- Email: [email protected]
In vivo evaluation of modified titanium implant surfaces produced using a hybrid plasma spraying processing
Materials Science and Engineering C 20 (2002) 117 – 124 www.elsevier.com/locate/msec
In vivo evaluation of modified titanium implant surfaces produced using a hybrid plasma spraying processing Yunzhi Yang a,*, Joo L. Ong a, Jiemo Tian b a
Department of Restorative Dentistry, Division of Biomaterials, University of Texas Health Science Center at San Antonio, MSC 7890, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA b State Key Laboratory of New Ceramics and Fine Processing, Tsinghua University, Beijing, 100084, China
Abstract In this study, the biological responses to surface-modified titanium (Ti) was investigated using a dog model. Titanium plasma spraying and ion implantation of amino (NH2+ ) groups were used as means of modifying Ti surfaces. Characterization of the modified Ti surfaces was performed using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and scanning Auger electron spectroscopy. In vivo evaluations were performed using fluorescence microscope, scanning electron microscope and energy disperse spectroscopy. It was observed in this study that ion-implanted porous-graded titanium coatings had a thick surface oxide layer, containing a small amount of nitride. In vivo study indicates direct bone contact between surface-modified Ti implants and osseous tissues. In addition, osseous tissues were observed to grow into the pores inside the coatings, thereby allowing the formation of a gradual calcium phosphate interface layer. It was concluded from this study that ion implantation of Ti surfaces with amino groups, induced higher concentration of calcium and phosphorus precipitation and more mineralization as compared to non-ion-implanted Ti surfaces. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Titanium; Surface modification; Plasma spraying; Ion implantation; Animal study
1. Introduction
2. Materials and methods
Titanium (Ti) and its alloys have been widely used as implants in dental and orthopedic applications. It is also known that the Ti surface plays an important role in governing the biological activity at the bone – implant interface [1,2]. To date, several surface modification methods, such as calcium phosphate or oxide coating [3– 5], ion implantation [6– 10] and alkali treatment [11 –16], have been employed as means to improve the biocompatibility, corrosion and wear and fatigue resistance of Ti. In addition to the reported methods for modifying Ti surfaces, our laboratory has also investigated and characterized the porous plasma-sprayed Ti coatings [17] and Ti surfaces ion-implanted with an amino group [18] produced. Our previous study using osteoblastlike cells had also suggested excellent biocompatibility when cultured on porous plasma-sprayed Ti coatings and Ti surfaces ion-implanted with an amino group [19]. In this study, the biological response to porous plasma-sprayed Ti coatings and Ti surfaces ion-implanted with an amino group was evaluated in vivo.
2.1. Materials preparation
*
Corresponding author. E-mail address: [email protected] (Y. Yang).
For this study, four different Ti surfaces were evaluated. The surfaces were: (1) sandblasted Ti (ST); (2) plasmasprayed porous Ti (PST); (3) sandblasted and ion-implanted Ti (SIT); and (4) plasma-sprayed and ion-implanted Ti (PSIT). The ST surfaces were prepared by sandblasting commercially pure titanium (99.3%, Chinese Nonferrous Metal Institute) with 46# Al2O3 particles. The samples were then cut into square plates (4 4 2 mm3) followed by ultrasonic cleaning in reagent-grade alcohol and acetone [17 –19]. The PST surfaces were prepared by sandblasting commercially pure titanium (99.3%, Chinese Nonferrous Metal Institute) with 46# Al2O3 particles. The sandblasted samples were then heat treated at 400– 500 jC for 3 –4 min, followed by sputter-cleaning with argon ions for 3 – 4 min. A porousgraded Ti coating was then deposited on the Ti surface using an A-3000S plasma spraying equipment and commercially pure Ti powder (99.3%, Chinese Nonferrous Metal Institute) of different sizes (100, 200 and 300 mesh). The base vacuum in the spraying chamber was maintained at 0.1 Pa. Spraying
0928-4931/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 ( 0 2 ) 0 0 0 2 1 - 8
118
Y. Yang et al. / Materials Science and Engineering C 20 (2002) 117–124
distance between the substrate and nozzle was at 300 mm, and the spraying pressure was maintained at 10 kPa. A mixture of argon (55 l/min) and hydrogen (5.5 l/min) was used as the plasma gas, and argon (1.2 l/min) was used as the transport gas for the powder. Using a spraying power of 30– 40 kW, graded pores of the coatings were produced by regulating the spraying powder and spraying power. Spraying time was used to regulate the coating thickness. After coating, the samples were cut into square plates (4 4 2 mm3) followed by ultrasonic cleaning in reagent-grade alcohol and acetone [17 – 19]. The SIT and PSIT surfaces were prepared by ion implanting an amine group (NH2 + ) to ultrasonically cleaned ST and PST surfaces. Ion implantation of the amine group was performed to a dose of 1017 cm 2 using a 400-keV ion implanter [18,19], a base pressure of V 10 4 Pa and a working pressure of f 1.0 10 3 Pa. Energy used during implantation was at 1017 cm 2 and 100 keV. 2.2. Materials characterization X-ray diffraction (XRD) analysis was used to characterize the structure of surface-modified Ti surfaces. With Cu Ka radiation, and an energy of 40 kV and 120 mA, the samples were analyzed using a Rigaku X-ray diffractometer (Rigaku D/max-RB). Using Al Ka radiation, X-ray photoelectron spectroscopy (XPS, Perkin-Elmer PHI 5300) was used to analyze the surface composition of the samples. At a sputtering rate of 4 nm/min using argon, XPS depth profiling was performed to evaluate the subsurface composition of the Ti. The sputtering rate was calibrated using thermal oxide SiO2. 2.3. In vivo study Three adult male beagle dogs (10 – 13 kg) were used. Prior to implantation, the four different Ti surfaces were sterilized in an autoclave. A total of 30 implants (6 ST, 6 PST, 9 SIT and 9 PSIT) were used in the study. During the surgery, the animals were anesthetized by intraperitoneal injection of a 3% nembutal (50 mg/kg). A longitudinal incision was then made on the medial surface of the leg. The femur was exposed, and five 4 2-mm holes were drilled up to the bone marrow space using intermittent
Fig. 1. Exposed femur showing implant sites.
