Ayako Oyane

Zinc-containing apatite layers on external fixation rods promoting cell activity

Zinc-containing apatite layers were successfully formed on commercially available anodically oxidized Ti external fixation rods using ZnCl(2)-containing supersaturated calcium phosphate solutions. With an increase in concentration of ZnCl(2) in the supersaturated calcium phosphate solutions, the amounts of zinc that precipitated on the Ti external fixation rods increased (from 0 to 0.195 + or - 0.020 microg cm(-2)); meanwhile, the amounts of calcium and phosphorus that precipitated on the Ti external fixation rods decreased (from 11.2 + or - 1.5 and 4.8 + or - 0.5 microg cm(-2) to 2.9 + or - 1.6 and 1.3 + or - 0.9 microg cm(-2), respectively). The zinc-containing apatite layers precipitated on the Ti external fixation rods caused a significant increase in fibroblastic proliferation, osteoblastic proliferation and differentiation in vitro. The Ti external fixation rods coated with zinc-containing apatite layers are expected to be more effective in accelerating the tissue regeneration around the surgical site than those coated with an apatite layer.

Mesoporous bioactive glass coatings on stainless steel for enhanced cell activity, cytoskeletal organization and AsMg immobilization

Mesoporous bioactive glass (MBG) coatings with SiO2:CaO:P2O5 mol ratio of 100:0:0, 80:15:5 and 70:25:5 and a tunable pore size and pore structure were prepared on a stainless steel plate by spin-coating sol solutions containing a triblock copolymer and the inorganic precursors. The calcium content in the MBG coatings affected the mesoporous structure. With the increase in calcium content, the crystallinity of the MBG coatings increased and thus the Brunauer–Emmett–Teller (BET) surface area and pore volume decreased. The MBG coatings were evaluated on the basis of protein adsorption, cell attachment, cell proliferation, cell differentiation, cytoskeletal organization and L-ascorbic acid phosphate magnesium salt n-hydrate (AsMg) immobilization for their potential in improving implant-bone integration. The results showed that the osteoblast MC3T3-E1 cells were stimulated by the mesoporous structure and chemical composition of the MBG coatings, with enhanced cell attachment, proliferation, differentiation and better developed cytoskeleton. Moreover, AsMg was successfully immobilized on the MBG coatings by using an AsMg-containing supersaturated calcium phosphate solution. The AsMg immobilized on the MBG coatings was not denatured and showed high activity enhancing the fibroblast NIH3T3 proliferation in vitro. An appropriate range of pore size and a preferred alignment of the mesochannels of the MBG coatings on stainless steel are promising to improve the implant-bone integration.

Fibronectin-calcium phosphate composite layer on hydroxyapatite to enhance adhesion, cell spread and osteogenic differentiation of human mesenchymal stem cells in vitro

Fibronectin (Fn) and type I collagen (Col) were immobilized on a surface of a hydroxyapatite (HAP) ceramic by coprecipitation with calcium phosphate in a supersaturated calcium phosphate solution prepared by mixing clinically approved infusion fluids. These proteins and the calcium phosphate precipitate formed a composite surface layer. As a result, the proteins were immobilized firmly as not to be released completely for 3 d in a physiological salt solution. When human mesenchymal stem cells (hMSCs) were cultured on a HAP ceramic in a differentiation medium supplemented with dexamethasone, beta-glycerophosphate and ascorbic acid, hMSCs spread well within 1 h. The alkaline phosphatase (ALP) activity of hMSCs cultured on the Fn-calcium phosphate composite layer significantly increased compared with that of hMSCs cultured on the untreated HAP ceramic. On the other hand, Col did not increase the ALP activity of hMSCs and no synergy between Fn and Col was observed. Therefore, the Fn-calcium phosphate composite layer formed on the HAP is useful for the enhancement of the spreading and osteogenic differentiation of hMSCs in vitro.

Fibroblast growth factor-2-apatite composite layers on titanium screw to reduce pin tract infection rate.

