Properties of calcium phosphate coatings before and after exposure to simulated biological fluid

Biomoteriols 18 (1997) 1271-1275 0 1997 Elsevier Science Limited Printed in Great Britain. All rights reserved PII:

ELSEVIER

SO142-9612

(97)

00074-4

014s9612/97/$17.00

Properties of calcium phosphate coatings before and after exposure to simulated biological fluid J.L. Ong, G.N. Raikar” and T.M. Smoot+ University of Texas Health Science Center at San Antonio, Department of Restorative Dentistry, Division of Biomaterials, 7703 Floyd Curl Drive, San Antonio, TX 78284-7890, USA; ‘University of Utah, Department of Chemistry, SUrfaCe Science and Optical Spectroscopy Facility, Salt Lake City, UT 84112, USA; tlJniversity of Alabama at Birmingham, Department of Biostatistics, University Station, Birmingham, AL 35294, USA The surface nature

qualities

and degree

tion. Thus,

of calcium of cellular

in this study,

sputter-deposited

the chemical

CaP coatings

characterized.

Significant

with coatings

heat treated

at 700°C (CA7). energy

were

crystallite statistically observed

after

the Ca/P ratio and surface

after energy

immersion were

indicated

no statistical

difference

and after

1 week.

1997 Elsevier

Science

crystallite

Calcium

phosphates,

magnetron

Limited.

compared carbon

immersion

An increase

between

to coatings

in a physiological in carbon

However,

heat treated and surface solution,

energy

surface

concentration

no significant

was observed

after

were

heat treatments,

concentration

the two coatings

All rights

sputtering,

solution

with different

size for the CA8 and CA7 coatings

in solution.

in surface

ratio,

of radiofrequency

i-week

the

remained was also

differences

in

immersion

for both samples

in

initially

reserved energy,

chemical

composition,

size

Received 24 September

1996; accepted 14 April 1997

properties of biomaterials needs to be addressed’s, lg. It is also known that tissues respond differently to CaP surfaces of different crystallinity/crystallite size20-27. Thus, as initial steps to elucidate the phenomena that control the performance of biomaterials in viva, the chemical composition and crystallographic properties of radiofrequency (RF] sputter-deposited CaP coatings prior to and after l-week immersion in a physiological solution were characterized in this study.

Calcium phosphate [Cap) coatings have been used primarily to alter implant surfaces, with the assumption that both improved osteointegration and long-term stability of the implants can be achievedlW3. Although numerous animal and clinical studies have been performed using Cap-coated implants, the coating qualities are either unknown, poorly known or left unstated4. As a result, it is common to see conflicting reports on tissue responses to Cap-coated implants, both in vitro and in vivo5-8. The lack of fully characterized Cap-coated implants makes comparison between conflicting studies equivocal. Implant success is directly influenced by biomaterial environment and the biological propertiesg-‘2. The surface qualities of CaP implants are important factors determining the nature and degree of cellular behaviour, especially cellular attachment, proliferation and differentiation10,13-‘7. Important biomaterial properties influencing the success of an imp1an.t include topography, chemical composition and structure of the biomaterials used. Since implant surfaces are bathed in biofluid prior to tissue contact, the influence of biofluid on the Correspondence

After

in solution.

immersion

In addition,

Keywords:

crystallites

the

and differentia-

in a physiological

observed

in the CalP

The crystallite

solution.

0

larger

determining

proliferation properties

immersion

size were

heat treatments.

l-week

factors

attachment,

and crystallographic

difference

increased.

are important

cellular

composition

at 850°C (CA8) having

l-week

implants

in crystallite

with different

for both samples

(Cap) especially

to and after in vitro

no statistical

size was significantly different

prior

differences

However,

observed

phosphate

behaviour,

MATERIALS

AND METHODS

RF sputtering Since the measurement of surface energy is dependent on the surface roughness of the substrate, mirror polished vicar glass discs (12.7 mm diameter x 1.6 mm thick) were used as substrates for CaP coatings. The glass discs were ultrasonically cleaned using Alconox detergent (Alconox, NY, USA) for 3Omin. The discs were then rinsed with deionized water and ethanol. Prior to sputtering, the discs were plasma cleaned for 2 min using a Plasma-Spreen II-973 system (Plasmatic Systems, Inc., NJ, USA). The discs were then placed in the RF sputtering NCR 3117 system (Vacuum