Fig. 2. Representative XRD patterns of different surface-modified Ti surfaces.
drilling and cooling saline. The distance between the holes was maintained at 15 mm. Implantation positions were shown in Fig. 1. Ten implants (2 ST, 2 PST, 3 SIT and 3 PSIT) were inserted in the left and right femur. Following the placement of implants, the soft tissues were closed. After implantation, penicillin was administered intramuscularly twice daily and for 3 days. Bone –implant interface was evaluated at 2, 4 and 8 weeks after implantation. Seven days prior to the sacrifice, tetracycline (50 mg/kg) was administered to label newly formed bone. After sacrificing the animals, the implants with their surrounding tissues were excised immediately followed by radiographs. After excision, the specimen blocks were fixed in 10% neutral formalin and dehydrated with serially graded alcohol. Using a diamond blade, the specimens were subsequently thick-sectioned transversely with a low-speed cutting machine (ISOMET, BUEHLER, USA). The specimens were then polished to approximately 100 Am and viewed using a fluorescence microscope (FM, UFX-II Nikon). Composition at the implant – bone interface was also evaluated using an energy dispersive spectroscopy (EDS, Hitachi PV9100) attached to a scanning electron microscope (SEM, Hitachi X-650). Prior to evaluation, the sections were sputter coated with aluminum. Aluminum coating was used instead of gold because of the overlapping of the phosphorus peak with the gold peak. Calcium, phosphorus and titanium ele-
Fig. 3. Representative XPS survey spectra of surface-modified Ti surfaces.
Y. Yang et al. / Materials Science and Engineering C 20 (2002) 117–124
119
To qualitatively evaluate bone –implant adhesion strength, bone – implant blocks were fixed, dehydrated and dried using a critical point dryer. The blocks were then fractured at the bone – titanium interface and divided into two groups. On one group of fractured blocks, the implants were removed and the bone block was evaluated using a scanning electron microscope. On the other group of fractured blocks, the two fractured surfaces (one fractured block containing bone and the other fractured block containing bone and implant) were then sputter-coated with gold and evaluated using a scanning electron microscope (SEM, AMRAY-1000).
3. Results 3.1. Materials characterization X-ray diffraction patterns of Ti powder, Ti substrates, ST, SIT, PST and PSIT are shown in Fig. 2. The diffraction peaks of PSIT were mainly oxide (Ti2O), whereas the other Ti materials were mainly metallic Ti in a hexagonal crystal. XRD also suggested that plasma spraying and ion implantation oxidized the Ti surface, thereby forming a thicker layer of oxide film. The other Ti surfaces were only slightly oxidized by the modification processing. A typical XPS survey spectrum of surface-modified Ti is shown in Fig. 3. Oxygen (O) and Ti were predominantly observed on the outermost surface of all Ti, tested with adventitious carbon (C) adsorbed on the surface. Fig. 4 shows the typical XPS depth profile of Ti and O on all surfacemodified Ti. Trace amounts of nitrogen (N) was observed for all ion-implanted Ti surfaces. The relative concentration of O was reduced as the relative concentration of Ti was increased during XPS depth profiling. Fig. 4. XPS depth profile of surface-modified Ti surfaces.
3.2. In vivo study ments were measured on three randomly selected interfaces per group. Differences in compositions were statistically compared using ANOVA and were considered statistically significant if p < 0.05.
Gross evaluation of the excised bone after 2 and 4 weeks implantation revealed an incomplete healing of the bone at the sites where the bone was drilled for implant placement. However, the implants were observed to be closely sur-
Fig. 5. Representative radiographs of implants in dog femora at (a) 2 weeks, (b) 4 weeks and (c) 8 weeks after implantation.
120
Y. Yang et al. / Materials Science and Engineering C 20 (2002) 117–124
rounded by newly formed bone at the implant site after 2 and 4 weeks implantation. Gross evaluation of the excised bone after 8 weeks implantation indicated a complete healing of the bone at the site where the bone was drilled for implant placement. The newly formed bone was similar to the old bone, and as a result of complete bone coverage at the implant site, the implants were completely covered by the bone after 8 weeks implantation. Representative radiographs at 2, 4 and 8 weeks after implantation are shown in Fig. 5a –c. As observed from the radiographs, shadows around the implants were undetectable, suggesting that the implants were in close contact with the newly formed bone. Since the implants were completely covered by the bone after 8 weeks implantation, drilling of the bone was performed to identify the location of the implants. As a result of the drilling, horizontal shadows were
Fig. 6. Representative fluorescence micrographs of a drilled bone site on the femur for implant placement and tissues around the implants at (a) 2 weeks, (b) 4 weeks and (c) 8 weeks after implantation (original magnification, 40). NB = newly formed bone, PB = pre-existing bone.
Fig. 7. Representative fluorescence micrograph of the PSIT implant – bone interface at 8 weeks after implantation (original magnification, 100 (arrowhead = NB).
observed for the radiograph of the femur after 8 weeks implantation (Fig. 5c). From the fluorescence microscopy, Ti implants within the same implantation period exhibited no significant differ-
Fig. 8. Interface morphology between specimen and bone at 8 weeks after implantation. (a) PST (original magnification, 250), with arrowheads indicating newly formed bone; (b) PSIT (original magnification, 250), with arrowheads indicating newly formed bone; (c) PST (original magnification, 500), with arrowheads indicating a thin gap at the bone – implant interface; (d) PSIT (original magnification, 500), with arrowheads indicating newly formed bone. M = implant; B = bone. The thin gap observed in the interface of PST (c) was an artifact produced during the preparation process. In all these micrographs, the parallel marks on the metallic Ti were attributed to the sectioning of the implant – bone blocks using a diamond blade.
Y. Yang et al. / Materials Science and Engineering C 20 (2002) 117–124
Fig. 9. SEM-EDS of the interface between PSIT and bone: (a) elemental analysis of Ca and Ti at 2 weeks after implantation, and (b) that of P at 2 weeks after implantation, and (c) that of Ca and Ti at 8 weeks after implantation, and (d) that of Ca and P at 8 weeks after implantation.
ence between the four groups of Ti tested. Fig. 6a – c shows the typical fluorescence micrographs of a drilled bone site for implant placement as well as the newly formed bone around the implant at 2, 4 and 8 weeks after implantation. At 2 and 4 weeks after implantation (Fig. 6a and b), newly
121
formed bone was observed around the implant. In addition, the newly formed bone and the pre-existing bone were distinctly different. At 8 weeks after implantation (Fig. 6c), there was no distinction between the newly formed bone and the pre-existing bone. The newly formed bone was observed to be concentrated on the Haversian canal after 8 weeks implantation, suggesting that the drilled bone site for implant placement had healed. The fluorescence data was in agreement with the gross observation and the radiographs. Fig. 7 shows the fluorescence micrograph of the interface between PSIT surface and the surrounding tissue at 8 weeks after implantation. At higher magnification, the newly formed bone was observed to grow into the pores of PSIT, thereby suggesting mechanical interlocking between the PSIT and bone. As shown in Fig. 8a and b, EDS analysis of the porous PST and PSIT surfaces indicates the presence of calcium (Ca) and phosphorus (P), suggesting bone ingrowth within the pores. The Ca/P ratio of the bone ingrowth with the pores of PST and PSIT surfaces was 1.87 and 1.74. The newly formed bone was observed to have reached pores in the depth of 100– 200 Am from the surface and provide mechanical interlock between the implants and the bone. This mechanical interlocking suggested the mechanism for stabilizing Ti implants evaluated in this study. Intimate bone contact with the surface-modified Ti was also shown in Fig. 8c and d. SEM-EDS analysis in Fig. 9 further confirmed that the newly formed bone was in intimate contact with implant surfaces as well as bone growth in the pores of PSIT. Interfacial line analysis from the implant to the bone revealed increasing concentration of Ca and P as the concentration of Ti decreased, suggesting the presence of a Ca – P layer at the PSIT implant – bone interface. The thickness of the Ca – P layer was observed to increase from about 4 –6 Am at 2 weeks
Fig. 10. A representative SEM micrograph showing the three different analyzed region (5 5 Am). On the right is a schematic drawing of interface microanalysis. 1 = implant; 2 = implant – bone interface; 3 = bone.
