- Email: [email protected]
Growth of bioactive surfaces on titanium and its alloys for orthopaedic and dental implants
Materials Science and Engineering C 22 (2002) 53 – 60 www.elsevier.com/locate/msec
Growth of bioactive surfaces on titanium and its alloys for orthopaedic and dental implants F.J. Gil a,*, A. Padro´s b, J.M. Manero a, C. Aparicio a, M. Nilsson a, J.A. Planell a a
Department of Materials Science and Metallurgy, E.T.S.E.I.B., Universidad Polite´cnica de Catalun˜a, Av Diagonal 647, 08028 Barcelona, Spain b Instituto Padro´s, Martı´ i Julia`, Barcelona, Spain Received 5 September 2000; accepted 3 June 2001
Abstract A simple chemical method was established for inducing bioactivity of titanium and its alloys. Recently, T. Kokubo demonstrated that an in vitro chemical-deposited bone-like apatite on Ti with bone-bonding ability could be induced. Following treatment, a dense bone-like apatite layer is formed on the surface of the titanium in simulated body fluid (SBF). Observation of the samples in wet state by means of the environmental scanning electron microscope (ESEM) enabled us to observe the calcium phosphate deposition process in situ over a number of days. One of the most important features of the study is that it was carried out on a single, unchanged titanium sample and the process was not at any stage interrupted. Moreover, it was demonstrated that human osteoblast adhesion and differentiation behaviour are better in bioactive titanium than in the titanium without the chemical treatment. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Biomaterials; Apatite; Bioactivity
1. Introduction Titanium and some of its alloys are now dominant biomaterials because of their good biocompatibility. Commercially pure titanium (cp Ti) implants are alloplastic materials used as the foundation for replacing teeth in dentistry and are also used for orthopaedics [1]. However, this interaction does not involve a chemical bond with bone [2]. The lack of ability to bond chemically may lead to slow fixation of cp Ti dental implants and to their gradual loosening over a long period [3]. For the last decade, different surface modifications have been employed to provide these implants with bone-bonding ability. These mainly consist of physical coating processes on the metals involving ceramic materials, for example, BioglassR [4], CeraboneR A/W glass –ceramic [5]. Cp Ti implants, which must work as load-bearing parts, are commonly coated with a plasma-sprayed hydroxyapatite layer so as to give their surfaces bioactive properties [6]. However, the plasma-spray technique does not permit ac-
curate control of the chemical composition, crystallographic structure and crystallinity of the coating. As a result, the hydroxyapatite layer is mechanically and chemically unstable [7]. It was recently demonstrated by Kokubo et al. [8] that an in vitro chemical-deposited bone-like apatite on cp Ti could be induced by an alkali and heat treatment process followed by a simulated body fluid (SBF) soaking. This apatite layer does not have the problems associated with the plasmaspray technique [9] and is an essential requirement for artificial materials to bond to living bone [10]. In this study, Kokubo’s conditions for the alkali and heat treatment on a cp Ti plate were reproduced in order to observe the continuous steps of the in vitro bone-like apatite chemical deposition during soaking of the treated cp Ti plate in SBF. The observations were carried out with an environmental scanning electron microscope (ESEM). It is a useful tool which allowed the observation of the evolution of the titanium surface in SBF.
2. Materials and methods *
Corresponding author. Tel.: +34-3-4016708; fax: +34-3-4016706. E-mail address: [email protected] (F.J. Gil).