to Dr JL. Ong. 1271

Biomaterials

1997, Vol. 18 No. 19

Sputtered

1272

Technology Associates, CO, USA) and the chamber was pumped down to a base pressure of 6 x lo-” Torr. High purity argon (99.999%) was backfilled into the chamber, bringing the pressure to about 10m4Torr. At an energy of 200 W and an RF voltage of 9OOV, CaP coatings were produced using a plasma sprayed hydroxyapatite (HA) target. At a rate of 0.2 pm per hour, a coating thickness of 0.4pm was achieved after 2 h sputtering. The coatings produced were amorphous and have a dissolution rate of up to 3 hz8. In order to decrease the dissolution rate, post-deposition heat treatments of at least 500°C have been reported”. Thus, in this study, post-deposition heat treatments of 700°C (CA7) and 850°C (CA8) were performed on the CaP coatings.

Immersion study In order to simulate implant surfaces in contact with biofluid, CaP coatings were immersed in alpha modification of minimum essential medium (aMEM) for 1 week. The c(-MEM is a balanced salt solution commonly used for cell culture studies. It contains CaC12, sodium salts (NaHC03, NaCl, NaH2P04.H20), KCl, MgSO,, amino acids, vitamins, glucose, lipoic acid, Phenol Red and sodium pyruvate. All immersed CaP coatings were kept in a sterile incubator at 37°C (95% air, 5% CO,). An initial stock solution of pH 7.4 was prepared by titrating the a-MEM with either hydrochloric acid or sodium hydroxide. In order to maintain the pH at 7.4, the media were changed every 2 days. At the end of l-week immersion, the samples were rinsed with double-distilled deionized water, air dried and stored in a desiccator prior to analyses.

X-ray dieaction X-ray diffraction (XRD) analyses were performed to evaluate the structure of the heat-treated CaP coatings prior to and after immersion in a physiological solution. A Siemens D500 diffractometer using CuK, radiation having energies of 40 keV and 30 mA was used. The incident X-rays passed through 3” and 1” slits before impinging upon the CaP coatings. Diffracted X-rays passed through l”, 0.6” and 0.05” slits at the X-ray counter. Initially and after a l-week interval, six samples for each treatment were analysed and the data were collected from 25” to 35” 28 at 0.1” per minute scan rate. Crystalline coatings were identified by matching the peaks with standard synthetic HA (JCPDS 9-00432). The crystallite size of the coatings was calculated based on the 002 reflections. At an CI level of 0.05, the crystallite sizes were statistically analysed using analysis of variance (ANOVA), with differences compared using the Student-NewmanKeuls test.

calcium

phosphate

coatings:

J.L. Ong et al.

of all elements of 45”. The atomic concentrations present were quantified. At an c1 value of 0.05, the atomic concentrations of all elements on CaP coatings and target were statistically analysed using ANOVA, with differences compared using the StudentNewman-Keuls test.

Contact angle In order to evaluate the surface energy of the CaP coatings, the wettabilities of CaP surfaces in various liquids were measured using a video contact angle VCA-2000 system (Advanced Surface Technology, MA, USA). The liquids used were double-distilled water, glycerol and methyl iodide. Three CaP surfaces were used for each liquid (a total of nine CaP surfaces used from each treatment). Five measurements were conducted using each flat specimen surface. The cosines of these angles were plotted against the surface tension values of the liquids as a Zisman plot. The critical surface tension or surface energy was determined by extrapolating to cosine = 1, i.e. complete wetting3’. At an c( value of 0.05, differences in the surface energies of CaP coatings prior to and after immersion were statistically analysed using ANOVA. Differences between groups (P < 0.05) were assessed by post-hoc pairwise comparisons of individual group means using Fisher’s Protected Least Significant Difference test.