122
Y. Yang et al. / Materials Science and Engineering C 20 (2002) 117–124
Fig. 11. Bar charts of EDS microanalysis in regions 1, 2 and 3 of ST, PST, SIT and PSIT surfaces after 4 and 8 weeks implantation. Asterisk showed significant differences between ion-implanted and non-ion-implanted surfaces ( p < 0.05).
after implantation, to about 30 Am at 8 weeks after implantation. In addition, the relative concentration of modified, porous implant surface and nonmodified titanium surface (Ti/(Ca + P + Ti)) at the bone – implant interface was 2 – 4 at.%, indicating no significant difference in the titanium ions released from the modified and nonmodified Ti surfaces. As shown in Fig. 10, elemental concentrations of Ca, P and Ti at three different bone –implant interfacial regions (region 1 = implant, region 2 = implant –bone interface and region 3 = bone) were quantified. Fig. 11 shows the elemental concentration of Ca, P and Ti at 4 and 8 weeks after implantation. The Ca/P ratio of the bone –implant interfaces after 4 and 8 weeks implantation is shown in Table 1. In agreement with the interfacial line analysis, the concentration of Ca and P increased, and the concentration of Ti decreased as the analysis progressed from regions 1 to 3 (implant to bone). No significant difference in the concentration of Ca and P in the ST implant and PST implant (region 1) was observed as the implantation period was increased from 4 to 8
weeks. However, a significant increase in the concentration of Ca and P in the SIT implant and PSIT implant (region 1) was observed as the implantation period was increased from 4 to 8 weeks. To qualitatively measure the adhesion strength between the implant and bone, the bone –implant blocks were subjected to fixation, dehydration and critical point drying, followed by fracturing into two halves. A representative scanning electron micrograph of the half with Ti removed is shown in Fig. 12. A thick, dense, newly formed bone layer
Table 1 The Ca/P ratio of the bone – implant interfaces after 4 and 8 weeks implantation Ca/P ratio (average F 1 SD)
ST PST SIT PSIT
4 8 4 8 4 8 4 8
weeks weeks weeks weeks weeks weeks weeks weeks
Region 1
Region 2
Region 3
– – – – – 1.14 F 0.31 – 1.16 F 0.46
1.69 F 0.56 1.88 F 0.41 1.88 F 0.41 1.77 F 0.34 1.18 F 0.48 1.37 F 0.42 1.48 F 0.38 1.38 F 0.41
1.82 F 0.47 1.86 F 0.40 1.78 F 0.21 1.80 F 0.34 1.74 F 0.41 1.56 F 0.34 1.78 F 0.31 1.77 F 0.44
Fig. 12. A representative scanning electron micrograph of a fractured half of the bone – implant block, with the implant removed. M = implant removed; NB = newly formed bone; PB = pre-existing bone.
Y. Yang et al. / Materials Science and Engineering C 20 (2002) 117–124
Fig. 13. Representative scanning electron micrographs of the two halves of the fractured bone – implant block after 8 weeks implantation. The two halves are: (a) block containing bone, and (b) block containing an implant embedded in the bone. Osteoblasts and fractured bone tips were observed on both fractured halves.
was observed between the pre-existing bone and implants. The newly formed bone was almost vertical to the preexisting bone, indicating bone rebuilding. Scanning electron micrographs of another fractured block, with one half containing the bone and the other half containing an implant embedded in the bone, are shown in Fig. 13a and b. Fracture was observed to occur within the bone instead of at the bone – implant interface. The surface of the two fractured halves was observed to have the similar features. Osteoblasts and fractured bone tips were observed on both fractured halves, suggesting that failure occurred within the newly formed bone region and that the implants were tightly bound to the bone.
4. Discussion In our previous research, the porous-graded Ti coatings on titanium substrates have been synthesized using plasma spraying. In this study, Ti oxide was observed on PSIT surfaces, whereas metallic Ti in a hexagonal crystal was observed in the PST, ST and SIT surfaces. Plasma spraying
123
and ion implantation was suggested to oxidize the Ti surface, thereby inducing a thicker layer of oxide film. From the XPS analysis, the outermost surface was predominantly Ti and O, with adventitious C adsorbed on the surface. Besides O and Ti, XPS depth profiling also indicates the presence of N in the subsurface. The implanted nitrogen element appeared to be in a typical ion implantation distribution. Moreover, the implanted N likely formed nitride under the effect of thermal peak [18]. The presence of N also suggested the presence of trace amounts of nitride in the oxide layer on all ion-implanted Ti surfaces. The roughness of the porous coatings (average roughness f 100 Am) and the diameter of the macropores (maximum diameter z 150 Am) were suggested to promote osseous ingrowth [20 – 22]. In this in vivo study, the newly formed bone was observed to grow into the pores within the Ti coatings, with the osseous ingrowth reaching a depth of 100– 200 Am, suggesting mechanical interlocking between the implant and the bone. On the basis of the relationship between bonding strength and pore depth reported in the literature [23 –25], observations from our fracture analysis suggested strong mechanical interlocking between the implant and the bone, which is critical for implant success. It was also known that porous surfaces will result in increased contact surface between implants and bone, which was beneficial for implant fixation. The increased surface was also reported to result in the increasing release of metal ions [26]. However, as observed in our study, the increase in surface area as a result of surface modification did not result in an increase in the titanium ions released. It was also reported that Ti and its alloys have good biocompatibility and formed direct contact with bone tissues under light microscopy [5,27]. In this study, fluorescence microscopy indicated direct bone contact on all four surfacemodified Ti tested. No significant difference in bone contact was observed at 8 weeks after implantation, and this observation was in agreement with studies by other investigators [28]. Light microscopic observations and morphometric analysis by other investigators reported no statistical difference between the commercially pure Ti implants and the nitrogen-ion implanted Ti implants at 3 months after implantation [28]. Increased Ca and P concentrations and a decreased Ti concentration were observed as the EDS analysis progressed from the implant region to the bone – implant interface and to the bone regions. In addition, no significant difference in the concentration of Ca and P in the ST implant and PST implant (region 1) was observed as the implantation was increased from 4 to 8 weeks. However, a significant increase in the concentration of Ca and P in the SIT implant and PSIT implant (region 1) was observed as the implantation period was increased from 4 to 8 weeks, suggesting the precipitation of Ca and P within the pores. In addition to the precipitation of Ca and P in region 1 of the SIT and PSIT implants, a change in the Ca/P ratio in region 1 was also observed as the implantation period was increased from 4 to 8 weeks, suggesting a
124
Y. Yang et al. / Materials Science and Engineering C 20 (2002) 117–124
tendency for gradual mineralization to occur into the pores of the implants. This was in agreement with other studies reporting the correlation between changes in the matrix Ca/P ratio and bone mineralization [6,29,30]. An increase in the surface bioactivity of the SIT and PSIT implants as compared to the ST and PST implants, as indicated by the occurrence of Ca and P precipitation and the gradual mineralization into the pores of the PSIT and SIT implants, was hypothesized to be the result of a thicker oxide layer and the presence of various microstructural defects created during ion implantation. However, further investigations will be needed to confirm these hypotheses.
