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Biomaterials 31 (2010) 3465–3470

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Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

The electron beam deposition of titanium on polyetheretherketone (PEEK)and the resulting enhanced biological properties

Cheol-Min Han a, Eun-Jung Lee a, Hyoun-Ee Kim a,*, Young-Hag Koh b, Keung N. Kim c,Yoon Ha c, Sung-Uk Kuh d

a Department of Materials Science and Engineering and Hybrid Materials Division in WCU Program, Seoul National University, Seoul, 151-744, Republic of Koreab Department of Dental Laboratory Science and Engineering, Korea University, Seoul, 136-706, Republic of Koreac Department of Neurosurgery, Spine and Spinal cord institute, College of Medicine, Yonsei University, 120-752, Seoul, Republic of Koread Department of Neurosurgery, College of Medicine, Yonsei University, Yongdong Severance Hospital, Seoul, Republic of Korea

a r t i c l e i n f o

Article history:Received 17 November 2009Accepted 11 December 2009Available online 13 February 2010

Keywords:Polyetheretherketone (PEEK)Ti coatingBiocompatibilitye-beam

* Corresponding author. Tel.: þ82 2 880 7161; fax:E-mail address: kimhe@snu.ac.kr (H.-E. Kim).

0142-9612/$ – see front matter � 2009 Elsevier Ltd.doi:10.1016/j.biomaterials.2009.12.030

a b s t r a c t

The surface of polyetheretherketone (PEEK) was coated with a pure titanium (Ti) layer using an electronbeam (e-beam) deposition method in order to enhance its biocompatibility and adhesion to bone tissue.The e-beam deposition method was a low-temperature coating process that formed a dense, uniformand well crystallized Ti layer without deteriorating the characteristics of the PEEK implant. The Ti coatinglayer strongly adhered to the substrate and remarkably enhanced its wettability. The Ti-coated sampleswere evaluated in terms of their in vitro cellular behaviors and in vivo osteointegration, and the resultswere compared to a pure PEEK substrate. The level of proliferation of the cells (MC3T3-E1) was measuredusing a methoxyphenyl tetrazolium salt (MTS) assay and more than doubled after the Ti coating. Thedifferentiation level of cells was measured using the alkaline phosphatase (ALP) assay and also doubled.Furthermore, the in vivo animal tests showed that the Ti-coated PEEK implants had a much higher bone-in-contact (BIC) ratio than the pure PEEK implants. These in vitro and in vivo results suggested that thee-beam deposited Ti coating significantly improved the potential of PEEK for hard tissue applications.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Polyetheretherketone (PEEK) is a semi-crystalline thermoplasticthat exhibits outstanding mechanical and chemical properties, aswell as a high thermal stability. Therefore, PEEK can widely be usedin diverse number of fields, such as the aerospace, automotive, andchemical process industries. PEEK, which was approved as a medicalgrade material by the U.S. FDA in the late 1990s, has recently beenstudied and used as a substitute for metallic implant materialsbecause of its appropriate biocompatibility and extremely lowelastic modulus (3w4 GPa) [1–5], which reduces the extent of stressshielding that is often observed in titanium-based metallic implants[6,7].

Despite its good mechanical properties, the adhesion of PEEKimplants to bone tissue proceeds slowly because of their relativelylow biocompatibility [3]. The biocompatibility of the implant isstrongly affected by its surface characteristics, including the surfaceroughness, wettability and chemical composition [8]. Therefore,

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All rights reserved.

considerable efforts have focused on modifying the surface of thePEEK implants. A representative example of the surface modifica-tions of the PEEK implant is the introduction of bioactive coatingmaterials using various physical and chemical methods, includingionic plasma deposition (IPD) [9], plasma spray deposition [10] andin vitro precipitation [11,12]. This coating layer must be biologicallybeneficial, physically stable and mechanically adherent to the PEEKsubstrate. Additionally, the coating process must be carried out atrelatively low temperatures in order to avoid thermal degradationof PEEK.