Commercially pure titanium plates of grade II (ASTM B 265-90) were used in this study. The titanium plates,
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 1 ) 0 0 3 8 9 - 7
54
F.J. Gil et al. / Materials Science and Engineering C 22 (2002) 53–60
Table 1 Chemical composition of the simulated body fluid and blood plasma (mM)
SBF Blood plasma
Na +
K+
Mg +
Ca2 +
Cl
HCO 3
HPO 4
SO 4
142.0 142.0
5.0 5.0
1.5 1.5
2.5 2.5
148.8 103.0
4.2 27.0
1.0 1.0
0.5 0.5
10 10 0.7 mm in size, were polished up to a 1 mm alumina suspension and washed with pure acetone and distilled water in an ultrasonic bath. They were then treated in accordance with the alkali and heat treatment described by Takadama et al. [11]. The plates were treated with 0.5 M NaOH aqueous solution at 60 C for 24 h, washed gently with distilled water and dried at 40 C for 24 h. Subsequently, they were heated up to 600 C at a rate of 5 C/min in an electric furnace, kept at 600 C for 1 h and then allowed to cool in the furnace. Finally, the treated titanium substrates were soaked in an acellular-simulated body fluid (SBF). The SBF is a solution with pH = 7.40 and an ion concentration nearly equal to those of human blood plasma (Table 1). Each plate was soaked in 40 ml of SBF at 37 C for 2 weeks. The SBF was renewed every 2 days. The samples were examined by environmental scanning electron mi-
croscopy, Electroscan 2020, with a thermoelectric cooling stage which allowed variations in temperature of F 20 C with respect to room temperature. The gas used was water vapour at 5 –10 Torr and the temperature was between 5 and 7 C in order to keep the samples hydrated as long as possible. The ESEM images were converted into TIFF format. Cell cultures were prepared with human osteoblast cells that were platted (12,100 cell/cm2) in a complete culture medium with wet atmosphere containing 5.5% of CO2 at 37 C for 24 h. Afterwards, that medium was changed by the other one with IMDM + K1, C and D3 vitamins, which induce osteoblastic differentiation. The cultures were kept in it for 72 h and, next, the number of present cells (adhesion) and the osteocalcine concentrations (differentiation) were quantified. This experimental method was based on NF S90-702 Standard. Discs of polyethylene and asmachined cp Ti were used as negative and positive controls, respectively.
3. Results and discussion Fig. 1 illustrates the appearance of a microporous layer made up of an alkaline titanate hydrogel formed during the alkaline/heat treatment described in the Materials and meth-
Fig. 1. Alkaline titanate hydrogel.
F.J. Gil et al. / Materials Science and Engineering C 22 (2002) 53–60
55
Fig. 2. X-ray diffraction of the alkaline titanate hydrogel obtained and Standard [8].
ods. This layer was characterised by a thin-film X-ray diffractometer as shown by the spectrum in Fig. 2. This spectrum shows that this surface layer is an amorphous
sodium titanate layer containing small amounts of a mixture of crystalline sodium titanates (Na2Ti5011) and rutile (TiO2). During the alkali treatment, the surface-passive TiO2 layer
Fig. 3. Apatite nucleus appeared after 3 days of immersion in SBF.
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F.J. Gil et al. / Materials Science and Engineering C 22 (2002) 53–60
partially dissolves into alkaline solution because of the corrosive attack of hydroxyl groups [8,12 – 14]. ! TiO2 þ OH HTiO 3 This reaction is assumed to proceed simultaneously with the following hydration of Ti metal [15,16].
release of alkali ions from the alkali titanate layer into SBF. The alkali release and the ion exchange with H3O + ions in simulated body fluid results in a pH increase in the surrounding fluid. The pH increase gives rise to an increase in the ionic activity product of apatite according to the following equilibrium in simulated body fluid [17,18]. 10Ca2þ þ 6PO3 4 þ 2OH ZCa10 ðPO4 Þ6 ðOHÞ2
! Ti þ 3OH TiðOHÞþ 3 þ 4e ! TiðOHÞþ 3 þ e TiO2 H2 O þ 0:5H2 ðgÞ TiðOHÞþ 3 þ OH ZTiðOHÞ4
A further hydroxyl attack to hydrated TiO2 will produce negatively charged hydrates on the surfaces of the substrates as follows. TiO2 nH2 O þ OH ZHTiO 3 nH2 O These negatively charged species are combined with alkali ions in the aqueous solution, resulting in the formation of an alkali titanate layer. During heat treatment, the hydrogel layer is dehydrated and has densified the titanate layer. When exposed to simulated body fluid, the alkali titanate layer is again hydrated to transform into TiO2 hydrogel via
The ESEM technique was used to observe the evolution of the surface appearance of a single sample in the SBF solution. The ESEM is an extremely useful tool as it enables the sample to be observed in wet state, and it is therefore not necessary to handle the sample and thus interrupt the apatite deposition reaction. When using a conventional scanning electron microscope, the sample tends to dry out as a result of the vacuum in the microscope’s chamber. Moreover, due to the low electric conductivity of the hydrogel layer, it must be covered with a gold conductive layer to prevent electrostatic charging. These factors mean that the sample could not be reused. Therefore, it could not be submerged again in SBF to allow the apatite precipitation process to continue. Fig. 3 shows how the first apatite nuclei appeared after 3 days. Fig. 4 shows that once the first nuclei had appeared, the growth of the apatite layer was very rapid. In
Fig. 4. Apatitic surface.