5. Conclusions In summary, osseointegration was observed on the ST, PST, SIT and PSIT surfaces. Osseous tissues were observed to grow into the pores inside the coatings, thereby allowing the formation of a gradual Ca – P interface layer. An increase in Ca and P concentrations and a decreased Ti concentration was also observed as the analysis progressed from the implant region to the bone – implant interface and to the bone regions. With an increase in implantation period from 4 to 8 weeks, an increase in the concentration of Ca and P was also observed on the subsurface of the SIT and PSIT implants. As such, it was concluded from this study that ion implantation of Ti surfaces with amino groups induced higher concentration of Ca and P precipitation and more mineralization as compared to non-ion-implanted Ti surfaces.
Acknowledgements This study was funded by the National Institute of Health (1RO1AR46581) and the Chinese 973 Hi-Tech Item (Grant Number: G19990647-02). The authors also thank Dr. Z. Chen, Dr. J. Zhang, Dr. M Zhang and J. Qiu for their help in the study.
References [1] S. Nishiguchi, H. Kato, M. Neo, M. Oka, H.-M. Kim, T. Kobuko, T. Nakamura, J. Biomed. Mater. Res. 54 (2001) 198.
[2] J. Lausmaa, J. Electron Spectrosc. Relat. Phenom. 81 (1996) 343. [3] J.L. Ong, D.C.N. Chan, Crit. Rev. Biomed. Eng. 28 (2000) 5. [4] J.G.C. Wolke, J.P.C. van der Waerden, K. de Groot, J.A. Jansen, Biomaterials 18 (1997) 483. [5] T. Kitsugi, T. Nakamura, M. Oka, W.-Q. Yan, T. Goto, T. Shibuya, T. Kokubo, S. Miyaji, J. Biomed. Mater. Res. 32 (1996) 149. [6] T. Hanawa, Mater. Sci. Eng. A267 (1999) 260. [7] R.G. Vardiman, R.A. Kant, J. Appl. Phys. 53 (1982) 690. [8] J.M. Williams, R.A. Buchanan, Mater. Sci. Eng. 69 (1985) 237. [9] J. Lausmaa, T. Rostlund, H. McKellop, Surf. Interface Anal. 15 (1990) 328. [10] M.T. Pham, W. Matz, H. Reuther, E. Richter, G. Steiner, S. Oswald, J. Mater. Sci. Lett. 19 (2000) 443. [11] T. Kokubo, F. Miyaji, H.-M. Kim, T. Nakamura, J. Am. Ceram. Soc. 79 (1996) 1127. [12] H.-M. Kim, F. Miyaji, T. Kokubo, T. Nakamura, J. Biomed. Mater. Res. 32 (1996) 409. [13] H.-M. Kim, F. Miyaji, T. Kokubo, T. Nakamura, J. Mater. Sci.: Mater. Med. 8 (1997) 341. [14] W.Q. Yan, T. Nakamura, K. Kawanabe, S. Nishiguchi, M. Oka, T. Kokubo, Biomaterials 18 (1997) 1185. [15] H.-M. Kim, H. Takadama, F. Miyaji, T. Kokubo, S. Nishiguchi, T. Nakamura, J. Mater. Sci.: Mater. Med. 11 (2000) 555. [16] H.-M. Kim, T. Kokubo, S. Fujibayashi, S. Nishiguchi, T. Nakamura, J. Biomed. Mater. Res. 52 (2000) 553. [17] Y.Z. Yang, J.M. Tian, J.T. Tian, Z.Q. Chen, X.J. Deng, D.H. Zhang, J. Biomed. Mater. Res. 52 (2000) 333. [18] Y.Z. Yang, J.M. Tian, J.T. Tian, Z.Q. Chen, J. Biomed. Mater. Res. 55 (2001) 442. [19] Y.Z. Yang, J.M. Tian, L. Deng, J.L. Ong, Biomaterials 23 (2002) 1383. [20] J. Klawitter, S. Hulbert, J. Biomed. Mater. Res. 2 (1971) 161. [21] T. Flatley, K. Lynch, M. Benson, Clin. Orthop. 179 (1983) 246. [22] J.C. Hollinger, G.C. Battistone, Clin. Orthop. 207 (1986) 290. [23] J.D. Bobyn, R.M. Pilliar, H.U. Cameron, G.C. Weatherly, Clin. Orthop. 150 (1980) 263. [24] S.D. Cook, K.A. Walsh , R.J. Haddad Jr., Clin. Orthop. 193 (1985) 271. [25] H.A. Luckey, E.G. Lamprecht, M.J. Walt, J. Biomed. Mater. Res. 26 (1992) 557. [26] P. Ducheyne, G. Willems, M. Martens, J. Helsen, J. Biomed. Mater. Res. 18 (1984) 283. [27] J.A. Jansen, J.P.C.M. van de Waerden, J.G.C. Wolke, K. De Groot, J. Biomed. Mater. Res. 25 (1991) 973. [28] C.B. Johansson, J. Lausmaa, T. Rostlund, P. Thomsen, J. Mater. Sci.: Mater. Med. 4 (1993) 132. [29] D. Korn, G. Soyez, G. Elssner, G. Petzow, E.F. Bres, B. d’Hoedt, W. Schulte, J. Mater. Sci.: Mater. Med. 8 (1997) 613. [30] M. Neo, S. Kotani, T. Nakamura, Yamamuro, C. Ohtsuki, T. Kokubo, Y. Bando, J. Biomed. Mater. Res. 26 (1992) 1419.