Titanium (Ti) is the most widely used implant material for load-bearing dental and orthopedic applications because of its excellentmechanical and biological properties [13,14]. Especially, in terms ofbiocompatibility, Ti is only surpassed by bioactive ceramics, such ashydroxyapatite or bioglass, and natural biopolymers, such ascollagen or its derivatives. Therefore, Ti is a strong candidate as thecoating material for PEEK implants. In this research, the electronbeam deposition (e-beam deposition) process was used to coat Tionto PEEK in order to enhance its biocompatibility. The e-beamdeposition method is a versatile coating technique that producesa dense, uniform film on any substrate at a low temperature [15,16].Even though the Ti was deposited onto PEEK at a relatively low

Fig. 1. Scanning electron microscopy (SEM) images of the surface of (A) as-machinedand (B) Ti-coated PEEK and (C) the cross-section of Ti-coated PEEK.

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temperature, the Ti was fully crystallized without the need for anypost-deposition heat treatment.

The physical properties of the Ti coating layer were evaluated interms of microstructure, crystallinity, wetting angle and bondingstrength with respect to the PEEK substrate. The effects of the Ticoating layer on the biological properties of PEEK were assessedthrough in vitro and in vivo tests. More specifically, the in vitrocellular responses of the samples were evaluated in terms of cellattachment, proliferation, and osteoblastic differentiation. The invivo bone conductivity was examined by measuring the bone-to-implant contact (BIC) ratio using a rabbit tibial defect model fora period of 4 weeks.

2. Materials and methods

2.1. Electron beam deposition (e-beam deposition)

Disc shaped PEEK (PEEK-OPTIMA�, Invibio, UK) substrates with a diameter of15 mm and a thickness of 2 mm were prepared, polished with a 2000-grit SiCabrasive paper, and then ultrasonically cleaned. Commercially pure Ti plates (KaheeMetal, Korea), with dimensions of 10 mm� 10 mm� 1 mm, were prepared asa target material, ground with a 220-grit SiC abrasive paper, and then ultrasonicallycleaned. All of the ultrasonic cleaning processes were successively conducted for3 min each in acetone, ethanol and distilled water. A thin Ti film was deposited ontothe PEEK surface using an e-beam evaporator (EVACO-EB800R, DR Vacuum Inc.,Korea). The prepared substrate was mounted on a rotating holder in a vacuumchamber and cleaned with an Ar ion beam with a voltage of 90 V and a current of1.5 A for 20 min before coating. Then the Ti film was coated on the PEEK substrate toa film thickness of 1 mm at a rate of w0.05 nm/s. The temperatures for the Ar ionbeam cleaning and titanium coating processes were approximately 90 �C and120 �C, respectively. During the coating process, the substrate holder was rotated at5 rpm to achieve a uniform thickness.

2.2. Characterization

The microstructures and phases of the specimens were evaluated using scan-ning electron microscopy (SEM; JSM-6360, JEOL, Tokyo, Japan) and X-ray diffraction(XRD; M18XHF-SRA, MacScience Co., Yokohama, Japan) analyses, respectively. Thewettability of both of the specimens was evaluated using the sessile drop method.Distilled water drops were applied onto the surface of both specimens and photo-graphed with a CCD camera that was connected to a goniometer (Phoenix 300,Surface Electro Optics Co. Ltd., Korea), so that the contact angle could be calculated.The bond strength between the Ti film and PEEK was determined using tensile tests(Sebastian V, Quad Group, Spokane, WA, USA). During these tests, an aluminum studwas mounted onto the specimen with an epoxy glue and hardened using a heattreatment for 1 h at 150 �C. The load at failure was measured to determine the bondstrength.