F.J. Gil et al. / Materials Science and Engineering C 22 (2002) 53–60
57
Fig. 5. New nucleation on the apatite structure.
this figure, the surface completely coated with the apatite can be observed. In Fig. 5, the new nucleation of apatite crystals on the apatite layer can be observed. It is possible to design the thickness of the apatite layer controlling the time of the treatment in SBF. Therefore, it is clear that the stage that controls the kinetics of the process is precisely
the nucleation stage. The nature of this deposited layer on the surface of the hydrogel is characterised by X-ray diffraction, as shown in Fig. 6. The spectrum shows a number of peaks at values around 25 – 34 and 46– 50 corresponding to the apatite; the titanate peaks were gradually disappearing.
Fig. 6. X-ray diffraction of the apatite layer.
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F.J. Gil et al. / Materials Science and Engineering C 22 (2002) 53–60
Table 2 Mean cell count and osteocalcine concentration values F standard deviation
Negative control Positive control Bioactive cp Ti
Cell count (n/0.01 cm2)
Osteocalcine concentration (ng/ml)
23.18 F 2.80 21.00 F 2.52 29.67 F 0.93
1.87 F 0.05 2.59 F 0.12 4.39 F 0.31
The main disadvantage of the convenctional methods (plasma spray) is related to the high temperature used. Due to the low thermal properties of titanium alloys, local heating of the surface is produced, thus changing the structure and the mechanical properties of the surface material. Another main problem concerns the control of the chemical composition and crystallinity of the apatite coating as well as its physical and mechanical properties during and after deposition [19 – 21]. Hydroxyapatite is thermically unstable during cooling, thus producing amorphous calcium phosphate phases. The presence of amorphous phases in the coating goes against the stability of the coating at long term after implantation. As it is known, amorphous calcium phosphates and even phases as tetra-
calcium and tricalcium phosphates, which are also formed during cooling, dissolve faster than hydroxyapatite, thus leading to complete mechanical disintegration of the coating and loosening of the implant fixation [21 – 23]. It has also been reported that adhesion of the coating to the implant is very limited, leading to early decohesion of the coating even after short implantation. This is related to the absence of any chemical link between the titanium surface and the hydroxyapatite coating. This is precisely the reason used by the new methods to improve adhesion by modifying the surface of the metal with different chemical agents so that apatite crystals could be deposited chemically. As an example, commercially pure titanium can be bioactive by attacking its oxide layer so that a gel of sodium titanate is produced, which is able to induce nucleation of apatite crystals. The human osteoblast cultures revealed that bioactive titanium surfaces had better cell response than positive and negative controls ( p-value < 0.05). The apatite layer has a significant effect on the adhesion (cell count) and differentiation (osteocalcine concentration) of osteoblast-like cells (Table 2). In Fig. 7, an osteoblast on the bioactive surface can be observed.
Fig. 7. Human osteoblast on the bioactive surface.
F.J. Gil et al. / Materials Science and Engineering C 22 (2002) 53–60
59
Fig. 8. Colony of bacilli and cocci appeared after 4 days of immersion in SBF.
One of the main drawbacks of this method was that the samples covered in apatite were susceptible to contamination by bacteria. SBF at 37 C is an ideal medium for bacterial growth unless very strict measures are taken to ensure aseptic conditions (autoclave, UV radiation, etc.). The ESEM permits easy analysis and determination of this contamination and the stage of the process at which it takes place. For example, Fig. 8 shows a colony of bacilli and cocci, which appeared after 4 days of immersion in SBF. The experiments performed on titanium samples revealed that it is after the third day of immersion in SBF, coinciding with the appearance of the apatite, that the sample is most susceptible to contamination. The fact that we can observe the deposition process in situ enables us, for instance, to study the effect of the incorporation of antibiotics such as vancomycin or gentamycin, and to see the influence of antibiotic concentration on bacteria removal. Acknowledgements This work was supported by CICYT project MAT-980415 and by Klockner Impants. The authors are specially grateful to Mercedes Rolda´n Chesa.