In vivo evaluation of modified titanium implant surfaces produced using a hybrid plasma spraying processing Yunzhi Yang a,*, Joo L. Ong a, Jiemo Tian b a
Department of Restorative Dentistry, Division of Biomaterials, University of Texas Health Science Center at San Antonio, MSC 7890, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900, USA b State Key Laboratory of New Ceramics and Fine Processing, Tsinghua University, Beijing, 100084, China
Abstract In this study, the biological responses to surface-modified titanium (Ti) was investigated using a dog model. Titanium plasma spraying and ion implantation of amino (NH2+ ) groups were used as means of modifying Ti surfaces. Characterization of the modified Ti surfaces was performed using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and scanning Auger electron spectroscopy. In vivo evaluations were performed using fluorescence microscope, scanning electron microscope and energy disperse spectroscopy. It was observed in this study that ion-implanted porous-graded titanium coatings had a thick surface oxide layer, containing a small amount of nitride. In vivo study indicates direct bone contact between surface-modified Ti implants and osseous tissues. In addition, osseous tissues were observed to grow into the pores inside the coatings, thereby allowing the formation of a gradual calcium phosphate interface layer. It was concluded from this study that ion implantation of Ti surfaces with amino groups, induced higher concentration of calcium and phosphorus precipitation and more mineralization as compared to non-ion-implanted Ti surfaces. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Titanium; Surface modification; Plasma spraying; Ion implantation; Animal study
1. Introduction
2. Materials and methods
Titanium (Ti) and its alloys have been widely used as implants in dental and orthopedic applications. It is also known that the Ti surface plays an important role in governing the biological activity at the bone – implant interface [1,2]. To date, several surface modification methods, such as calcium phosphate or oxide coating [3– 5], ion implantation [6– 10] and alkali treatment [11 –16], have been employed as means to improve the biocompatibility, corrosion and wear and fatigue resistance of Ti. In addition to the reported methods for modifying Ti surfaces, our laboratory has also investigated and characterized the porous plasma-sprayed Ti coatings [17] and Ti surfaces ion-implanted with an amino group [18] produced. Our previous study using osteoblastlike cells had also suggested excellent biocompatibility when cultured on porous plasma-sprayed Ti coatings and Ti surfaces ion-implanted with an amino group [19]. In this study, the biological response to porous plasma-sprayed Ti coatings and Ti surfaces ion-implanted with an amino group was evaluated in vivo.
2.1. Materials preparation
*
Corresponding author. E-mail address: [email protected] (Y. Yang).
For this study, four different Ti surfaces were evaluated. The surfaces were: (1) sandblasted Ti (ST); (2) plasmasprayed porous Ti (PST); (3) sandblasted and ion-implanted Ti (SIT); and (4) plasma-sprayed and ion-implanted Ti (PSIT). The ST surfaces were prepared by sandblasting commercially pure titanium (99.3%, Chinese Nonferrous Metal Institute) with 46# Al2O3 particles. The samples were then cut into square plates (4 4 2 mm3) followed by ultrasonic cleaning in reagent-grade alcohol and acetone [17 –19]. The PST surfaces were prepared by sandblasting commercially pure titanium (99.3%, Chinese Nonferrous Metal Institute) with 46# Al2O3 particles. The sandblasted samples were then heat treated at 400– 500 jC for 3 –4 min, followed by sputter-cleaning with argon ions for 3 – 4 min. A porousgraded Ti coating was then deposited on the Ti surface using an A-3000S plasma spraying equipment and commercially pure Ti powder (99.3%, Chinese Nonferrous Metal Institute) of different sizes (100, 200 and 300 mesh). The base vacuum in the spraying chamber was maintained at 0.1 Pa. Spraying
0928-4931/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 4 9 3 1 ( 0 2 ) 0 0 0 2 1 - 8
118
Y. Yang et al. / Materials Science and Engineering C 20 (2002) 117–124
distance between the substrate and nozzle was at 300 mm, and the spraying pressure was maintained at 10 kPa. A mixture of argon (55 l/min) and hydrogen (5.5 l/min) was used as the plasma gas, and argon (1.2 l/min) was used as the transport gas for the powder. Using a spraying power of 30– 40 kW, graded pores of the coatings were produced by regulating the spraying powder and spraying power. Spraying time was used to regulate the coating thickness. After coating, the samples were cut into square plates (4 4 2 mm3) followed by ultrasonic cleaning in reagent-grade alcohol and acetone [17 – 19]. The SIT and PSIT surfaces were prepared by ion implanting an amine group (NH2 + ) to ultrasonically cleaned ST and PST surfaces. Ion implantation of the amine group was performed to a dose of 1017 cm 2 using a 400-keV ion implanter [18,19], a base pressure of V 10 4 Pa and a working pressure of f 1.0 10 3 Pa. Energy used during implantation was at 1017 cm 2 and 100 keV. 2.2. Materials characterization X-ray diffraction (XRD) analysis was used to characterize the structure of surface-modified Ti surfaces. With Cu Ka radiation, and an energy of 40 kV and 120 mA, the samples were analyzed using a Rigaku X-ray diffractometer (Rigaku D/max-RB). Using Al Ka radiation, X-ray photoelectron spectroscopy (XPS, Perkin-Elmer PHI 5300) was used to analyze the surface composition of the samples. At a sputtering rate of 4 nm/min using argon, XPS depth profiling was performed to evaluate the subsurface composition of the Ti. The sputtering rate was calibrated using thermal oxide SiO2. 2.3. In vivo study Three adult male beagle dogs (10 – 13 kg) were used. Prior to implantation, the four different Ti surfaces were sterilized in an autoclave. A total of 30 implants (6 ST, 6 PST, 9 SIT and 9 PSIT) were used in the study. During the surgery, the animals were anesthetized by intraperitoneal injection of a 3% nembutal (50 mg/kg). A longitudinal incision was then made on the medial surface of the leg. The femur was exposed, and five 4 2-mm holes were drilled up to the bone marrow space using intermittent
Fig. 1. Exposed femur showing implant sites.
