Materials Letters 63 (2009) 1995–1998

Contents lists available at ScienceDirect

Materials Letters

j ourna l homepage: www.e lsev ie r.com/ locate /mat le t

Highly porous titanium (Ti) scaffolds with bioactive microporous hydroxyapatite/TiO2 hybrid coating layer

Jong-Hoon Lee a, Hyoun-Ee Kim a, Young-Hag Koh b,⁎a Department of Materials Science and Engineering, Seoul National University, Seoul, 151-742, Republic of Koreab Department of Dental Laboratory Science and Engineering, Korea University, Seoul, 136-703, Republic of Korea

⁎ Corresponding author. Tel.: +82 940 2844.E-mail address: kohyh@korea.ac.kr (Y.-H. Koh).

0167-577X/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.matlet.2009.06.023

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 April 2009Accepted 14 June 2009Available online 21 June 2009

Keywords:Metals and alloysPorosityMechanical propertiesTitanium (Ti)Hydroxyapatite (HA)

Highly porous Ti scaffolds with a bioactive microporous hydroxyapatite (HA)/TiO2 hybrid coating layer werefabricated using the sponge replication process and micro-arc oxidation (MAO) treatment to produce theporous Ti scaffold and hybrid coating layer, respectively. In particular, the morphology and chemicalcomposition of the hybrid coating layer were controlled by carrying out the MAO treatment in electrolytesolutions containing various concentrations of HA, ranging from 0 to 30 wt.%. The fabricated sample showedhigh porosity of approximately 70 vol.% with interconnected pores and reasonably high compressive strengthof 18±0.3 MPa. Furthermore, the surfaces could be coated successfully with a bioactive microporous HA/TiO2 hybrid layer. The amount of HA particles in the hybrid coating layer increased with increasing HAcontent in the electrolyte solution, while preserving the microporous morphology. This hybrid coatingimproved the osteoblastic activity of the porous Ti scaffolds significantly.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Titanium (Ti) and its alloys are well recognized as one of the mostattractive biomaterials that have been extensively used for dental andorthopedic implants over the past few decades, owing to theirexcellent mechanical properties (e.g., high strength and toughness),biocompatibility, and chemical stability [1]. Recently, considerableeffort has been made to produce porous Ti scaffolds to match theirmechanical properties (particularly, elastic modulus and stiffness) tothose of the surrounding bone and provide a favorable environmentfor bone ingrowth [2,3].

Currently, there are a variety of manufacturing methods availablefor producing porous Ti scaffolds, including the sintering of metalpowders [4], space holder method [5], rapid prototyping method [6],and freeze casting method [7,8]. Recently, the sponge replicationmethod has also been proven to be a very useful method, since it canproduce porous Ti scaffolds with high porosity and good interconnec-tions between the pores [9,10,11]. These characteristics are believed tobe quite beneficial to the bone ingrowth and vascularization of newlyformed tissue [12]. In addition, the biocompatibility of the porous Tiscaffolds can be improved significantly by coating their surfaces withbioactive materials [10,11,13,14,15].

Therefore, in this study, we fabricated highly porous Ti scaffoldsusing the polymeric sponge replication method and coated theirsurfaces with a bioactive, microporous HA/TiO2 hybrid coating layer

ll rights reserved.

using the micro-arc oxidation (MAO) treatment. In particular, the MAOtreatment was carried out in an electrolyte solution containing HAparticles to incorporate them into the coating layer in an in-situmanner[16]. In order to the control morphology and chemical composition ofthe hybrid coating layer, HA was added to the electrolyte solution atvarious concentrations (0, 10, 20, and 30 wt.%). The porous Ti scaffoldspreparedwere characterized in termsof their pore structures, crystallinephases, chemical compositions, and compressive strengths. The pre-liminary osteoblastic activity of the samples was also evaluated using invitro tests.

