Int. J. Oral Maxillofac. Surg. 2012; 41: 13041309 doi:10.1016/j.ijom.2011.12.035, available online at http://www.sciencedirect.

com

Research Paper Biomaterials and Tissue Engineering

Hydroxyapatite coating for titanium bre mesh scaffold enhances osteoblast activity and bone tissue formation
Makoto Hirota, Tohru Hayakawa, Masao Yoshinari, Akihiro Ametani, Takaki Shima, Yuka Monden, Tomomichi Ozawa, Mitsunobu Sato, Chika Koyama, Naoto Tamai, Toshinori Iwai, Iwai Tohnai: Hydroxyapatite coating for titanium bre mesh scaffold enhances osteoblast activity and bone tissue formation. Int. J. Oral Maxillofac. Surg. 2012; 41: 13041309. # 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Makoto Hirotaa, Tohru Hayakawab, Masao Yoshinaric, Akihiro Ametanid, Takaki Shimad, Yuka Mondena, Tomomichi Ozawaa, Mitsunobu Satoe, Chika Koyamaa, Naoto Tamaia, Toshinori Iwaia, Iwai Tohnaia
a Department of Oral and Maxillofacial Surgery, Yokohama City University Graduate School of Medicine, 3-9 Fuku-ura, Kanazawaku, Yokohama 236-0004, Japan; bDepartment of Dental Engineering, Tsurumi University School of Dentistry, 2-1-3 Tsurumi, Tsurumiku, Yokohama 230-8501, Japan; cDivision of Oral Implants Research, Oral Health Science Center, Tokyo Dental College, 1-2-2 Masago, Mihama-ku, Chiba 261-8502, Japan; dMedical Device Department, HI-LEX Corporation, Inc., 1-12-28 Sakae-cho, Takarazuka 665-0845, Japan; eCoordination Engineering Laboratory, Faculty of Engineering, Kogakuin University, 2665-1 Nakano, Hachioji, Tokyo 192-0015, Japan

Abstract. This study investigated the bone regeneration properties of titanium bre mesh as a tissue engineering material. A thin hydroxyapatite (HA) coating on the titanium bre web was created using the developed molecular precursor method without losing the complex interior structure. HA-coated titanium bre mesh showed apatite crystal formation in vitro in a human osteoblast culture. Titanium bre 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 signicantly higher in the HAcoated titanium bre mesh than in the non-coated titanium bre mesh 2 weeks after implantation. These results suggest that thin HA coating enhances osteoblast activity and bone regeneration in the titanium bre mesh scaffold. Thin HA-coating improved the ability of titanium bre mesh to act as a bone regeneration scaffold.

Keywords: Titanium bre mesh; hydroxyapatite coating; molecular precursor method; bone regeneration; scaffold; tissue engineering. Accepted for publication 22 December 2011 Available online 17 April 2012

Titanium bre mesh is a porous material made of titanium bres with a diameter of 50 mm; the web form can be processed to any shape. Titanium bre mesh shows sufcient biological compatibility and strength, and research into titanium bre mesh-based bone regenerative materials have been reported.13 Cell culture research using titanium bre mesh has reported that a type I collagen coating on titanium bre mesh accelerated the differentiation of rat bone marrow cells into osteoblasts,4 that a
0901-5027/01001304 + 06 $36.00/0

TiO2 coating accelerates differentiation of rat bone marrow stromal cells into osteoblasts5 and that uniform bone formation in rat bone defects was seen.6 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.7,8 Uniformly coating a thin layer of HA onto a scaffold such as titanium bre mesh is difcult using these methods.

