This study investigated the bone regeneration properties of titanium fibre mesh as a tissue engineering material. A thin hydroxyapatite (HA) coating on the titanium fibre web was created using the developed molecular precursor method without losing the complex interior structure. HA-coated titanium fibre mesh showed apatite crystal formation in vitro in a human osteoblast culture. Titanium fibre mesh discs with or without a thin HA coating were implanted into rat cranial bone defects, and the animals were killed at 2 and 4 weeks. The in vivo experience revealed that the amount of newly formed bone was significantly higher in the HA-coated titanium fibre mesh than in the non-coated titanium fibre mesh 2 weeks after implantation. These results suggest that thin HA coating enhances osteoblast activity and bone regeneration in the titanium fibre mesh scaffold. Thin HA-coating improved the ability of titanium fibre mesh to act as a bone regeneration scaffold.
Titanium fibre mesh is a porous material made of titanium fibres with a diameter of 50 μm; the web form can be processed to any shape. Titanium fibre mesh shows sufficient biological compatibility and strength, and research into titanium fibre mesh-based bone regenerative materials have been reported. Cell culture research using titanium fibre mesh has reported that a type I collagen coating on titanium fibre mesh accelerated the differentiation of rat bone marrow cells into osteoblasts, that a TiO 2 coating accelerates differentiation of rat bone marrow stromal cells into osteoblasts and that uniform bone formation in rat bone defects was seen.
Various techniques, including magnetron sputtering, plasma spray methods, and similar physical vapor deposition, are used to add a thin coat of hydroxyapatite (HA) to titanium materials. Uniformly coating a thin layer of HA onto a scaffold such as titanium fibre mesh is difficult using these methods.
Recently, the molecular precursor method has been described as a new technology for providing a thin coating of HA. In this method, the titanium fibre mesh is simply immersed into the molecular precursor solution and then heated, resulting in a thin HA coating on the surface and inside the titanium fibre mesh. Using this method, formation of trabecular bone into a titanium fibre mesh has been accelerated after the implantation of titanium fibre mesh into the trabecular bone defects of rabbits. The authors also observed that bone formation into the titanium fibre mesh scaffold in rat cranial bone defect was enhanced by a thin HA coating using the usual molecular precursor method. Hayakawa et al. developed the molecular precursor method with continuous oxygen gas introduction during the heating processes to raise the stability of the thin HA film coated on the scaffold. They obtained a sufficiently apatite coated titanium fibre mesh.
The present study evaluated the functional activity of human osteoblasts and the bone response of titanium fibre mesh with or without HA coating, using the developed molecular precursor method. HA crystal formation by osteoblasts cultured in the titanium fibre mesh and new bone formation within the mesh implanted into a rat cranial bone defect were investigated.
Material and methods
Sintered titanium fibre mesh (Hi-Lex, Kobe, Japan) was processed into a 3-dimensional scaffold structure with an average internal pore size of 250 μm. The diameter of each titanium fibre mesh was 50 μm and the porosity of the titanium fibre mesh was 87%. Porosity was set by adjusting the titanium fibre mesh weight for unit area. The titanium fibre mesh was adjusted until the weight became 900 g/m 2 , it was then trimmed to the required shape using a cutting machine. Two shapes were made: a 10 mm × 10 mm square, 3 mm thick for in vitro study ( Fig. 1 ); and a disc 5 mm in diameter and 1.5 mm thick for in vivo study.
The mesh was given a thin HA coating using the molecular precursor method according to Hayakawa et al. A precursor solution was prepared by mixing Ca-EDTA/amine complex and dibutylammonium diphosphate salt in ethanol at a Ca/P ratio of 1.67; the calcium ion concentration was 0.470 mmol/g. The titanium fibre mesh was soaked in the precursor solution for 20 min with ultrasonic treatment. After immersion, the titanium fibre mesh was pre-heated in a muffle kiln at 60 °C for 20 min, then heated at 600 °C for 2 h under atmospheric conditions. During the heating process, oxygen gas was continuously added.
Electron probe microanalysis
The thin HA layer on the titanium fibre surface was observed by electron probe microanalysis (EPMA) (JXA-8200; JEOL, Tokyo, Japan) using an accelerating voltage of 25 kV by detecting the X-ray intensities of Ca-Kα, P-Kα and Ti-Kα. The sample was embedded in epoxy resin and cut with a micro-cutting machine (Finecut HS-100; Heiwa Tech, Tokyo, Japan) to observe the interior of the titanium fibre mesh scaffold. Before EPMA, the sample was ground, polished with alumina and cleaned with 70% ethanol. The HA coating on the titanium fibre mesh was evaluated by the elementary mapping of Ca, P and Ti.