2.3. Biological properties

2.3.1. In vitro testsThe biological properties were evaluated using both in vitro cell tests with an

MC3T3-E1 cell line (ATCC, CRL-2593) and in vivo animal tests. For the in vitro celltests, the specimens were sterilized overnight using UV irradiation. The pre-incu-bated cell line was placed onto the specimens at densities of 5�104, 2�104 and1.5�104 cells/cm2 for the cell attachment, proliferation and differentiation tests,respectively, and then the cells were cultured in a humidified incubator with 5% CO2

at 37 �C. An alpha-minimum essential medium (a-MEM, Welgene Co., Ltd., Korea)that was supplemented with 5% fetal bovine serum (FBS) and 1% penicillin-strep-tomycin was used as the culturing medium, and 10 mM b-GP and 50 mg/ml ascorbicacid were added for the alkaline phosphatase (ALP) test. The attached cells wereobserved using confocal laser scanning microscopy (CLSM, Zeiss-LSM510, Carl ZeissInc., NY, USA). After 3 h of culturing on the specimens, the cells were fixed with 4%paraformaldehyde in phosphate buffered saline (PBS) for 10 min, washed in PBS, andpermeabilized with 0.1% Trion X-100 in PBS for 5 min. Then the cells were rewashedin PBS and stained with fluorescent phalloidin for 20 min. The proliferation behaviorof the cells was determined over a 5-day period using the MTS (methoxyphenyltetrazolium salt) method. The quantity of the formazan product was directlyproportional to the number of living cells and was quantified with the absorbancemeasurements that were taken at 490 nm using a micro-reader (Biorad, Model 550,USA). The degree of differentiation of the cells was assessed by measuring the ALPactivity using p-nitrophenyl phosphate (pNPP) (Sigma–Aldrich, UK). During thistest, pNPP was converted into p-nitrophenol (pNP) in the presence of ALP at a ratethat was proportional to the ALP activity. The production of pNP was determinedusing the absorbance that was measured at 405 nm using a micro-reader after 7 and

14 days of incubation. The experimental data for the biological tests were expressedas means� standard deviations (SD) for n¼ 3.

2.3.2. In vivo testsThe in vivo animal tests were carried out on five male New Zealand white rabbits

(12 weeks, average weight 3 kg). During the in vivo tests, PEEK screws witha diameter of 3.4 mm, thread length of 4.5 mm and total length of 6 mm wereprepared. Only one side of the PEEK screw was coated with a 1 mm Ti film in order tocompare the osteointegration of the two materials in the same defect. A combina-tion of 1.5 cc of 2% Xylazine HCl (Rompun, Bayer Korea, Korea) and 0.5 cc of Tilet-amine HCl (Zoletil, Virbac lab, France) was used as the general anesthesia, andLidocaine (Yuhan Corporation, Korea) with 1:100,000 epinephrine was injected asthe local anesthesia. Tibial defects, with a diameter of 3.4 mm, were created on eachof the hind legs using a hand piece drill. The half-coated PEEK screws wereimplanted into both of the defects for each of the five rabbits. After surgery, the

Fig. 2. X-ray diffraction (XRD) patterns of (A) as-machined and (B) Ti-coated PEEK: C

stands for PEEK and - for Ti.

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wounds were sutured with Surgisorb (Samyang Ltd, Korea) and then cephradine(Bayer Korea, Korea), an antibiotic, was injected into the rabbits for 3 days. Fourweeks after implantation, the rabbits were sacrificed, the extracted defects werefixed in a 10% neutral formaldehyde solution, and blocks were made using the resin.The digital images of the resin block sections that were stained with Masson-Trichrome and haematoxylin–eosin were obtained using Axioskop microscopy(Olympus BX51, Olympus Corporation, Tokyo, Japan) The bone-in-contact (BIC)ratios were calculated from the images using a digital image analysis program (SPOT,Diagnostic instrument, Inc., MI, USA) with two different analytical techniques; a) theabsolute BIC ratios of the bare and Ti-coated specimens and b) a relative BIC ratio ofthe Ti-coated PEEK specimen with respect to bare PEEK. In this system, themeasurement area of the BIC ratio was limited to the region where the originalcortical bone existed. The statistical analysis was performed using one-way analysisof variance (ANOVA) test, with a level of significance of p< 0.05.

