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Ti was used as commercially pure titanium (Hyundai, Grade 2). Sponge titanium (purity 99.99%) and granular silver (Yesbio, purity 99.99%) were used as the raw materials for alloy production. Titanium-silver alloys containing silver (2.0 at%) were designed. After weighing the designed composition, a 30 g quantity was melted four times in an arc melter to promote chemical homogeneity. The chamber was evacuated to 0.66 Pa and high-purity argon gas was introduced until the pressure reached 26 kPa before melting. After melting, the alloys were arc melted, homogenized at 950℃ for 72 h, and hot-rolled to 1 mm thickness. The alloys were then solution annealed at 950℃ for 1 h and quenched in water.

cp-Ti and Ti-Ag alloy were sectioned using a cutting machine (10×10×0.15 mm) and made holes by using an engine lathe. The hole diameter was 0.7 mm.

In order to obtain a micro-metric rough surface, the specimens were grit-blasted for 20 seconds using a 110 ㎛ aluminum oxide grit (Renfert, Germany) at a pressure of 4 bar and a distance of 10 mm from the sandblaster nozzle tip (2 mm in diameter). After grit-blasting, the specimens were washed for 10 minutes in distilled water using an ultrasonic cleaner. A nano-metric rough surface was obtained by anodizing the grit blasted specimens at 20 V for 60 min, in a 0.5% HF aqueous solution at room temperature using a DC power supply (Genesys 600-2.6, Densi-Lambda, japan). After anodic oxidation, the specimens were rinsed in distilled water. The specimens were annealed at 350 ˚C for 3 hours.

The simple machined and porous surfaces were coated uniformly with gold for the electric conductivity, and their surface morphology was observed using scanning electron microscopy (SEM, Model S-800, Hitachi, Japan).

The in vivo study was performed under the guidelines of the Animal Care and Use Committee, Yonsei Medical Center, Seoul, Korea. Twelve adult male rabbits weighting 3.20 ± 0.25 ㎏ were used (Samtako, Seoul, Korea). A midline incision was performed down to the bone surface of the calvarial bone, and a skin periosteum flap was raised to expose the calvarial bone on both sides of the midline. The outer cortical layer of calvarium bone was removed by trephine burr, and a 8 mm diameter cortical bone defect was made in each side of the calvarial bone; thus, a total of two bone defects were made in each animal (Figure 1). The meshes or without mesh on hole were covered for healing. Six of the twelve animals were euthanized after a healing period of 2 weeks and the other six animals after 4 weeks for histomorphometrical evaluation. Computed tomography (CT) images of the calvarial bone were taken for each rabbit (Model skyscan 1076, Skyscan, Belgium). CT analysis was used to analyze differences in the percentage areas of newly mineralized bone after 2 weeks and 4 weeks of healing.

Figure 1. 
(a) A cutaneous flap was demarcated, and then the periosteum was removed and the calvarial bone exposed on both sides of the midline. On each side, a circle 8 mm in diameter was made with a trephine bur. (b) Titanium meshes either with or without surface treatment of between cp-Ti and Ti-Ag mesh placed in the bone and the mesh fixed by sewing.

Figure 2 shows a photomicrograph of porous layer mesh. There is made by grit-blasting and anodic oxidation. The surface consisted of nano-metric pores on the micro-metric surface with a hemisphere shape. The diameter of the micro-metric rough surface ranged from 1 to 10 ㎛, and micro-metric pores were separated irrelgularly on the mesh. The diameter of the nano-metric pores ranged from 40 to 100 nm, and nano-metric pores were well separated uniformly over the micro-metric pores. The initial attachment of cells is important for implantation of dental implant. The implant surface with micrometer-ordered pore structure increases the surface area and consequently increases the osseointegration efficiency(Abron et al., 2001). The adhesion of cells such as osteoblasts is a prerequisite to subsequent cell functions such as the synthesis of extracellular matrix proteins and the formation of mineral deposits. The introduction of nanostructured ceramics significantly improves osteoblast adhesion (Webster et al., 2000; Webster et al., 2001b)

Figure 2. 
SEM photographs show a nano porous layer formed on Ti-Ag mesh. (a) ×200, (b) ×5000, (c) ×50000.

Figure 3 shows newly regenerated bone formed after a mesh implanted to calvaria bone. In the hole without mesh, regenerated bone didn’t entirely fill for 4 weeks. But, the newly formed bone with mesh was more formed than without mesh.

Figure 3. 
Computed tomography (CT) images for histomorphometrical evaluation of implanted meshes (10×10×0.15 mm). Newly generated bone tissue formed on the mesh was observed by 3-D scanning image. (Gray: newly generated bone, blue: mesh, in side: inner of the mesh, outside: outer of the mesh) the images were observed about implantation periods between inside and outside each of 2 weeks and 4 weeks.

Figure 4 shows the volume of mineralized bone in newly generated tissue on the mesh. Ti-Ag mesh produced more bone than the hole case without mesh in rabbit calvaria experiment (P<0.05). And in containing mesh on hole, we observed that results of 4 weeks were occurred newly bone better than 2 weeks. We thought a mesh might affect to formation of regenerated bone. We observed newly formed bone at the outer mesh. In this case, growth factor might transfer from hole of mesh.

Figure 4. 
Volume of mineralized bone in newly generated tissue on the mesh (White graphs are 2 weeks result and grey graphs are 4 weeks result).

The excellent biocompatibility of titanium and the easy handling of the titanium micro-mesh systems allowed their application for three-dimensional reconstruction of large bony defects. The most likely hypothesis that can explain the apparent benefits of the mesh lies in its probable protective effect during the healing time following bone grafting, as already found with non-resorbable membranes (Antoun et al., 2001). This study used a Ti–Ag alloy to overcome mechanical and biological problems of existing Ti or Ti alloys. Because the binary alloy containing Ti and Ag is an alloy with high corrosion resistance (Moon et al., 2012). Ti–Ag alloys are believed to have enhanced corrosion resistance due to the formation of a stable passive oxide film that is more easily formed as the silver (Ag) is added to the Ti. Ag, which has a much higher electromotive force than Ti, facilitates the dissolution of Ti in artificial saliva, which then forms a stable Ti oxide film over the Ti–Ag alloy surface (Oh et al., 2005; Shim et al., 2005; Shim et al., 2006).

Nano structure formation of titanium surface suggests that anodization might be a quick and inexpensive technique to modify surfaces of titanium-based implants to improve bone cell functions required for improved orthopedic applications (Yao et al., 2008). The micro-nanoporous layer was the most hydrophilic surface. Such a highly hydrophilic implant surface would result in better cell attachment in the initial stage of implant placement, and consequently result in improved osseointegration (Kilpadi and Lemons, 1994). In previous study, we found that the expressions of bone-related genes were greater for cells grown on the porous Ti-Ag alloy layer compared to that of cells cultured on a simple-machined surface which is important in terms of a faster rate of osseointegration (Moon et al., 2012).

Therefore, the porous mesh of micro-nanometric structures including superior corrosion resistance and mechanical property used in this study may have advantages as Guided Bone Regeneration membrane for damaged maxillofacial reconstruction.