Recently, the molecular precursor method has been described as a new technology for providing a thin coating of HA.9 In this method, the titanium bre 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 bre mesh.9 Using this method, formation of trabecular bone into a titanium bre mesh has been accelerated after the implantation of titanium bre mesh into the trabecular bone

# 2012 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.

Hydroxyapatite coating for titanium ber mesh scaffold

defects of rabbits.9,10 The authors also observed that bone formation into the titanium bre mesh scaffold in rat cranial bone defect was enhanced by a thin HA coating using the usual molecular precursor method.11 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 lm coated on the scaffold.12 They obtained a sufciently apatite coated titanium bre mesh. The present study evaluated the functional activity of human osteoblasts and the bone response of titanium bre mesh with or without HA coating, using the developed molecular precursor method.12 HA crystal formation by osteoblasts cultured in the titanium bre mesh and new bone formation within the mesh implanted into a rat cranial bone defect were investigated.
Material and methods

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Fig. 1. (a) Titanium bre web and (b) HA-coated titanium bre web using human osteoblast culture. Materials are 10 mm 10 mm and 3 mm thick, and porosity is 87%.

Sintered titanium bre mesh (Hi-Lex, Kobe, Japan) was processed into a 3dimensional scaffold structure with an average internal pore size of 250 mm. The diameter of each titanium bre mesh was 50 mm and the porosity of the titanium bre mesh was 87%. Porosity was set by adjusting the titanium bre mesh weight for unit area. The titanium bre mesh was adjusted until the weight became 900 g/ m2, 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.
HA coating

(EPMA) (JXA-8200; microanalysis JEOL, Tokyo, Japan) using an accelerating voltage of 25 kV by detecting the Xray intensities of Ca-Ka, P-Ka and Ti-Ka. 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 bre mesh scaffold. Before EPMA, the sample was ground, polished with alumina and cleaned with 70% ethanol. The HA coating on the titanium bre mesh was evaluated by the elementary mapping of Ca, P and Ti.
Osteoblast culture in titanium bre mesh

days 7 and 14 after dissemination, titanium bre meshes were xed 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 eld-emission scanning electron microscopy (JSM-6340F; JEOL, Tokyo, Japan) at an accelerating voltage of 5 kV.
Animal experiments

The mesh was given a thin HA coating using the molecular precursor method according to Hayakawa et al.12 A precursor solution was prepared by mixing CaEDTA/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 bre mesh was soaked in the precursor solution for 20 min with ultrasonic treatment. After immersion, the titanium bre mesh was pre-heated in a mufe kiln at 60 8C for 20 min, then heated at 600 8C 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 bre surface was observed by electron probe

Human osteoblasts (CC-2538; Takara Bio, Ohtsu, Japan) were disseminated in a 75 cm2 ask at a concentration of 5000 cells/cm2. 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% conuence, osteoblasts were detached by trypsinization and suspended in culture medium. The cells were suspended in osteoblast differentiation-inducing medium at a concentration of 5 104 cells/ml. This medium was prepared by adding OGMTM Differentiation SingleQuots1 (Takara Bio) to an osteoblast growth medium. Square-shaped titanium bre 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 (5 104 cells/well) and cultured at 37 8C under 5% CO2. The medium was changed the day after dissemination and every 3 days thereafter. On culture

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 912 weeks; body weight 200300 g) were used. After sufcient 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 bre 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 bre mesh samples embedded in the cranial bone were removed and xed 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-decalcied 25 mm thick sections were prepared

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cover glass were attached to a slide glass and evaluated under light microscopy. New bone formation within the titanium bre 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 bre mesh disc was calculated as the bone formation ratio. The mean of measured values on newly formed bone in the titanium bre mesh was calculated and compared for each group using the MannWhitney U-test. Values of p < 0.05 were considered statistically signicant.
Results

using a microtome (Leica, Tokyo, Japan).13,14 Sections were made using the middle part of the titanium bre mesh disc. The section was double stained with basic fuchsin and methylene blue. The surface of the section was rst 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

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 bres was maintained. Crystals were deposited in the HAcoated titanium bre mesh, but no crystals were seen in the non-coated titanium bre web (Figs. 3 and 4). In the HA-coated

Fig. 2. Elemental analysis of HA-coated titanium bre surface using an EPMA. Thin layers of phosphorus (a) and calcium (c) are seen on (b) titanium (arrowheads). Scale bar 20 mm.