Fig. 2. Representative XRD patterns of different surface-modified Ti surfaces.
drilling and cooling saline. The distance between the holes was maintained at 15 mm. Implantation positions were shown in Fig. 1. Ten implants (2 ST, 2 PST, 3 SIT and 3 PSIT) were inserted in the left and right femur. Following the placement of implants, the soft tissues were closed. After implantation, penicillin was administered intramuscularly twice daily and for 3 days. Bone –implant interface was evaluated at 2, 4 and 8 weeks after implantation. Seven days prior to the sacrifice, tetracycline (50 mg/kg) was administered to label newly formed bone. After sacrificing the animals, the implants with their surrounding tissues were excised immediately followed by radiographs. After excision, the specimen blocks were fixed in 10% neutral formalin and dehydrated with serially graded alcohol. Using a diamond blade, the specimens were subsequently thick-sectioned transversely with a low-speed cutting machine (ISOMET, BUEHLER, USA). The specimens were then polished to approximately 100 Am and viewed using a fluorescence microscope (FM, UFX-II Nikon). Composition at the implant – bone interface was also evaluated using an energy dispersive spectroscopy (EDS, Hitachi PV9100) attached to a scanning electron microscope (SEM, Hitachi X-650). Prior to evaluation, the sections were sputter coated with aluminum. Aluminum coating was used instead of gold because of the overlapping of the phosphorus peak with the gold peak. Calcium, phosphorus and titanium ele-
Fig. 3. Representative XPS survey spectra of surface-modified Ti surfaces.
Y. Yang et al. / Materials Science and Engineering C 20 (2002) 117–124
119
To qualitatively evaluate bone –implant adhesion strength, bone – implant blocks were fixed, dehydrated and dried using a critical point dryer. The blocks were then fractured at the bone – titanium interface and divided into two groups. On one group of fractured blocks, the implants were removed and the bone block was evaluated using a scanning electron microscope. On the other group of fractured blocks, the two fractured surfaces (one fractured block containing bone and the other fractured block containing bone and implant) were then sputter-coated with gold and evaluated using a scanning electron microscope (SEM, AMRAY-1000).
3. Results 3.1. Materials characterization X-ray diffraction patterns of Ti powder, Ti substrates, ST, SIT, PST and PSIT are shown in Fig. 2. The diffraction peaks of PSIT were mainly oxide (Ti2O), whereas the other Ti materials were mainly metallic Ti in a hexagonal crystal. XRD also suggested that plasma spraying and ion implantation oxidized the Ti surface, thereby forming a thicker layer of oxide film. The other Ti surfaces were only slightly oxidized by the modification processing. A typical XPS survey spectrum of surface-modified Ti is shown in Fig. 3. Oxygen (O) and Ti were predominantly observed on the outermost surface of all Ti, tested with adventitious carbon (C) adsorbed on the surface. Fig. 4 shows the typical XPS depth profile of Ti and O on all surfacemodified Ti. Trace amounts of nitrogen (N) was observed for all ion-implanted Ti surfaces. The relative concentration of O was reduced as the relative concentration of Ti was increased during XPS depth profiling. Fig. 4. XPS depth profile of surface-modified Ti surfaces.
3.2. In vivo study ments were measured on three randomly selected interfaces per group. Differences in compositions were statistically compared using ANOVA and were considered statistically significant if p < 0.05.
Gross evaluation of the excised bone after 2 and 4 weeks implantation revealed an incomplete healing of the bone at the sites where the bone was drilled for implant placement. However, the implants were observed to be closely sur-
Fig. 5. Representative radiographs of implants in dog femora at (a) 2 weeks, (b) 4 weeks and (c) 8 weeks after implantation.
120
Y. Yang et al. / Materials Science and Engineering C 20 (2002) 117–124
rounded by newly formed bone at the implant site after 2 and 4 weeks implantation. Gross evaluation of the excised bone after 8 weeks implantation indicated a complete healing of the bone at the site where the bone was drilled for implant placement. The newly formed bone was similar to the old bone, and as a result of complete bone coverage at the implant site, the implants were completely covered by the bone after 8 weeks implantation. Representative radiographs at 2, 4 and 8 weeks after implantation are shown in Fig. 5a –c. As observed from the radiographs, shadows around the implants were undetectable, suggesting that the implants were in close contact with the newly formed bone. Since the implants were completely covered by the bone after 8 weeks implantation, drilling of the bone was performed to identify the location of the implants. As a result of the drilling, horizontal shadows were
Fig. 6. Representative fluorescence micrographs of a drilled bone site on the femur for implant placement and tissues around the implants at (a) 2 weeks, (b) 4 weeks and (c) 8 weeks after implantation (original magnification, 40). NB = newly formed bone, PB = pre-existing bone.
Fig. 7. Representative fluorescence micrograph of the PSIT implant – bone interface at 8 weeks after implantation (original magnification, 100 (arrowhead = NB).
observed for the radiograph of the femur after 8 weeks implantation (Fig. 5c). From the fluorescence microscopy, Ti implants within the same implantation period exhibited no significant differ-
Fig. 8. Interface morphology between specimen and bone at 8 weeks after implantation. (a) PST (original magnification, 250), with arrowheads indicating newly formed bone; (b) PSIT (original magnification, 250), with arrowheads indicating newly formed bone; (c) PST (original magnification, 500), with arrowheads indicating a thin gap at the bone – implant interface; (d) PSIT (original magnification, 500), with arrowheads indicating newly formed bone. M = implant; B = bone. The thin gap observed in the interface of PST (c) was an artifact produced during the preparation process. In all these micrographs, the parallel marks on the metallic Ti were attributed to the sectioning of the implant – bone blocks using a diamond blade.
Y. Yang et al. / Materials Science and Engineering C 20 (2002) 117–124
Fig. 9. SEM-EDS of the interface between PSIT and bone: (a) elemental analysis of Ca and Ti at 2 weeks after implantation, and (b) that of P at 2 weeks after implantation, and (c) that of Ca and Ti at 8 weeks after implantation, and (d) that of Ca and P at 8 weeks after implantation.
ence between the four groups of Ti tested. Fig. 6a – c shows the typical fluorescence micrographs of a drilled bone site for implant placement as well as the newly formed bone around the implant at 2, 4 and 8 weeks after implantation. At 2 and 4 weeks after implantation (Fig. 6a and b), newly
121
formed bone was observed around the implant. In addition, the newly formed bone and the pre-existing bone were distinctly different. At 8 weeks after implantation (Fig. 6c), there was no distinction between the newly formed bone and the pre-existing bone. The newly formed bone was observed to be concentrated on the Haversian canal after 8 weeks implantation, suggesting that the drilled bone site for implant placement had healed. The fluorescence data was in agreement with the gross observation and the radiographs. Fig. 7 shows the fluorescence micrograph of the interface between PSIT surface and the surrounding tissue at 8 weeks after implantation. At higher magnification, the newly formed bone was observed to grow into the pores of PSIT, thereby suggesting mechanical interlocking between the PSIT and bone. As shown in Fig. 8a and b, EDS analysis of the porous PST and PSIT surfaces indicates the presence of calcium (Ca) and phosphorus (P), suggesting bone ingrowth within the pores. The Ca/P ratio of the bone ingrowth with the pores of PST and PSIT surfaces was 1.87 and 1.74. The newly formed bone was observed to have reached pores in the depth of 100– 200 Am from the surface and provide mechanical interlock between the implants and the bone. This mechanical interlocking suggested the mechanism for stabilizing Ti implants evaluated in this study. Intimate bone contact with the surface-modified Ti was also shown in Fig. 8c and d. SEM-EDS analysis in Fig. 9 further confirmed that the newly formed bone was in intimate contact with implant surfaces as well as bone growth in the pores of PSIT. Interfacial line analysis from the implant to the bone revealed increasing concentration of Ca and P as the concentration of Ti decreased, suggesting the presence of a Ca – P layer at the PSIT implant – bone interface. The thickness of the Ca – P layer was observed to increase from about 4 –6 Am at 2 weeks
Fig. 10. A representative SEM micrograph showing the three different analyzed region (5 5 Am). On the right is a schematic drawing of interface microanalysis. 1 = implant; 2 = implant – bone interface; 3 = bone.