2. Experimental procedure

Highly porous Ti scaffolds were produced by coating a~15×15×20mmpolyurethane sponge (25 pores per inch, Jeil UrethaneCo., Korea) with a titanium hydride (TiH2) slurry that was prepared bydispersing 60 g TiH2 powders (Alfa Aesar,WardHill, MA, USA) in 200mlof ethanol containing3 gof triethyl phosphate (TEP; (C2H5)3PO4, Sigma-Aldrich, USA) as a dispersant, and 3 g of polyvinylbutyl (PVB, Sigma-Aldrich, USA) as a binder. The TiH2-coated sponges were then heat-treated at 800 °C for 3 h to remove the polymeric sponges and at 1300 °Cfor 2 h in a vacuum to convert TiH2 to Ti metal and to densify Ti walls.This procedurewas repeated again to reduce the porosity of the sample.

The surfaces of the prepared porous Ti scaffolds were coated with abioactive, microporous HA/TiO2 hybrid layer using the micro-arcoxidation (MAO) treatment [16]. The as-received HA powders (NT-HA,OssGen Co., Korea) were highly crystallized, determined by X-raydiffraction (XRD,M18XHF-SRA,MacScienceCo., Yokohama, Japan),with

Fig.1. SEMmicrographs of the porous Ti scaffolds showing (A) a highly porous structureand (B) sintered Ti walls.

Fig. 2. Typical XRD patterns of the porous Ti scaffold after heat-treatment in a vacuum at1300 °C for 2 h. (Ti: JCPDS file No. 44-1294)

1996 J.-H. Lee et al. / Materials Letters 63 (2009) 1995–1998

a range of particle size of 50–100 nm, observed by field emissionscanning electron microscopy (FE-SEM, JSM-6330F, JEOL Techniques,Tokyo, Japan). The HA powders were dispersed in a mixture of 75 vol.%de-ionizedwater and 25-vol.% ethanol. The amount of HApowder in thesuspensionwas varied from 0 to 30wt.%. Subsequently, the suspensionswere mixed with 0.15 M calcium acetate (Ca(CH3COO)2∙H2O, Sigma-Aldrich, USA) and 0.02 M glycerol phosphate calcium salt (CH2(OH)CH(OH)CH2OPO3Ca, Sigma-Aldrich, USA). Ammoniumhydroxide (NH4OH,Sigma-Aldrich, USA) was then added to adjust the pH to 11. The MAOtreatment was carried out in the prepared electrolyte solution byapplying a pulsed DC field (375 V) to the specimen for 3 min using apulsed DC power supply (Model-P6214, Auto Electric Co., Seoul, Korea).

The prepared samples were evaluated using several analysistechniques. The porosity of the samples was calculated by measuringtheir weight and dimensions. The pore structures andmicrostructureswere characterized by field emission scanning electron microscopy(FE-SEM). The crystalline phases and chemical compositions werecharacterized by X-ray diffraction (XRD) and energy dispersivespectroscopy (EDS) attached to the scanning electron microscope,respectively. For the compressive strength test, ~10×10×15 mmsamples were loaded axially at a crosshead speed of 5 mm/min usinga screw driven load frame (Instron 5565, Instron Corp., Canton, MA,USA). Seven samples were tested to obtain the average values and itsstandard deviation.

The biological properties of the samples produced in the electrolytesolution with various HA contents (0, 10, 20, and 30 wt.%) wereexamined using in vitro cell tests. The MC3T3-E1 cell line (ATCC, CRL-2593) was used to characterize the proliferation and differentiationbehavior of the cells. The pre-incubated cell lines were placed onto thesamples at a cell density of 1.5×104 cells/cm2, and cultured in ahumidified incubator containing 5% CO2 ant 37 °C. Minimum essentialmedium (α-MEM:Welgene Co., Ltd., Seoul, Korea) supplemented with10% fetal bovine serum (FBS, Life Technologies, Inc., USA) was usedas the culturing medium. The proliferation behavior was determinedusing 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, Promega, Madison, USA) for

mitochondrial reduction after culturing the sample for 5 days. Thequantity of the formazan product, which is measured by the absorbanceat 490 nm using a micro reader (Biorad, Model 550, USA), is directlyproportional to the number of living cells in the culture.