RESULTS X-ray difbaction Representative XRD patterns of CA7 and CA8 are shown in Figure I. An apatite-type structure, oriented in the 00.2 plane, was exhibited by both CA7 and CA8 samples. The coating positions matched JCPDS 09432. The crystallite sizes of CaP coatings initially

3500

-

:

3000. -

z

2500

-

2000

-

1500

-

% E

1000 -

500 -

X-ray photoelectron spectroscopy X-ray photoelectron spectroscopy (XPS) was used to evaluate the chemical composition of the CaP coating surfaces prior to and after immersion in a physiological solution. All surface spectra over the range of O1100eV were obtained using Mg K, radiation at 15 kV and 20mA. Initially and after a l-week interval, duplicate samples were analysed using a take-off angle Biomaterials

1997,

Vol. 18 No. 19

O25

I

I

I

1

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,

26

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33

34

35

2 Theta Figure 1 Representative XRD scans of CA7 and CA8 samples prior to and after immersion in a physiological solution. XRD analyses show an apatite-type structure, oriented in the 002 plane for all samples.

Sputtered

calcium

phosphate

coatings:

Table 1 Crystallite sizes of calcium to and after immersion in solution CA7 coatings Initial 1 week

(nm)

J.L. Ong et al.

phosphate

coatings

CA8 coatings

1273 prior

(nm)

58.9It1.1 63.8zt1.5

54.7f 0.6 59.5 * 0.8

CA7, CaP coatings heat treated at 700°C; CA8, CaP coatings heat treated at 850°C.

and at 1 week are summarized in Table 1. Prior to immersion, the crystallite size was observed to be statistically different for CA7 coatings (54.7 f 0.6 nm) and CA8 coatings (58.9 f 1.1 nm). Significant growth in the crystallite size between the initial and l-week for CA7 coatings samples was also observed (59.5 f 0.8 nm) and CA8 coatings (63.8 f 1.5 nm) after l-week immersion.

H 3 4000 s 8

X-ray photoelectron spectroscopy Representative XPS spectra of the initial CA7 and CA8 surfaces are provided in Figure 2. Carbon, phosphorus, oxygen and calcium were observed on all surfaces. The carbon peak observed at 284.8 eV was attributed to and C-H bonded carbon) hydrocarbon (C-C adsorption. As shown in Table 2, the carbon concentration was observed to increase after l-week immersion in solution. The surface Ca/P ratio of the sputtering target was observed to be 1.58 f 0.06. Table 2 also summarizes the Ca/P ratio for CA7 and CA8 surfaces. Initially, no significam difference in the Ca/P ratio was observed between CA7 and CA8 surfaces (c( value of 0.05). However, they were statistically different from the target. After immersion in solution, no significant change in the Ca/P ratio for CA7 and CA8 surfaces was observed.

Wettability

0

200

400

600

800

1000

Binding energy (ev) Figure 2 and target

Representative XPS surface scans prior to immersion in physiological

of CA7, solution.

CA8

0.9 0.8

---. El

0.7

CA7 CA8

d.6 d

0.5 -

f

0.4-

.t 2 0.3 "

0.2 0.0-

The Zisman plots for the CA7 and CA8 coatings initially and after 1 week are shown in Figures 3 and 4. Initially, no significant difference in the surface between CA7 observed was energy (36.8 f 0.1 dyncm-‘) and CA8 (36.4 f 0.2 dyncm-l) coatings. Similarly, at 1 week, the surface energy of CA8 (37.2 f 0.1 dyn cm-‘) coatings was not significantly different from CA7 (34.5 f 0.4 dyn cm-‘) coatings.

35

40

45

50

55

60

65

70

75

Surface Tension (dyn/cm)

DISCUSSION

Figure 3 Zisman plot of initial CaP coatings. CA7, CaP coatings heat treated at 700°C; CA8, CaP coatings heat treated at 850°C. The error bars represent the standard error (s.e.) of the contact angles at various surface tension. bars in bold. &AT = error

Depending on the surface properties of biomaterials, interactions between an implant and adjacent tissues are dependent, in part, on the surface properties of the implant materials. Different rates of cellular responses have been observed in vitro, and these differences

have been attributed to varying surface properties, such as surface chemistries and crystallinitieszO27,31,32. In this study, the chemical composition and crystallographic properties of RF sputter-deposited

Table 2

coatings

Carbon

concentration

and CalP ratio of calcium

phosphate

Initial 1 week

cont.

24.3 f 0.8 32.3 f 3.0

in solution

CA8 coatings

CA7 coatings Carbon

prior to and after immersion

(at %)

CalP ratio

Carbon

cont.