Fibroblast growth factor-2 (FGF-2)-apatite composite layers were formed on anodically oxidized titanium screws to improve bone-screw interface strength and to reduce pin tract infection rate through enhanced skin tissue healing in external fixation. A calcium-containing solution supplemented with FGF-2, a phosphate-containing solution, and a sodium bicarbonate solution were mixed at a Ca/P molar ratio of 2.0 to prepare a calcium phosphate solution supersaturated with respect to calcium phosphates. Screws were individually immersed in 10 mL of the calcium phosphate solution at 37 degrees C for 2 days. Low-crystalline apatite layers incorporating FGF-2 were formed on the screw surface at FGF-2 concentrations in the supersaturated calcium phosphate solution equal to or lower than 10 mug/mL. The amounts of FGF-2 immobilized on the screws ranged from 2.3- to 2.4-mug per screw. The immobilized FGF-2 retained biological activity, as demonstrated by NIH3T3 cell proliferation. Titanium screws with the composite layer were percutaneously implanted into the bilateral proximal tibial metaphyses in rabbits for 4 weeks. The titanium screws with the composite layer formed at the optimum FGF-2 concentration showed a significantly higher bone-screw interface strength and a lower pin tract infection rate than those without the composite layer: the extraction torque and infection rates were respectively 0.230 +/- 0.073 Nm and 43.8% for the screws with the composite layer, and 0.170 +/- 0.056 Nm and 93.8% for those without the composite layer. Therefore, titanium screws with the FGF-2-apatite composite layer are useful for improving bone-screw interface strength and infection resistance in external skeletal fixation.

Formation of a FGF-2 and calcium phosphate composite layer on a hydroxyapatite ceramic for promoting bone formation

Fibroblast growth factor-2 (FGF-2) was immobilized on a hydroxyapatite (HAP) ceramic in supersaturated calcium phosphate solution prepared using solutions corresponding to clinically approved infusion fluids. To avoid the risk of FGF-2 denaturation, FGF-2 immobilization was carried out at 25 degrees C. FGF-2 was successfully immobilized on HAP ceramic surfaces by deposition with calcium phosphate to form a FGF-2 and calcium phosphate composite layer. A maximum of 2.72 +/- 0.01 microg cm(-2) of FGF-2 was immobilized in the composite layer formed on the HAP ceramic under the optimum condition. A FGF-2-immobilized HAP ceramic is likely to have the ability to release a sufficient amount of FGF-2 to promote bone formation. FGF-2 released from a FGF-2-immobilized HAP ceramic maintained its biological activity, since the proliferation of fibroblastic NIH3T3 was promoted. Therefore, the FGF-2-immobilized HAP ceramic is expected to be a useful material for promoting new bone formation.

Ultra-Structural Study of the Laminin-Apatite Composite Layer Formed on Ethylene-Vinyl Alcohol Copolymer by a Biomimetic Process

Mechanism of formation of a laminin-apatite composite layer on the surface of an ethylene-vinyl alcohol copolymer (EVOH) using a liquid phase coating process was investigated by transmission electron microscopy (TEM). In this coating process, an EVOH substrate is alternately dipped in calcium and phosphate solutions, and then immersed in a laminin-containing calcium phosphate (LCP) solution. From the results obtained by the present study, formation of the laminin-apatite composite layer on EVOH is likely to proceed via the following events. By the alternate dipping process, particulate amorphous calcium phosphate, which is a precursor of apatite, was deposited onto the EVOH surface. When the specimen was subsequently immersed in the LCP solution, the amorphous calcium phosphate on the specimen transformed itself into needle-like apatite crystal, and then grew into a layer. During this process, laminin molecules contained in the LCP solution were incorporated into a matrix of the apatite crystals to produce a laminin-apatite composite layer on the EVOH surface.