References [1] S.L. Carlsson, T.R. Rostlund, B. Albrektsson, T. Albrektsson, P.-I. Bra˚nemark, Osseointegration of titanium implant, Acta Orthop. Scand. 57 (1986) 285 – 289. [2] L. Sennerby, P. Thomsen, L.E. Ericson, Ultrastructure of the bonetitanium interface in rabbits, Mater. Med. 3 (1992) 262 – 271. [3] K. Takatsuka, T. Yamamuro, T. Nakamura, T. Kokubo, Bone-bonding behaviour of titanium alloy evaluated mechanically with detaching failure load, J. Biomed. Mater. Res. 29 (1995) 157 – 164. ¨ . Anderson, Bioactive glass coating, in: L.L. Hench, J. [4] L.L. Hench, O Wilson (Eds.), An Introduction to Bioceramics, World Science Singapore, Singapore, 1993, pp. 239 – 260. [5] K. Takatsuka, T. Yamamuro, T. Kitsugi, T. Nakamura, T. Shibuya, T. Goto, A new bioactive glass – ceramic as a coating material on titanium alloy, J. Appl. Biomater. 4 (1993) 317 – 329. [6] K. De Groot, R.G.T. Geesink, C.P.A.T. Klein, P. Serekian, Plasma sprayed coatings of hydroxyapatite, J. Biomed. Mater. Res. 21 (1987) 1375 – 1387. [7] C.Y. Yang, R.M. Lin, B.C. Wang, T.M. Lee, E. Chang, Y.S. Hang, P.Q. Chen, In vitro and in vivo mechanical evaluations of plasmasprayed hydroxyapatite coatings on titanium implants: the effect of coating characteristics, J. Biomed. Mater. Res. 37 (1997) 335 – 345. [8] T. Kokubo, F. Mijyaji, H.-M. Kim, Spontaneous formation of bonelike apatite layer on chemically treated titanium metals, J. Am. Ceram. Soc. 79 (1996) 1127 – 1129. [9] W.Q. Yan, T. Nakamura, M. Kobayashi, H.-M. Kim, F. Miyaji, T.
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[10] [11]
[12] [13] [14]
[15]
[16]
[17]
F.J. Gil et al. / Materials Science and Engineering C 22 (2002) 53–60 Kokubo, Bonding of chemically treated titanium implants to bone, J. Biomed. Mater. Res. 37 (1997) 267 – 275. T. Kokubo, Novel bioactive materials, An. Quim. Int. Ed., (1997) S49 – S55. H. Takadama, H.M. Kim, F. Miyaji, T. Kokubo, T. Nakamura, How does alkali-treated titanium induce apatite formation, in: R.Z. LeGeros, J.P. LeGeros (Eds.), Bioceramics vol. 11, (1998) 663 – 666. T. Hurlen, W. Wihelmsen, Passive behaviour of titanium, Electrochim. Acta 31 (1986) 1139 – 1146. K.E. Healy, P. Ducheyne, Passive dissolution kinetics of titanium in vitro, J. Mater. Sci.: Mater. Med. 4 (1993) 117 – 126. K.E. Healy, P. Ducheyne, The mechanism of passive dissolution kinetics of titanium in a model physiological environment, J. Biomed. Mater. Res. 36 (1992) 319 – 338. L.D. Arsov, C. Kormman, W. Plieth, In situ Raman spectra of anodically formed titanium dioxide layer in solution of H2SO4, KOH, and HNO3, Electrochem. Soc. 138 (1991) 2964 – 2970. H.M. Kim, F. Miyaji, T. Kokubo, T. Nakamura, Preparation of bioactive Ti and its alloys via simple surface treatment, J. Biomed. Mater. Res. 32 (1996) 409 – 417. J. Gamble, Chemical Anatomy Physiology and Pathology of Extrac-
[18] [19] [20]
[21]
[22]
[23]
ellular Fluid, 6th edn., Harvard University Press, Cambridge, MA, 1967. W. Neuman, M. Neuman, The Chemical Dynamics of Bone Mineral, University of Chicago Press, Chicago, IL, 1958. L. Cleries, In vitro studies of calcium phosphate coatings obtained by laser ablation, PhD Thesis, University of Barcelona, Spain, 1999. H. Hero, H. Wie, R.B. Jorgensen, I.E. Ruyter, Hydroxyapatite coating on titanium produced by isostatic pressing, J. Biomed. Mater. Res. 28 (1994) 344 – 348. K. De Groot, C.P. Klein, J.G. Wolke, J.M. Blieck-Hogervorst, Plasma sprayed coatings of calcium phosphate, in: T. Yamamuro, L. Hench, J. Wilson (Eds.), CRC Handbook of Bioactive Ceramics, Calcium phosphate and hydroxyapatite ceramics, vol. II, CRC Press, Boca Raton, FL, 1990, pp. 133 – 142. A.S. Posner, N.C. Blumenthal, F. Betts, in: C. Eon (Ed.), Proceedings of the 2nd International Conference on Phosphorous Compounds, Imphos, Paris, 1980, p. 25. E. Park, S.R. Condrate, D.T. Hoelzer, G.S. Fischman, Interfacial characterisation of plasma-spray coated calcium phosphate on Ti6Al4V, J. Mater. Sci. Med. 9 (1998) 643 – 649.