1.9 f 0.1 1.6fO.l

22.6 f 0.2 33.6 I’L 3.8

Ca/P ratio

(at %)

1.8fO.l 1.6 ?c 0.2

CA7. CaP coatings heat treated at 700°C: CA8, CaP coatings heat treated at 850°C.

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No. 19

Sputtered calcium phosphate

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0.0 -

-0.1 -0.2 -

35

40

45

50

55

60

65

70

75

Surface Tension (dynkm) Figure 4 Zisman plot of CaP coatings. CA7, CaP coatings heat treated at 700°C; CA8, CaP coatings heat treated at 850°C. The error bars represent the standard error (s.e.) of the contact angles at various surface tension. SECA7 = error bars in bold.

CaP coatings prior to and after in vitro immersion in a physiological-like solution were characterized. From the XRD analyses, an apatite-type structure, oriented in the 002 plane, was exhibited by both CA7 and CA8 coatings. The coating positions matched JCPDS 09-432. The crystallite size of CA8 coatings was observed to be statistically larger compared to CA7 coatings. As observed in previous studies, crystallinity increases with temperature2g*33. Significant growth in crystallite sizes was observed after l-week immersion. It has been suggested that the apatite formed by these heat treatments was poorly crystallized34. Thus, the increase in crystallinity after immersion in solution was suggested to be due to the dissolution of the amorphous phase. The dissolution of the coatings may result in the supersaturation of calcium and phosphorus ions in the physiological media, therefore resulting in the reprecipitation of a crystallized coating3523”. It was also hypothesized that the presence of water molecules promoted the conversion of the amorphous phase into crystalline HA and significantly enhanced the crystallinity of the coating37. Besides the change in crystallite size, no significant difference in the Ca/P ratio was observed between the initial CA7 and CA8 surfaces (a value of 0.05). However, the Ca/P ratios observed from the sputtered coatings are different from those of the plasma-sprayed HA target. Preferential sputtering of calcium was observed in the sputtered coatings, probably due to the possibility of the phosphorus ions being pumped away before they are deposited at the substrate38. It was also suggested by other investigators that the phosphorus ions may be weakly bound to the growing film and may be sputtered away by incoming ions or electrons3’. As reported in previous studies, the presence of carbonate was also evident in the sputtered coatingszg. After immersion in solution, no significant change in the Ca/P ratio was observed for CA7 and CA8 surfaces. Biomaterials 1997,Vol. 18 No. 19

coatings: J.L. Ong et al.

However, as shown in Table 2, the carbon concentration was observed to increase after l-week immersion in solution. Besides the presence of calcium and phosphate salts present in the cell culture media, carbonate salts such as sodium bicarbonate were also observed. Thus, the increase in the carbon concentration after l-week immersion was suggested to be due to the incorporation of carbonate into the coatings. Initially, no significant difference in the surface energy was observed between CA7 and CA8 surfaces. Similarly, at 1 week, the surface energy of CA8 samples was not significantly different from CA7 samples, indicating that the surface energy of the samples was independent of the crystallite size. Using titanium substrates, no influence of crystallite size on surface energy was observed by other investigators4’. However, the influence of surface energies may be affected by other properties, such as surface roughness and variation in composition, and further studies are needed to determine their effect on surface energy and cellular responses. Thus, it was concluded from this study that the surface energy of CaP coatings was not influenced by the crystallite size.

ACKNOWLEDGEMENTS The authors would like to acknowledge Foundation for funding this study.

The Whitaker

REFERENCES 1.

2.

3.

4.

5. 6.

7.

8.

9.

10.

