activity. Endothelialization was observed only on the collagen-immobilized or VEGF FN-co-immobilized film surfaces. We proposed that the VEGF with FN-co-immobilized biomaterials could be used for artificial vessels and for other tissue engineering scaffolds.

Apatite-Silica Gel Composite Materials Prepared by a New Alternate Soaking Process

Novel apatite/silica composite materials were synthesized by modifying the surface of silica gel beads or plates with apatite. An “alternate soaking” process, which involves alternate soaking in a CaCl2/tris-HCl aqueous solution (pH 7.4) and a Na2HPO4 aqueous solution, was utilized to prepare a composite of silica gel and apatite. The characteristics of apatite formed on the surface of silica gel have been studied using X-ray diffraction, Fourier-transform infrared spectrophotometry (FT-IR) and scanning electron microscopy (SEM). Data clearly showed surface modification of silica with the crystalline apatite. Pore volume and specific total surface area, which were measured using nitrogen gas adsorption apparatus, were decreased after apatite deposition on the surface of silica gel beads, whereas the external surface area was increased drastically. The alternate soaking process made it possible to prepare apatite/silica gel composites in a remarkably short period of time, i.e. several hours.

In vitro calcification model (2) : Apatite formation on segmented polyurethane thin films by using an alternate soaking process : The effect of adsorbed serum proteins on calcification

In the present study, the effect of serum proteins on the calcification (apatite deposition) of commercially available polyurethane (PU) films, Pellethane® 2363-80AE, Pellathane® 2363-55DE, and K-III, was evaluated by using an alternating soaking process. Bovine serum albumin, γ-globulin and fibrinogen were used as typical serum proteins. After the proteins were adhered to PU films, the films were used in the apatite formation process. The thin-film X-ray diffraction spectra of a fibrinogen adhered K-III sample, after 200 calcification sequences, showed HAp formation. The calcium content on the surface of all films to which protein adhered was greater than that of films incubated in protein free solutions. The films to which fibrinogen adhered showed remarkable apatite formation, though the amount of adhered fibrinogen was much smaller than the amounts of the other proteins. K-III films to which fibrinogen had adhered showed small globular apatite aggregations, whereas the Pellethane® 2363 series films showed confetti-shaped apatite. Different morphologies exhibited by the deposits depended on the types of serum protein and on the substrate chemistry. The reason for the different apatite morphology on different PU films is not clear, however, the kind of serum protein seems to play an important role in the calcification of a PU.