3. Results and discussion

The sponge replication method allowed the production of highlyporous Ti scaffolds with good interconnections between pores, asshown in Fig. 1 (A). In addition, it was observed that Ti walls could bedensified well without noticeable defects, such as pores and cracks, asshown in Fig. 1 (B). These results suggest that the sponge replicationmethod using a TiH2 slurry is quite useful for producing highly porousTi scaffolds. The sample had a reasonably high compressive strength of18±0.2 MPa at a porosity of approximately 70 vol.%.

The typical XRD result of the sample after heat-treatment at1300 °C for 2 h in a vacuum is shown in Fig. 2. Only peaks associatedwith the crystalline Ti phase (JCPDS file No. 44-1294) were observed,indicating the successful conversion of the TiH2 powders to Ti metallicphase via a two-step dehydrogenation process. However, it should benoted that secondary phases, including phosphate from TEP used asthe dispersant for example, might be present, though their amountwould be less than the detection limit of XRD.

The surfaces of the porous Ti scaffold were coated with a bioactive,microporous HA/TiO2 hybrid layer using the micro-arc oxidation(MAO) treatment. In particular, the MAO treatment was carried out inan electrolyte solution containing HA particles. The surface morphol-ogy was affected by the HA content used in the electrolyte solution, asshown in Fig. 3 (A)–(D). Without the addition of HA particles to theelectrolyte solution, the sample showed a typical morphology of asample treated by the MAO process, namely, a microporous TiO2 layerwas formed on the surface of the Ti strut (Fig. 3 (A)) [16,17]. On theother hand, when the HA content in the electrolyte was 10 wt.%, someHA particles appeared as the bright particles were deposited on thesurface of the Ti strut, while the microporous structure was preserved(Fig. 3 (B)). The deposition of HA particles becamemore vigorous withincreasing the HA content (Fig. 3 (C) and (D)).

Fig. 4 (A) shows a typical cross-sectional view of the HA/TiO2

hybrid coating layer produced with a HA content of 20 wt.% in theelectrolyte solution. All of the prepared samples showed a similarmorphology. The chemical composition across the HA/TiO2 hybridcoating layer, which is marked by an arrow in Fig. 4 (A), was examinedclosely by energy dispersive spectroscopy (EDS) line scan, as shown inFig. 4 (B). Three distinctively different layers were observed. In otherwords, a relatively strong peak for Ti was observed at the region nearthe Ti strut, indicating the formation of a TiO2 layer with negligible

Fig. 3. SEM micrographs of the samples after micro-arc oxidation (MAO) treatment in electrolyte solutions with HA contents of (A) 0, (B) 10, (C) 20, and (D) 30 wt.%, showing thesurface morphology.

1997J.-H. Lee et al. / Materials Letters 63 (2009) 1995–1998

incorporation of the HA particles (zone “I”). Beyond this region, strongpeaks for Ca, P, and O appeared as well as a peak for Ti, which indicatesthe considerable incorporation of the HA particles within the coating

Fig. 4. (A) Typical SEM image of the cross-section of the sample after the micro-arcoxidation (MAO) treatment in the electrolyte solution with a HA content of 20 wt.% and(B) EDS line scan crossing the coating layer, as indicated by the arrow in Fig. 3 (A).

layer (zone “II”). However, as the distance from the Ti strut wasincreased, the intensity of the peak for Ti decreased gradually, whilethe intensity of the peaks for Ca and P did not change significantly.This suggests that the outmost region of the coating layer consistsmainly of a HA phase with a small amount of TiO2 (zone “III”). Thisgradient in chemical composition of the HA/TiO2 hybrid coating layeris quite beneficial to bonding between the Ti strut and the coatinglayer.