Growth of bioactive surfaces on titanium and its alloys for orthopaedic and dental implants F.J. Gil a,*, A. Padro´s b, J.M. Manero a, C. Aparicio a, M. Nilsson a, J.A. Planell a a
Department of Materials Science and Metallurgy, E.T.S.E.I.B., Universidad Polite´cnica de Catalun˜a, Av Diagonal 647, 08028 Barcelona, Spain b Instituto Padro´s, Martı´ i Julia`, Barcelona, Spain Received 5 September 2000; accepted 3 June 2001
Abstract A simple chemical method was established for inducing bioactivity of titanium and its alloys. Recently, T. Kokubo demonstrated that an in vitro chemical-deposited bone-like apatite on Ti with bone-bonding ability could be induced. Following treatment, a dense bone-like apatite layer is formed on the surface of the titanium in simulated body fluid (SBF). Observation of the samples in wet state by means of the environmental scanning electron microscope (ESEM) enabled us to observe the calcium phosphate deposition process in situ over a number of days. One of the most important features of the study is that it was carried out on a single, unchanged titanium sample and the process was not at any stage interrupted. Moreover, it was demonstrated that human osteoblast adhesion and differentiation behaviour are better in bioactive titanium than in the titanium without the chemical treatment. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Biomaterials; Apatite; Bioactivity
1. Introduction Titanium and some of its alloys are now dominant biomaterials because of their good biocompatibility. Commercially pure titanium (cp Ti) implants are alloplastic materials used as the foundation for replacing teeth in dentistry and are also used for orthopaedics [1]. However, this interaction does not involve a chemical bond with bone [2]. The lack of ability to bond chemically may lead to slow fixation of cp Ti dental implants and to their gradual loosening over a long period [3]. For the last decade, different surface modifications have been employed to provide these implants with bone-bonding ability. These mainly consist of physical coating processes on the metals involving ceramic materials, for example, BioglassR [4], CeraboneR A/W glass –ceramic [5]. Cp Ti implants, which must work as load-bearing parts, are commonly coated with a plasma-sprayed hydroxyapatite layer so as to give their surfaces bioactive properties [6]. However, the plasma-spray technique does not permit ac-
curate control of the chemical composition, crystallographic structure and crystallinity of the coating. As a result, the hydroxyapatite layer is mechanically and chemically unstable [7]. It was recently demonstrated by Kokubo et al. [8] that an in vitro chemical-deposited bone-like apatite on cp Ti could be induced by an alkali and heat treatment process followed by a simulated body fluid (SBF) soaking. This apatite layer does not have the problems associated with the plasmaspray technique [9] and is an essential requirement for artificial materials to bond to living bone [10]. In this study, Kokubo’s conditions for the alkali and heat treatment on a cp Ti plate were reproduced in order to observe the continuous steps of the in vitro bone-like apatite chemical deposition during soaking of the treated cp Ti plate in SBF. The observations were carried out with an environmental scanning electron microscope (ESEM). It is a useful tool which allowed the observation of the evolution of the titanium surface in SBF.
2. Materials and methods *
Corresponding author. Tel.: +34-3-4016708; fax: +34-3-4016706. E-mail address: [email protected] (F.J. Gil).