Fig. 3. Crystal formation of calcication by human osteoblast culture in the titanium bre web. Stereoscope ndings of non-coated (a and c) and HA-coated (b and d) titanium bre webs in which human osteoblasts had been cultured for 7 days (a and b) and 14 days (c and d). In the HA-coated titanium bre web, small crystals (arrowheads) are seen on titanium bres after 7 days of culture (b) and larger crystals (arrowheads) are seen on titanium bres after 14 days of culture (d). No crystals are seen on non-coated titanium bre webs at 7 days (a) or 14 days (c). Scale bar 1.0 mm.

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Fig. 4. (a) Stereoscope nding of apatite crystal in HA-coated titanium bre web after 14 days of human osteoblast culture. Apatite crystals are deposited on titanium bre (arrows). Scale bar 200 mm. (b) Scanning electron microscope ndings of human osteoblast cultured in HA-coated titanium bre web for 7 days. Human osteoblast extends projections to adhere to titanium bres, secreting calcication matrix (arrows). Scale bar 20 mm.

Fig. 5. Histological analysis of titanium bre mesh implanted to rat cranial bone defect. (a) Titanium bre mesh disc (diameter 5 mm; thickness 1 mm) is implanted in a rat cranial bone defect (arrowhead). (b) Four weeks after implantation, the titanium disc is embedded in the tissue (arrowhead). Histologically, newly formed bone is seen in the HA-coated titanium bre mesh disc (d), while little newly formed bone is seen in the non-coated titanium bre mesh disc (c) 2 weeks after implantation. Four weeks after implantation, both non-coated (e) and HA-coated (f) titanium bre mesh disks are lled with newly formed bone. (g) Magnication of (b). Newly formed bone penetrated into titanium bre mesh from remaining bone. Scale bar 1 mm.

titanium bre mesh, small crystals were seen on titanium bres after 7 days of culture (Fig. 2b), with larger crystals evident on titanium bres after 14 days of culture (Fig. 2d). The large magnication stereoscopic view revealed that crystals appeared as assemblies of needle-like crystal structures (Fig. 3a). Scanning electron microscopy of human osteoblasts cultured in HA-coated titanium bre mesh for 7 days revealed that human osteoblast extended the projection and adhered to titanium bres, secreting calcication matrix (Fig. 3b). Histological bone formation into the titanium bre mesh implanted to rat cra-

nial bone defect was examined (Fig. 5a and b). In the early stage, 2 weeks after implantation, new bone formation was more marked in the HA-coated titanium bre mesh than in the non-coated titanium bre web (Fig. 5c and d). In the late stage, 4 weeks after implantation, new bone formation in the HA-coated and noncoated titanium bre mesh was almost even. The newly formed bone ratio in the titanium bre mesh was 7.1% in the non-coated group and 35.8% in the HAcoated group 2 weeks after implantation (Table 1). The ratio was signicantly higher in the HA-coated group at 2 weeks than in the non-coated group (p < 0.05).

Four weeks after implantation, the newly formed bone ratio in the titanium bre mesh was 45.1% in the non-coated group and 56.2% in the HA-coated group (Table 1).

Table 1. New bone formation ratio of noncoated (control) and hydroxyapatite (HA)coated titanium bre web in the rat cranial bone defect. New bone formation ratio (%) (SD) Group 2 weeks 4 weeks
*

Control 7.1 (10.9)* 45.1 (35.5)

HA coating 35.8 (22.4)* 56.2 (20.9)

p < 0.05.