Fig. 3. Confocal laser scanning microscopy (CLSM) images of the MC3T3-E1 cellscultured on (A) as-machined and (B) Ti-coated PEEK for 3 h.

Fig. 4. Cell viability of the MC3T3-E1 cells cultured on as-machined and Ti-coatedPEEK for 5 days (**: p< 0.001).

3. Results

3.1. Ti coating layer

Fig. 1(A–C) show that a dense, uniform Ti layer was coated onthe surface of the PEEK substrate using e-beam deposition. Thesurface of the as-machined PEEK substrate exhibited some sharpmachining grooves, in Fig. 1(A). The surface of the Ti coating layerwas relatively smooth without any noticeable defects, such ascracks and large voids, in Fig. 1(B). Furthermore, the Ti coating layerwas very uniform with a thickness of 1 mm throughout the sampleand exhibited a strong adhesion to the PEEK substrate, in Fig. 1(C).Additionally, the thickness of the Ti coating layer was controllableby simply adjusting the coating time.

The crystalline phases of the as-machined PEEK and Ti-coatedPEEK substrates were examined using XRD analysis, in Fig. 2(A) and(B), respectively. As expected, the pure PEEK substrate onlyexhibited peaks associated with the crystalline PEEK phase(Fig. 2(A)) [17]. After the Ti coating, the PEEK substrate showedadditional peaks at 2q¼ 35�, 38� and 40�, which corresponded tothe Ti peaks, suggesting that the high crystalline Ti phase wasformed on the surface of the PEEK substrate even without any postheat treatment, which was one of the most striking advantages ofe-beam deposition. The Ti coating layer had a preferred orientationfor the (002) plane (2q¼ 38�), presumably, because of its lowsurface energy characteristics [18,19].

The wettability of the samples was evaluated by measuring thecontact angle with a goniometer. The contact angles of theas-machined and Ti-coated PEEK substrates were 71�5.1� and

54� 2.4�, respectively, which matched well with the results foundin previous literature [20,21]. These observations indicated that thewettability of the PEEK substrate was significantly improved by theTi coating layer.

Fig. 5. Degree of differentiation of the MC3T3-E1 cells cultured on bare and Ti-coatedPEEK for 7 and 14 days (**: p< 0.001).

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3.2. Biological properties

The biological properties of the as-machined and Ti-coatedPEEK were evaluated using in vitro cell tests and in vivo animal tests.The in vitro cellular responses of the samples were assessed interms of the initial cell attachment, proliferation, and osteoblasticdifferentiation. The CLSM images of the MC3T3-E1 cells cultured onthe samples for 3 h are shown in Fig. 3(A) and (B). Both of thesamples revealed good cell attachment, where the red colorrepresents the actin in the cells, indicating good biocompatibility.However, the cells appeared to grow and spread more actively onthe Ti-coated PEEK substrate (Fig. 3(B)) than the pure PEEKsubstrate (Fig. 3(A)).

The level of proliferation of the cells that were cultured for 5days was determined using the MTS method, in Fig. 4. The twospecimens exhibited a significant difference in the levels of prolif-eration (p< 0.001). The number of living cells on the Ti-coatedPEEK surface was more than twice of that on the as-machined PEEKsurface. The ALP activities of the cells that were cultured for 7 and14 days on the samples are shown in Fig. 5. The Ti-coated PEEKsubstrate showed a significantly higher ALP level (p< 0.001) thanthe as-machined PEEK substrate. The ALP activity of cells that werecultured on Ti-coated PEEK for 7 days was even higher than thecells cultured on as-machined PEEK for 14 days. These observationsclearly indicated that the Ti coating layer remarkably improved thecellular response of the PEEK substrate.