Tracy, B.M. and Doremus, R. H., Direct electron studies microscopy of the bone-hydroxyapatite interface. I. Biomed. Mater. Res., 1984,18,719-726. Cooley, D. R., van Dellen, A. F., Burgess, J. 0. and Windeler, A. S., The advantages of coated titanium implants prepared by radiofrequency sputtering from hydroxyapatite. J. Prosthet. Dent., 1992,67,93-99. Niki, M., Ito, G., Matsuda, T. and Ogino, M., Comparative push-out data of bioactive and non-bioactive materials of similar rugosity. In The Bone-Biomaterial Interface, ed. J.E. Davies. University of Toronto Press, Toronto, 1991,pp. 350-356. Larson, F. G., Hydroxyapatite coatings for medical implants. Med. Dev. Diag. Ind., 1994, April Issue, 3440. Evans, E. J., Toxicity of hydroxyapatite in vitro: the effect of particle size. Biomateriak, 1991,12,574-576. Puleo, D.A., Holleran, L. A., Doremus, R.H. and Bizios, R., Osteoblast responses to orthopedic implant materials in vitro. J. Biomed. Mater. Res., 1991,25,711-723. Cooley, D.R., Van Dellen, A.F., Burgess, 7.0. and Windeler, A.S., The advantages of coated titanium implants prepared by radio frequency sputtering from hydroxyapatite. J. Prosthet. Dent., 1992, 67,93-110. Dalton, J.E. and Cook, S.D., In viva mechanical and histological characteristics of HA-coated implants vary with coating vendor. J. Biomed. Mater. Res., 1995, 29, 239-245. Kasemo, B. and Lausmaa, J., The biomaterial-tissue interface and its analogues in surface science and technology. In The Bone-Biomaterial Inteqface, ed. J. E. Davies. University of Toronto Press, Toronto, 1991, pp. 19-32. Hanawa, T. and Ota, M., Calcium phosphate naturally formed on titanium in electrolyte solution. Biomateriak,1991,12, 767-773.

Sputtered calcium phosphate 11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

coatings: J.L. Ong et al.

Michaels, C.M., Keller, J. C. and Stanford, C. M., In vitro periodontal ligament fibroblast attachment to plasma cleaned Ti surfaces. 1. Oral Implant., 1991, 17,132-139. Keller, J. C., Douglherty, W. M. J., Grotendorst, G. R. and Wightman, J. P., In vitro cell attachment to characterized cp Ti surfaces. I. Adhesion, 1989, 28, 115-133. van Bitterswijk, C. A., Leenders, H., v.d. Brink, J. et al., Degradation and .interface reactions of hydroxyapatite coatings: effect of crystallinity. 19th Annual Meeting of the Society for Riomaterials, 28 April-2 May 1993, Birmingham, AL, USA, 1993, p. 337. Keller, J. C., Zaharias, R., Chang, Y. L. and Hurson, S., Osteoblast responses to HA coatings of varying crystallinity. J. Dent. Res., 1995, 74,111. Curtis, A. S. G. and Clark, P., The effects of topographic and mechanical properties of materials on cell behaviour. Crit. Rev. Biocompat., 1990, 5, 343-363. Hanein, D., Sabanay, H., Addadi, L. and Geiger, B., Selective interactions of cells with crystal surfaces. J. Cell Sci., 1993, 104,275-288. Albrektsson, T., Elranemark, P.I., Hansson, H.A. and Lindstrom, J., Osseointegrated titanium implants. Requirements for ensuring a long-lasting direct boneto-implant anchorage in man. Acta Orthop. &and., 1981,52,155-170. Jarcho, M., Kay, J. F., Gumaer, K. I., Doremus, R. H. and Drobeck, H.P., Tissue, cellular, and subcellular events at a bone-ceramic hydroxyapatite interface. J. Bioeng., 1977,1, 79-92. Hench, L. L. and F’aschall, H. A., Direct chemical bond of bioactive glass-ceramic materials to bone and muscle. J, Biomed. Mater. Res., 1973, 7, 25-42. Keller, J.C., Zaharias, R., Chang, T.L. and Hurson, S., Osteoblast responses to HA coatings of varying crystallinity. J. Dent. Res., 1995, 74, 111. van Bitterswijk, C. A., Leenders, H., v.d. Brink, J. et al., Degradation and interface reactions of hydroxyapatite coatings: effect o,F crystallinity. Transactions of the 19th Annual Meering of the Society for Biomaterials, 1993, p. 337. Ong, J. L., Hoppe, C. A., Cardenas, H. L. et nl., Osteoblast precursor cell activity on HA surfaces of different treatments. J. Biomed. Mater. Res. (in press). Hanein, D., Sabanay, H., Addadi, L. and Geiger, B., Selective interactions of cells with crystal surfaces. J. Cell Sci., 1993, 104,275-288. Dhert, W. J. A., Klem, C.P.A. T., Wolker, J. G. C., van der Velde, A. A., de Groot, K. and Rozing, P.M., Fluorapamagnesiumwhitlockite-, and hydroxyapatitetite-, coated titanium plugs: mechanical bonding and the effect of different implantation sites. In Ceramics in Surgery, ed. Substitutive and Reconstructive P. Vincenzi. Elsevier, Amsterdam, 1991, pp. 385-394. Gabbi, C., Borghetti, P., Cacchioli, A., Antoletti, N. and Pitteri, S., Physical, chemical and biological characterization of hydroxyapatite coatings of differentiated crystallinity. Transactions of the 4th World Biomaterials Congress, 1992, p. 5. Maxian, S. H., Zawadski, J. P. and Dunn, M. G., Evalua-