122
Y. Yang et al. / Materials Science and Engineering C 20 (2002) 117–124
Fig. 11. Bar charts of EDS microanalysis in regions 1, 2 and 3 of ST, PST, SIT and PSIT surfaces after 4 and 8 weeks implantation. Asterisk showed significant differences between ion-implanted and non-ion-implanted surfaces ( p < 0.05).
after implantation, to about 30 Am at 8 weeks after implantation. In addition, the relative concentration of modified, porous implant surface and nonmodified titanium surface (Ti/(Ca + P + Ti)) at the bone – implant interface was 2 – 4 at.%, indicating no significant difference in the titanium ions released from the modified and nonmodified Ti surfaces. As shown in Fig. 10, elemental concentrations of Ca, P and Ti at three different bone –implant interfacial regions (region 1 = implant, region 2 = implant –bone interface and region 3 = bone) were quantified. Fig. 11 shows the elemental concentration of Ca, P and Ti at 4 and 8 weeks after implantation. The Ca/P ratio of the bone –implant interfaces after 4 and 8 weeks implantation is shown in Table 1. In agreement with the interfacial line analysis, the concentration of Ca and P increased, and the concentration of Ti decreased as the analysis progressed from regions 1 to 3 (implant to bone). No significant difference in the concentration of Ca and P in the ST implant and PST implant (region 1) was observed as the implantation period was increased from 4 to 8
weeks. However, a significant increase in the concentration of Ca and P in the SIT implant and PSIT implant (region 1) was observed as the implantation period was increased from 4 to 8 weeks. To qualitatively measure the adhesion strength between the implant and bone, the bone –implant blocks were subjected to fixation, dehydration and critical point drying, followed by fracturing into two halves. A representative scanning electron micrograph of the half with Ti removed is shown in Fig. 12. A thick, dense, newly formed bone layer
Table 1 The Ca/P ratio of the bone – implant interfaces after 4 and 8 weeks implantation Ca/P ratio (average F 1 SD)
ST PST SIT PSIT
4 8 4 8 4 8 4 8
weeks weeks weeks weeks weeks weeks weeks weeks
Region 1
Region 2
Region 3
– – – – – 1.14 F 0.31 – 1.16 F 0.46
1.69 F 0.56 1.88 F 0.41 1.88 F 0.41 1.77 F 0.34 1.18 F 0.48 1.37 F 0.42 1.48 F 0.38 1.38 F 0.41
1.82 F 0.47 1.86 F 0.40 1.78 F 0.21 1.80 F 0.34 1.74 F 0.41 1.56 F 0.34 1.78 F 0.31 1.77 F 0.44
Fig. 12. A representative scanning electron micrograph of a fractured half of the bone – implant block, with the implant removed. M = implant removed; NB = newly formed bone; PB = pre-existing bone.
Y. Yang et al. / Materials Science and Engineering C 20 (2002) 117–124
Fig. 13. Representative scanning electron micrographs of the two halves of the fractured bone – implant block after 8 weeks implantation. The two halves are: (a) block containing bone, and (b) block containing an implant embedded in the bone. Osteoblasts and fractured bone tips were observed on both fractured halves.
was observed between the pre-existing bone and implants. The newly formed bone was almost vertical to the preexisting bone, indicating bone rebuilding. Scanning electron micrographs of another fractured block, with one half containing the bone and the other half containing an implant embedded in the bone, are shown in Fig. 13a and b. Fracture was observed to occur within the bone instead of at the bone – implant interface. The surface of the two fractured halves was observed to have the similar features. Osteoblasts and fractured bone tips were observed on both fractured halves, suggesting that failure occurred within the newly formed bone region and that the implants were tightly bound to the bone.
4. Discussion In our previous research, the porous-graded Ti coatings on titanium substrates have been synthesized using plasma spraying. In this study, Ti oxide was observed on PSIT surfaces, whereas metallic Ti in a hexagonal crystal was observed in the PST, ST and SIT surfaces. Plasma spraying
123
and ion implantation was suggested to oxidize the Ti surface, thereby inducing a thicker layer of oxide film. From the XPS analysis, the outermost surface was predominantly Ti and O, with adventitious C adsorbed on the surface. Besides O and Ti, XPS depth profiling also indicates the presence of N in the subsurface. The implanted nitrogen element appeared to be in a typical ion implantation distribution. Moreover, the implanted N likely formed nitride under the effect of thermal peak [18]. The presence of N also suggested the presence of trace amounts of nitride in the oxide layer on all ion-implanted Ti surfaces. The roughness of the porous coatings (average roughness f 100 Am) and the diameter of the macropores (maximum diameter z 150 Am) were suggested to promote osseous ingrowth [20 – 22]. In this in vivo study, the newly formed bone was observed to grow into the pores within the Ti coatings, with the osseous ingrowth reaching a depth of 100– 200 Am, suggesting mechanical interlocking between the implant and the bone. On the basis of the relationship between bonding strength and pore depth reported in the literature [23 –25], observations from our fracture analysis suggested strong mechanical interlocking between the implant and the bone, which is critical for implant success. It was also known that porous surfaces will result in increased contact surface between implants and bone, which was beneficial for implant fixation. The increased surface was also reported to result in the increasing release of metal ions [26]. However, as observed in our study, the increase in surface area as a result of surface modification did not result in an increase in the titanium ions released. It was also reported that Ti and its alloys have good biocompatibility and formed direct contact with bone tissues under light microscopy [5,27]. In this study, fluorescence microscopy indicated direct bone contact on all four surfacemodified Ti tested. No significant difference in bone contact was observed at 8 weeks after implantation, and this observation was in agreement with studies by other investigators [28]. Light microscopic observations and morphometric analysis by other investigators reported no statistical difference between the commercially pure Ti implants and the nitrogen-ion implanted Ti implants at 3 months after implantation [28]. Increased Ca and P concentrations and a decreased Ti concentration were observed as the EDS analysis progressed from the implant region to the bone – implant interface and to the bone regions. In addition, no significant difference in the concentration of Ca and P in the ST implant and PST implant (region 1) was observed as the implantation was increased from 4 to 8 weeks. However, a significant increase in the concentration of Ca and P in the SIT implant and PSIT implant (region 1) was observed as the implantation period was increased from 4 to 8 weeks, suggesting the precipitation of Ca and P within the pores. In addition to the precipitation of Ca and P in region 1 of the SIT and PSIT implants, a change in the Ca/P ratio in region 1 was also observed as the implantation period was increased from 4 to 8 weeks, suggesting a
124
Y. Yang et al. / Materials Science and Engineering C 20 (2002) 117–124
tendency for gradual mineralization to occur into the pores of the implants. This was in agreement with other studies reporting the correlation between changes in the matrix Ca/P ratio and bone mineralization [6,29,30]. An increase in the surface bioactivity of the SIT and PSIT implants as compared to the ST and PST implants, as indicated by the occurrence of Ca and P precipitation and the gradual mineralization into the pores of the PSIT and SIT implants, was hypothesized to be the result of a thicker oxide layer and the presence of various microstructural defects created during ion implantation. However, further investigations will be needed to confirm these hypotheses.