The in vivo bioactivity was assessed using the rabbit tibial defectmodel. In Fig. 6(A), half of a PEEK screw was coated with Ti ata thickness of 1 mm in order to closely examine the efficacy of the Ticoating layer. The Ti-coated region appeared dark in contrast,which made it easy to distinguish between the two halves of thescrew even after implantation into rabbits, in Fig. 6(B).

Fig. 7(A) and (B) shows the histological images of the stainedsections of the bare PEEK and the Ti-coated sides of the screw,respectively, where the pink colored areas corresponded to the bonetissue, and the dark areas were the implant material. Boundariesbetween the new bone (indicated as ‘NB’) and the old bone (indi-cated as ‘OB’) was marked with dashed lines. Both sides showedsimilar regeneration behavior, that is, the spaces between screwthreads were almost completely filled with new bone. However, thebare PEEK side showed gaps between the new bone and the implantmaterial surface, which were indicated by arrows, whereas theTi-coated side exhibited rather tight contact with the bone. Basedon these images, the bone-to-implant contact (BIC) ratios werecalculated using an image analysis program and statistically treated

Fig. 6. Optical images of the half-coated PEEK screws

in Fig. 8. The absolute BIC ratios of the bare and Ti-coated specimensare shown in Fig. 8 (A). The Ti-coated side showed a significantlyhigher BIC ratio (p< 0.05) compared to the bare PEEK side., Addi-tionally, a relative BIC ratio (the BIC ratio of Ti-coated side comparedto the bare side of the same specimen) was calculated to minimizethe statistical uncertainties that originated from the individualanimals. In Fig. 8(B), the relative BIC ratio of the Ti-coated specimenwith respect to the bare PEEK more clearly exhibited the effect thatthe Ti coating had on PEEK. The relative BIC ratio was almost 2, witha p value less than 0.001. These in vitro and in vivo tests suggestedthat e-beam coating of Ti was a very promising method forenhancing the biocompatibility of PEEK implant materials.

4. Discussion

PEEK is recognized as a good implant material in hard tissueengineering because of its good mechanical properties, low densityand good chemical resistance. However, its relatively low adhesionto bone tissue has limited its wider application. Many studies havebeen steadily conducted to overcome the limitations of PEEK.Coating PEEK with a bioactive substance is a very effective tech-nique for enhancing the biocompatibility of PEEK, while main-taining its other advantages [9–12]. In this study, a thin Ti layer wascoated onto a PEEK substrate using the e-beam deposition method,and the effects of the coating on the biomedical performance ofPEEK were evaluated through in vivo and in vitro tests.

(A) before implantation and (B) after harvesting.

Fig. 7. Histological images of the stained sections of the (A) bare and (B) Ti-coatedsides of the half-coated PEEK screws that were harvested from rabbits 4 weeks afterimplantation. M stands for material, OB for old bone and NB for new bone. The dashedlines indicate the boundary between the new and old bone, and the arrows indicatethe interfaces between the new bone and the implant.

Fig. 8. A) Bone-to-implant contact (BIC) ratio of the bare and Ti-coated PEEK sides ofthe screws that were removed from the rabbits 4 weeks after implantation (*p< 0.05)and B) relative BIC ratio of the Ti-coated side with respect to the bare side of the samePEEK screw specimen.

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In hard tissue engineering, bioceramics, such as HA, tricalciumphosphate (TCP) and bioactive glasses, are representative coatingmaterials for enhancing the biocompatibility of metallic or poly-meric implants. However, in most cases, these materials requirea post-coating heat treatment to ensure their stability through thecrystallization [15,16]. Although PEEK has a certain degree ofthermal resistance, required heat treatments are very likely toadversely affect the PEEK substrate. In contrast, Ti crystallizesperfectly during the deposition process without any heat treatment[18,19]. The simple crystal structure of metallic Ti is directly relatedto the ease of the crystallization. Therefore, coating Ti throughe-beam deposition is a plausible approach for PEEK.