1275

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

tion of amorphous versus crystalline hydroxyapatite coatings on smooth and rough titanium: in vitro and in vivo studies. Transactions of the 4th World Biomaterials Congress, 1992, p. 101. de Bruijn, J.D., Klein, C.P.A. T., de Groot, K. and van Blitterswijk, CA., Influence of crystal structure on the establishment of the bone calcium phosphate interface in vitro. Cell Mater., 1993, 3, 407-417. Ong, J. L., Lucas, L.C., Lacefield, W.R. and Rigney, E.D., Structure, solubility and bond strength of thin calcium phosphate coatings produced by ion beam sputter deposition. Biomaterials, 1992,13, 249-254. Ong, J. L. and Lucas, L.C., Post-deposition heat treatments for ion beam sputter deposited calcium phosphate coatings. Biomaterials, 1994, 15, 337-341. Zisman, W. A., Relation of the equilibrium contact angle to liquid and solid constitution. In Contact Angle, Wettability and Adhesion, ed. R. Gould. American Chemical Society, Washington, DC, 1964, pp. l-51. Bjursten, L.M., Gould, T.R. L., Skalak, R. et al., Basic science committee. In Tissue Integration in Oral and Maxilla-facial Reconstruction, ed. D. van Steenberghe, T. Albrektsson, P.-I. Branemark, P. J. Henry, R. Holt and G. Liden. Proceedings of an International Congress, Brussels, May 1985. Excerpta Medica, Princeton, 1986, pp. 511-513. Hoppe, CA., Ong, J. L., Carries, D.L., Cardenas, H. L. and Sogal, A., Osteoblast response to HA ceramics of different crystallinity. J. Dent. Res., 1996, 75,78. Jarcho, M., Bolen, C. H., Thomas, M. B., Bobick, J., Kay, J. F. and Doremus, R. H., Hydroxylapatite synthesis and characterization in dense polycrystalline form. J. Mater. Sci., 1976,11,2027-2035. Ong, J. L., Lucas, L.C., Raikar, G.N., Weimer, J. J. and Gregory, J. C., Surface characterization of ion-beam sputter-deposited Ca-P coatings after in vitro immersion. Colloid and Surfaces, 1994, 87,151-162. LeGeros, R.Z., Formation of calcium phosphates in vitro. In Calcium Phosphates in Oral Biology and Medicine. ed. M. M. Myers, Karger, New York, 1991, pp. 46-67. Leung, Y., Walters, M. A., Blumenthal, N. C., Ricci, J. L. and Spivak, J. M., Determination of the mineral phases and structure of the bone-implant interface using Raman spectroscopy. 1. Biomed. Mater. Res., 1995, 29, 591-594. Cao, Y., Weng, J., Chen, J., Feng, J., Yang, Z. and Zhang, X., Water vapour-treated hydroxyapatite coatings after plasma spraying and their characteristic. Biomaterials, 1996,17,419-424. Zalm, P. C., Quantitative sputtering. In Handbook of Ion Beam Processing Technology, ed. J. J. Cuomo, S.M. Rossnagel and H.R. Kaufman. Noyes, Park Ridge, NJ, 1989, pp. 78-111. van Dijk, K., Schaeken, H. G., Wolke, J. G. G. and Jansen, J. A., Influence of annealing temperature on RF magnetron sputtered calcium phosphate coatings. Biomaterials, 1996, 17,405-410. Kilpadi, D. V. and Lemons, J. E., Surface energy characterization of unalloyed titanium implants. J. Biomed. Mater. Res., 1994, 28,1419-1425.

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