5. Conclusions In summary, osseointegration was observed on the ST, PST, SIT and PSIT surfaces. Osseous tissues were observed to grow into the pores inside the coatings, thereby allowing the formation of a gradual Ca – P interface layer. An increase in Ca and P concentrations and a decreased Ti concentration was also observed as the analysis progressed from the implant region to the bone – implant interface and to the bone regions. With an increase in implantation period from 4 to 8 weeks, an increase in the concentration of Ca and P was also observed on the subsurface of the SIT and PSIT implants. As such, it was concluded from this study that ion implantation of Ti surfaces with amino groups induced higher concentration of Ca and P precipitation and more mineralization as compared to non-ion-implanted Ti surfaces.
Acknowledgements This study was funded by the National Institute of Health (1RO1AR46581) and the Chinese 973 Hi-Tech Item (Grant Number: G19990647-02). The authors also thank Dr. Z. Chen, Dr. J. Zhang, Dr. M Zhang and J. Qiu for their help in the study.
References [1] S. Nishiguchi, H. Kato, M. Neo, M. Oka, H.-M. Kim, T. Kobuko, T. Nakamura, J. Biomed. Mater. Res. 54 (2001) 198.
[2] J. Lausmaa, J. Electron Spectrosc. Relat. Phenom. 81 (1996) 343. [3] J.L. Ong, D.C.N. Chan, Crit. Rev. Biomed. Eng. 28 (2000) 5. [4] J.G.C. Wolke, J.P.C. van der Waerden, K. de Groot, J.A. Jansen, Biomaterials 18 (1997) 483. [5] T. Kitsugi, T. Nakamura, M. Oka, W.-Q. Yan, T. Goto, T. Shibuya, T. Kokubo, S. Miyaji, J. Biomed. Mater. Res. 32 (1996) 149. [6] T. Hanawa, Mater. Sci. Eng. A267 (1999) 260. [7] R.G. Vardiman, R.A. Kant, J. Appl. Phys. 53 (1982) 690. [8] J.M. Williams, R.A. Buchanan, Mater. Sci. Eng. 69 (1985) 237. [9] J. Lausmaa, T. Rostlund, H. McKellop, Surf. Interface Anal. 15 (1990) 328. [10] M.T. Pham, W. Matz, H. Reuther, E. Richter, G. Steiner, S. Oswald, J. Mater. Sci. Lett. 19 (2000) 443. [11] T. Kokubo, F. Miyaji, H.-M. Kim, T. Nakamura, J. Am. Ceram. Soc. 79 (1996) 1127. [12] H.-M. Kim, F. Miyaji, T. Kokubo, T. Nakamura, J. Biomed. Mater. Res. 32 (1996) 409. [13] H.-M. Kim, F. Miyaji, T. Kokubo, T. Nakamura, J. Mater. Sci.: Mater. Med. 8 (1997) 341. [14] W.Q. Yan, T. Nakamura, K. Kawanabe, S. Nishiguchi, M. Oka, T. Kokubo, Biomaterials 18 (1997) 1185. [15] H.-M. Kim, H. Takadama, F. Miyaji, T. Kokubo, S. Nishiguchi, T. Nakamura, J. Mater. Sci.: Mater. Med. 11 (2000) 555. [16] H.-M. Kim, T. Kokubo, S. Fujibayashi, S. Nishiguchi, T. Nakamura, J. Biomed. Mater. Res. 52 (2000) 553. [17] Y.Z. Yang, J.M. Tian, J.T. Tian, Z.Q. Chen, X.J. Deng, D.H. Zhang, J. Biomed. Mater. Res. 52 (2000) 333. [18] Y.Z. Yang, J.M. Tian, J.T. Tian, Z.Q. Chen, J. Biomed. Mater. Res. 55 (2001) 442. [19] Y.Z. Yang, J.M. Tian, L. Deng, J.L. Ong, Biomaterials 23 (2002) 1383. [20] J. Klawitter, S. Hulbert, J. Biomed. Mater. Res. 2 (1971) 161. [21] T. Flatley, K. Lynch, M. Benson, Clin. Orthop. 179 (1983) 246. [22] J.C. Hollinger, G.C. Battistone, Clin. Orthop. 207 (1986) 290. [23] J.D. Bobyn, R.M. Pilliar, H.U. Cameron, G.C. Weatherly, Clin. Orthop. 150 (1980) 263. [24] S.D. Cook, K.A. Walsh , R.J. Haddad Jr., Clin. Orthop. 193 (1985) 271. [25] H.A. Luckey, E.G. Lamprecht, M.J. Walt, J. Biomed. Mater. Res. 26 (1992) 557. [26] P. Ducheyne, G. Willems, M. Martens, J. Helsen, J. Biomed. Mater. Res. 18 (1984) 283. [27] J.A. Jansen, J.P.C.M. van de Waerden, J.G.C. Wolke, K. De Groot, J. Biomed. Mater. Res. 25 (1991) 973. [28] C.B. Johansson, J. Lausmaa, T. Rostlund, P. Thomsen, J. Mater. Sci.: Mater. Med. 4 (1993) 132. [29] D. Korn, G. Soyez, G. Elssner, G. Petzow, E.F. Bres, B. d’Hoedt, W. Schulte, J. Mater. Sci.: Mater. Med. 8 (1997) 613. [30] M. Neo, S. Kotani, T. Nakamura, Yamamuro, C. Ohtsuki, T. Kokubo, Y. Bando, J. Biomed. Mater. Res. 26 (1992) 1419.