The cell viability on the samples was measured using an MTS assayafter culturing for 5 days, as shown in Fig. 5. It was observed that thelevels of cell growth on the samples after the MAO treatment in theelectrolyte solutions containing the HA particles were higher than thaton the pure Ti scaffold. In addition, the cell growth level was increasedwith increasing the HA content in the electrolyte solution. Theseresults indicate that the bioactivity of the highly porous Ti scaffoldscould be improved significantly by creating the microporous HA/TiO2

Fig. 5. Cell viabilitymeasuredbyMTS assay after culturing 5 days on the samples producedby theMAO treatment in the electrolyte solutions with various HA contents (0,10, 20, and30 wt.%). For the purpose of comparison, the pure Ti scaffold was also tested.

1998 J.-H. Lee et al. / Materials Letters 63 (2009) 1995–1998

hybrid coating layer using the MAO treatment in the electrolytesolutions containing the HA particles.

4. Conclusions

Highly porous Ti scaffolds with a bioactive, microporous HA/TiO2

hybrid coating layer were fabricated. The porous Ti scaffolds producedusing the polymeric sponge replication method had a highly porousstructure with interconnected pores and a reasonably high compres-sive strength of 18±0.3 MPa at a porosity of approximately 70 vol.%.In addition, the surfaces of the Ti struts were coated successfully withthemicroporous HA/TiO2 hybrid layer using theMAO treatment in theelectrolyte solution containing HA particles. The hybrid coating layerconsistedmainly of three distinctively different regions, i.e. a TiO2-richregion, HA/TiO2 hybrid region, and HA-rich region, which promotedbonding between the Ti strut and the coating layer. As the HA contentwas increased, the incorporation of HA particles within the coatinglayer was enhanced. This led to significant improvement in theosteoblastic activity of the porous Ti scaffolds, which would make thepresent hybrid coating technique worthy of further investigation.

Acknowledgments

This researchwas supported by a grant from the Fundamental R&DProgram for Core Technology of Materials funded by the Ministry ofKnowledge Economy, Republic of Korea.

References

[1] Long M, Rack HJ. Biomaterials 1998;19:1621–39.[2] Niinomi M. Mater Sci Eng A 1998;243:231–6.[3] Wen CE, Yamada Y, Shimojima K, Chino Y, Asahina T, Mabuchi M. J Mater Sci: Mater

Med 2002;13:397–401.[4] Oh IH, Nomura N, Masahashi N, Hanada S. Scripta Mater 2003;49:1197–202.[5] BramM, Stiller C, Buchkremer HP, Stöver D, Baur H. Adv Eng Mater 2000;2:196–9.[6] Li JP, Habibovic P, van den Doel M, Wilson CR, de Wijn JR, van Blitterswijk CA, et al.

Biomaterials 2007;28:2810–20.[7] Chino Y, Dunand DC. Acta Mater 2008;56:105–13.[8] Yook SW, Yoon BH, Kim HE, Koh YH, Kim YS. Mater Lett 2008;62:4506–8.[9] Cachinho SCP, Correia RN. Powder Technol 2007;178:109–13.[10] Cachinho SCP, Correia RN. J Mater Sci: Mater Med 2008;19:451–7.[11] Zhao J, Lu X, Weng J. Mater Lett 2008;62:2921–4.[12] Karageorgiou V, Kaplan D. Biomaterials 2005;26:5474–91.[13] Reiner T, Kababya S, Gotman I. J Mater Sci: Mater Med 2008;19:583–9.[14] Negroiu G, Piticescu RM, Chitanu GC, Mihailescu IN, Zdrentu L, Miroiu M. J Mater

Sci: Mater Med 2008;19:1537–44.[15] Lopez-Heredia MA, Sohier J, Gaillard C, Quillard S, Dorget M, Layrolle P. Biomaterials

2008;29:2608–15.[16] Kim DY , Kim M, Kim HE, Koh YH, Kim HW, Jang JH. Acta Biomater (doi:10.1016/j.

actbio.2009.02.021).[17] Li LH, Kong YM, KimHW, KimYW,KimHE, Heo SJ, et al. Biomaterials 2004;25:2867–75.