The main benefits of the e-beam deposition of Ti noted in thisstudy were the undamaged substrate and the good stability of thecoating layer, which were confirmed using SEM, XRD and bondingstrength analysis. SEM revealed that the Ti layer coated onto PEEKwas dense, smooth, uniform and crack-free without any trace ofsubstrate damage. The as-machined PEEK substrate showed thetypical XRD patterns of partially crystallized PEEK peaks. After Ti

coating, the PEEK peaks remained unchanged and no secondaryphase was observed. The bond strength between the Ti layer andthe PEEK substrate was in the range of 20 MPa, which was highenough for use as a biomedical coating layer.

The deposition rate also played an important role during thecoating process because it affected the temperature inside thevacuum chamber. In this system, the deposition rate was controlledusing the electron emission current, and the chamber temperatureincreased with increasing current. The crystallinity and mechanicalproperties of the PEEK substrate degraded if the deposition ratewas too high because the temperature inside the chamber excee-ded the glass transition temperature of PEEK (about 145 �C) [22].Care was taken to keep the temperature inside the chamber below80 �C by controlling the deposition rate.

All of the in vitro test results showed significant improvementsin the biocompatibility after Ti coating. First, the cells more widelyspread on the Ti-coated PEEK than as-machined PEEK. Similartendencies were observed for the level of proliferation that wasmeasured using the MTS method and the level of differentiationthat was evaluated from the ALP activity. Both the MTS and ALPactivities of the cells were more than doubled after Ti coating. TheALP activity of the cells cultured on the Ti-coated PEEK for 7 dayswas even higher than the cells cultured on the as-machined PEEKcultured for 14 days. In general, the wettability and cellularbehavior have a close relationship [23,24]. In this study, the contactangle changed from 72� to 54� after Ti coating, suggesting that not

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only the chemical composition of the PEEK surface, but also itswettability contributed to the enhanced biocompatibility.

The effects of the Ti layer on the osteoconductivity of theimplant were investigated through in vivo animal tests using therabbit tibia model. The half-coated PEEK screws exhibited differentdegrees of contact with the bone depending on the side in thehistological images. The Ti-coated surface showed a significantlyhigher BIC ratio than the bare PEEK surface (P< 0.05). The effects ofTi coating on the osteoconductivity were even more conspicuouswhen the relative BIC ratio was calculated. The contact between thenew bone and the implant material was influenced by the implantsurface characteristics [25–27]. In this present research, the twosides of the screw were identical except for the Ti coating. There-fore, the relative BIC ratio in Fig. 8(B) only represented the effects ofthe Ti coating only. These in vivo test results, along with thewettability and in vitro test results, confirmed the biocompatibilityof the PEEK materials was improved significantly by the Ti coatinglayer using e-beam deposition.

5. Conclusions

The biocompatibility of PEEK was remarkably improved aftera Ti coating was added to the surface using the e-beam depositiontechnique. The Ti coating layer was dense, uniform and stronglyadhered to the PEEK substrate. The wettability of PEEK was alsoenhanced because of the Ti coating. The level of in vitro cellularresponses, determined using MTS and ALP assays, more thandoubled after Ti was coated onto the PEEK substrate. The in vivoanimal tests also showed that the Ti coating enhanced the osteo-conductivity of PEEK. These in vitro and in vivo results suggestedthat the e-beam deposited Ti coating significantly improved thepotential of PEEK for hard tissue applications.

Acknowledgments

This research was supported by a grant from the FundamentalR&D Program for Core Technology of Materials funded by theMinistry of Knowledge Economy, Republic of Korea.

Appendix

Figures with essential color discrimination. Figs. 3, 6 and 7 inthis article are difficult to interpret in black and white. The full colorimages can be found in the on-line version, at doi:10.1016/j.biomaterials.2009.12.030.

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