Osteoblast culture in titanium fibre mesh
Human osteoblasts (CC-2538; Takara Bio, Ohtsu, Japan) were disseminated in a 75 cm 2 flask at a concentration of 5000 cells/cm 2 . An osteoblast culture medium kit (Takara Bio) was used for cell proliferation, and the medium was changed the day after dissemination and every 3 days thereafter. After achieving 80% confluence, osteoblasts were detached by trypsinization and suspended in culture medium. The cells were suspended in osteoblast differentiation-inducing medium at a concentration of 5 × 10 4 cells/ml. This medium was prepared by adding OGM™ Differentiation SingleQuots ® (Takara Bio) to an osteoblast growth medium.
Square-shaped titanium fibre mesh with or without HA coating ( Fig. 1 ) was placed on a 24-well plate, and the primary cultured cell suspension was disseminated at 1 ml/well (5 × 10 4 cells/well) and cultured at 37 °C under 5% CO 2 . The medium was changed the day after dissemination and every 3 days thereafter. On culture days 7 and 14 after dissemination, titanium fibre meshes were fixed for 15 min in citric acid buffer (pH 5.4) and 60% acetone/10% methanol, and apatite crystal formation was observed under stereomicroscopy (M80; Leica, Solms, Germany). Osteoblasts were also observed under field-emission scanning electron microscopy (JSM-6340F; JEOL, Tokyo, Japan) at an accelerating voltage of 5 kV.
All animal experiments were approved by the ethics committee at Yokohama City University (No. 08-110) and performed in accordance with the national guidelines for the care and use of laboratory animals. Male Wister rats (age 9–12 weeks; body weight 200–300 g) were used.
After sufficient anaesthesia was achieved with pentobarbital injection (dose, 50 mg/kg), the skin and periosteum were incised to expose the cranial bone. A circular bone defect was then created with a 5.2 mm diameter trephine bur, and a defect size-matched titanium fibre disc with or without HA coating was placed into the defect. Following consistent hemostasis, the periosteum and skin were sutured tightly.
Five animals from each group were killed 2 and 4 weeks after implantation. The titanium fibre mesh samples embedded in the cranial bone were removed and fixed in 4% paraformaldehyde for 1 week. The samples were then serially dehydrated with 70%, 80%, 90% and 100% alcohol and embedded in methyl methacrylate resin. Non-decalcified 25 μm thick sections were prepared using a microtome (Leica, Tokyo, Japan). Sections were made using the middle part of the titanium fibre mesh disc. The section was double stained with basic fuchsin and methylene blue. The surface of the section was first etched with 2 N HCl, then drops of 2% methylene blue solution were applied to the surface and left for 1 min. The sections were rinsed with water and drops of 2% basic aqueous fuchsin solution were similarly applied and left for 30 s. After rinsing with water and drying, a cover glass was placed on the surface. The thin samples attached to the cover glass were attached to a slide glass and evaluated under light microscopy.
New bone formation within the titanium fibre mesh disc was histomorphometrically evaluated. Using an image analysis system (NIH Image; National Institutes of Health, Bethesda, MD, USA), the ratio of the area of newly formed bone to the porosity area within the titanium fibre mesh disc was calculated as the bone formation ratio. The mean of measured values on newly formed bone in the titanium fibre mesh was calculated and compared for each group using the Mann–Whitney U -test. Values of p < 0.05 were considered statistically significant.
EPMA showed a thin layer of phosphorus and calcium (arrowheads) on the titanium surface ( Fig. 2 ). As these elemental layers are thin, porosity between the titanium fibres was maintained.
Crystals were deposited in the HA-coated titanium fibre mesh, but no crystals were seen in the non-coated titanium fibre web ( Figs. 3 and 4 ). In the HA-coated titanium fibre mesh, small crystals were seen on titanium fibres after 7 days of culture ( Fig. 2 b), with larger crystals evident on titanium fibres after 14 days of culture ( Fig. 2 d). The large magnification stereoscopic view revealed that crystals appeared as assemblies of needle-like crystal structures ( Fig. 3 a). Scanning electron microscopy of human osteoblasts cultured in HA-coated titanium fibre mesh for 7 days revealed that human osteoblast extended the projection and adhered to titanium fibres, secreting calcification matrix ( Fig. 3 b).