Enhanced bone formation using hydroxyapatite ceramic coated with fibroblast growth factor-2

Our objective was to develop a bone substitute coated with fibroblast growth factor-2 (FGF-2) that subsequently releases FGF-2. We investigated the use of our system of bone substitutes to induce bone formation. Hydroxyapatite ceramic buttons (HAP-CBs) were coated with FGF-2 by precipitation in supersaturated calcium phosphate solution. HAP-CBs were coated with high or low doses of FGF-2, denoted as FGF-H and FGF-L. The release of FGF-2 from FGF-H and FGF-L was evaluated using its release profile and bioactivity. The efficacy of the subsequent bone formation was quantified using rats with round-shaped bone defects (5mm in diameter) of the right parietal bone. Group 1 was treated only with HAP-CBs, group 2 with HAP-CBs and drops of FGF-2 solution, group 3 with FGF-L and group 4 with FGF-H. To detect the release of FGF-2 in vivo, the expression of bone morphogenic protein-2 (BMP-2) was measured in the defective bone tissue. FGF-2 was released in vitro from FGF-H and FGF-L, and maintained its bioactivity. Rats treated with FGF-L showed better bone formation than rats from the other groups. BMP-2 expression was detected in the defective bone tissues of group 3 at 14 days, which might indicate in vivo FGF-2 release during this period. A specific FGF-2 concentration may be needed for bone formation, and our system can release FGF-2 at adequate concentrations to induce bone formation.

Dissolution rate of zinc-containing β-tricalcium phosphate ceramics

The dissolution rates of ceramic tricalcium phosphate (TCP) and zinc-containing tricalcium phosphate (ZnTCP) ceramics at pH 5.5 decrease with increasing zinc content. The relative dissolution rates of ceramic ZnTCP with zinc contents of 0.5 and 1.0 mol% are more than three times higher than the relative resorbed volumes of these ZnTCP ceramics which were reported previously. The dissolution rates are likely controlled by only one mechanism, that is polynucleation, in the zinc content range from 0 to 1.0 mol%. Since the surface area of ceramic TCP or ZnTCP is much lower than that of powdered TCP or ZnTCP, the initial dissolution flux of ceramic TCP or ZnTCP remains unchanged for a longer period than that of powdered TCP. As a result, no change in dissolution kinetics is expected during one cycle of osteoclastic resorption. Since neither the dissolution rate nor the change in dissolution kinetics accounts for the previously reported drastic reduction in osteoclastic resorption associated with ZnTCP, it is concluded that the drastic reduction in resorption arises from the suppressive effects of ZnTCP on osteoclast formation or osteoclastic activity.

Highly efficient gene transfer system using a laminin–DNA–apatite composite layer

We have recently developed a safe and efficient gene transfer system using a laminin–DNA–apatite composite layer. The objectives of the present study were to fully characterize and optimize the laminin–DNA–apatite composite layer in relation to the efficiency of gene transfer and to demonstrate the feasibility of the composite layer in the induction of cell differentiation.

Optimization of poly(ε-caprolactone) surface properties for apatite formation and improved osteogenic stimulation†

A biodegradable polymer with surface properties that promotes cell attachment and host integration is widely recognized as a useful three-dimensional construct for bone tissue engineering applications. In this work, studies were carried out to correlate surface properties of modified polycaprolactone (PCL) films with cell-material interactions. PCL film substrates were subjected to various degrees of chemical hydrolysis using different pretreatment solutions to introduce different densities of carboxylate groups onto the surfaces. The extent of hydrolysis on the films was optimized to allow the deposition of a dense and uniform bone-like apatite layer by an alternate soak treatment, followed by subsequent incubation in simulated body fluid (SBF). The hydrolyzed and apatite-coated PCL films were investigated using scanning electron microscopy, thin film X-ray diffractometer (TF-XRD), water contact angle, and Alizarin red staining. Surface wettability, roughness, and chemistry of various PCL substrates were correlated with cell attachment, proliferation, viability, and alkaline phosphatase activity. Results demonstrated that cell attachment increased with increasing surface hydrophilicity and roughness. The apatite-coated films showed significantly improved surface wettability and enhanced surface roughness, which subsequently led to better cell attachment potential, high-cell viability, and enhanced bone formation capability. Thus, surface modification with an apatite coating layer is a promising approach for enhancing the efficacy of the polymeric scaffold for bone tissue engineering applications.