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using continuous oxygen gas induction was not clear. Although a precise comparison was difcult because of differences in the observation period, there was little difference in the bone formation rate in the HA-coating titanium bre mesh between the two methods. The bone formation rate of HA-coating titanium bre mesh at 2 weeks in the present study was around 35%, while that rate was only about 20% at 3 weeks in the previous study.11 Titanium bre mesh reportedly has a potential role for activation of osteogenic cells.2 In addition, in the present study, HA coating enhanced the bone formation activity of the titanium bre mesh. The HA coating of titanium bre mesh was possible using the molecular precursor method to enable a uniform thin coat to be applied without losing the 3-dimensional structure. In other words, higher osteoconductivity could be produced in the titanium bre mesh scaffold by HA coating with the molecular precursor method. Titanium bre mesh needs further mechanical and functional assessment, but a potential role as a bone formation scaffold can be expected. HA-coated titanium bre mesh may nd applications as a research material or clinical biomaterial, for example in new types of bone substitutes, dental implants, or scaffolds for reconstruction and tissue engineering.
Competing interests
4. van den Dolder J, Jansen JA. The response of osteoblast-like cells towards collagen type I coating immobilized by p-nitrophenylchloroformate to titanium. J Biomed Mater Res A 2007;83:7129. 5. Meretoja VV, De Ruijter AE, Peltola TO, rhi TO. Osteoblast differentiaJansen JA, Na tion with titania and titaniasilica-coated titanium ber meshes. Tissue Eng 2005;11: 148997. ritalo V, Walboomers 6. Meretoja VV, Tirri T, Aa rhi TO. Titania and titania XF, Jansen JA, Na silica coatings for titanium: comparison of ectopic bone formation within cell-seeded scaffolds. Tissue Eng 2007;13: 85563. 7. Hacking SA, Zuraw M, Harvey EJ, Tanzer M, Krygier JJ, Bobyn JD. A physical vapor deposition method for controlled evaluation of biological response to biomaterial chemistry and topography. J Biomed Mater Res 2007;82:17987. 8. Takemoto M, Fujibayashi S, Neo M, Suzuki J, Kokubo T, Nakamura T. Mechanical properties and osteoconductivity of porous bioactive titanium. Biomaterials 2005;26:601423. 9. Hayakawa T, Takahashi K, Okada H, Yoshinari M, Hara H, Mochizuki C, et al. Effect of thin carbonate-containing apatite (CA) coating of titanium ber mesh on trabecular bone response. J Mater Sci Mater Med 2008;19: 208796. 10. Hayakawa T, Takahashi K, Yoshinari M, Okada H, Yamamoto H, Sato M, et al. Trabecular bone response to titanium implants with a thin carbonate-containing apatite coating applied using the molecular precursor method. Int J Oral Maxillofac Implants 2006;21:8518. 11. Hirota M, Ametani A, Monden Y, Noishiki Y, Hayakawa T, Tohnai I. Molecular precursor method facilitates thin hydroxyapatite coating of titanium ber web scaffold and enhances bone formation: experimental study in rat cranial bone defects. Int J Oral Maxillofac Implants 2010;25:88892. 12. Hayakawa T, Ametani A, Kuboki Y, Sato M. Thin cabonate-containing apatite coating of titanium web using molecular precursor method under oxygen gas introduction. J Oral Tissue Eng 2009;6:20110. 13. van den Dolder J, Farber E, Spauwen PHM, Jansen JA. Bone tissue reconstruction using titanium ber mesh combined with rat bone marrow stromal cells. Biomaterials 2003;24: 174550. 14. van der Lubbe HB, Klein CP, de Groot K. A simple method for preparing thin (10 mM) histological sections of undecalcied plastic embedded bone with implants. Stain Technol 1988;63:1716. 15. Lavos-Valereto IC, Wolynec S, Deboni MCZ, Koenig Jr B. In vitro and in vivo biocompatibility testing of Ti6AI7Nb alloy with and without plasma-sprayed

Discussion

HA coatings are widely used on titanium biomaterials to improve biocompatibility2 and bone formation.1518 Irregular HA coating decreases cell activity on the material.19 In the present study, HA coating was performed using a molecular precursor method.9,10 As the method was a simple solution technique, HA coating was easy and pretreatment with titanium was unnecessary. Using the molecular precursor method, a uniform coating of thin HA on the titanium bre mesh was readily achieved without affecting the architecture. As a result, osteoconductivity of the titanium bre mesh was maintained and the ability was accelerated by the thin HA coating. In the present in vivo study, a signicantly higher amount of newly formed bone penetrated the HAcoated titanium bre mesh scaffold 2 weeks after implantation. The new bone formation rate was signicantly higher for the HA-coated titanium bre mesh than for the non-coated titanium bre mesh. The authors previously reported that HA-coating titanium bre mesh implanted in rat cranial bone defect showed 10-fold bone formation compared with non-coating titanium bre mesh 3 weeks after implantation, while the bone formation was only 4-fold in HA-coating titanium bre mesh 6 weeks after implantation.11 The uniformly thin HA coating of titanium bre mesh was considered to have accelerated the bone formation process at the early stage of bone healing. Human osteoblast culture in the titanium bre mesh revealed that a thin HA coating on the titanium bres enhanced crystal formation. These crystals were produced by osteoblasts and could be considered to represent HA crystal. Osteoblasts are osteogenic cells associated with bone formation by producing osteoid and mineralization of the osteoid matrix for calcication. HA is considered effective as a cell culture substrate,2 and osteoblasts show enhanced maturation and functional activity for apatite formation by HA coating.2022 In the last stage of osteoblast maturation, osteocalcin is expressed,23,24 and osteocalcin expression by osteoblasts is reportedly enhanced by HA coating on titanium alloy.25 In the present study, obvious apatite crystal formation and crystal matrix secretion could be observed only in HAcoated titanium bre mesh. This nding suggests that the HA coating enhanced maturation and the functional activity of osteoblasts cultured in titanium bre mesh. The obvious efcacy of the developed procedure of molecular precursor method

None declared.
Acknowledgments. This study was supported in part by a Grant-in-Aid for Young Scientists (B) (no. 23792366) from Japan Society for the Promotion of Science.

Animal experiments were approved by the ethics committee at Yokohama City University (no. 08-110).
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hydroxyapatite coating. J Biomed Mater Res 2001;58:72733. Narasaraju TSB, Phoebe DE. Review of some physico-chemical aspects of hydroxyapatite. J Mater Sci 1996;31:121. Yang Y, Kim KH, Agrawal CM, Ong JL. Inuence of post-deposition heating time and the presence of water vapor on sputter-coated calcium phosphate crystallinity. J Dent Res 2003;82:8337. Yang Y, Kim KH, Agrawal CM, Ong JL. Effect of post-deposition heating temperature and the presence of water vapor during heat treatment on crystallinity of calcium phosphate coatings. Biomaterials 2003;24: 51317. Takahashi K, van den Beucken JJ, Wolke JG, Hayakawa T, Nishiyama N, Jansen JA. Characterization and in vitro evaluation of biphasic calcium pyrophosphate-tri calcium phosphate radio frequency magnetron sputter coatings. J Biomed Mater Res A 2008; 84:68290. 20. Hulshoff JE, van Dijk K, de Ruijter JE, Rietveld FJ, Ginsel LA, Jansen JA. Interfacial phenomena: an in vitro study of the effect of calcium phosphate (CaP) ceramic on bone formation. J Biomed Mater Res 1998;40:46474. 21. Perizzolo D, Laceeld WR, Brunette DM. Interaction between topography and coating in the formation of bone nodules in culture for hydroxyapatite- and titanium-coated micromachined surfaces. J Biomed Mater Res 2001;56:494503. 22. Siebers MC, Walboomers XF, Leeuwenburgh SC, Wolke JG, Jansen JA. The inuence of the crystallinity of electrostatic spray deposition-derived coatings on osteoblastlike cell behavior, in vitro. J Biomed Mater Res A 2006;78:25867.

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Corresponding author Tel.: +81 45 787 2659 fax: +81 45 785 8438 E-mail: mhirota@med.yokohama-cu.ac.jp