Commercially pure titanium plates of grade II (ASTM B 265-90) were used in this study. The titanium plates,
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 1 ) 0 0 3 8 9 - 7
54
F.J. Gil et al. / Materials Science and Engineering C 22 (2002) 53–60
Table 1 Chemical composition of the simulated body fluid and blood plasma (mM)
SBF Blood plasma
Na +
K+
Mg +
Ca2 +
Cl
HCO 3
HPO 4
SO 4
142.0 142.0
5.0 5.0
1.5 1.5
2.5 2.5
148.8 103.0
4.2 27.0
1.0 1.0
0.5 0.5
10 10 0.7 mm in size, were polished up to a 1 mm alumina suspension and washed with pure acetone and distilled water in an ultrasonic bath. They were then treated in accordance with the alkali and heat treatment described by Takadama et al. [11]. The plates were treated with 0.5 M NaOH aqueous solution at 60 C for 24 h, washed gently with distilled water and dried at 40 C for 24 h. Subsequently, they were heated up to 600 C at a rate of 5 C/min in an electric furnace, kept at 600 C for 1 h and then allowed to cool in the furnace. Finally, the treated titanium substrates were soaked in an acellular-simulated body fluid (SBF). The SBF is a solution with pH = 7.40 and an ion concentration nearly equal to those of human blood plasma (Table 1). Each plate was soaked in 40 ml of SBF at 37 C for 2 weeks. The SBF was renewed every 2 days. The samples were examined by environmental scanning electron mi-
croscopy, Electroscan 2020, with a thermoelectric cooling stage which allowed variations in temperature of F 20 C with respect to room temperature. The gas used was water vapour at 5 –10 Torr and the temperature was between 5 and 7 C in order to keep the samples hydrated as long as possible. The ESEM images were converted into TIFF format. Cell cultures were prepared with human osteoblast cells that were platted (12,100 cell/cm2) in a complete culture medium with wet atmosphere containing 5.5% of CO2 at 37 C for 24 h. Afterwards, that medium was changed by the other one with IMDM + K1, C and D3 vitamins, which induce osteoblastic differentiation. The cultures were kept in it for 72 h and, next, the number of present cells (adhesion) and the osteocalcine concentrations (differentiation) were quantified. This experimental method was based on NF S90-702 Standard. Discs of polyethylene and asmachined cp Ti were used as negative and positive controls, respectively.
3. Results and discussion Fig. 1 illustrates the appearance of a microporous layer made up of an alkaline titanate hydrogel formed during the alkaline/heat treatment described in the Materials and meth-
Fig. 1. Alkaline titanate hydrogel.
F.J. Gil et al. / Materials Science and Engineering C 22 (2002) 53–60
55
Fig. 2. X-ray diffraction of the alkaline titanate hydrogel obtained and Standard [8].
ods. This layer was characterised by a thin-film X-ray diffractometer as shown by the spectrum in Fig. 2. This spectrum shows that this surface layer is an amorphous
sodium titanate layer containing small amounts of a mixture of crystalline sodium titanates (Na2Ti5011) and rutile (TiO2). During the alkali treatment, the surface-passive TiO2 layer
Fig. 3. Apatite nucleus appeared after 3 days of immersion in SBF.
56
F.J. Gil et al. / Materials Science and Engineering C 22 (2002) 53–60
partially dissolves into alkaline solution because of the corrosive attack of hydroxyl groups [8,12 – 14]. ! TiO2 þ OH HTiO 3 This reaction is assumed to proceed simultaneously with the following hydration of Ti metal [15,16].
release of alkali ions from the alkali titanate layer into SBF. The alkali release and the ion exchange with H3O + ions in simulated body fluid results in a pH increase in the surrounding fluid. The pH increase gives rise to an increase in the ionic activity product of apatite according to the following equilibrium in simulated body fluid [17,18]. 10Ca2þ þ 6PO3 4 þ 2OH ZCa10 ðPO4 Þ6 ðOHÞ2
! Ti þ 3OH TiðOHÞþ 3 þ 4e ! TiðOHÞþ 3 þ e TiO2 H2 O þ 0:5H2 ðgÞ TiðOHÞþ 3 þ OH ZTiðOHÞ4
A further hydroxyl attack to hydrated TiO2 will produce negatively charged hydrates on the surfaces of the substrates as follows. TiO2 nH2 O þ OH ZHTiO 3 nH2 O These negatively charged species are combined with alkali ions in the aqueous solution, resulting in the formation of an alkali titanate layer. During heat treatment, the hydrogel layer is dehydrated and has densified the titanate layer. When exposed to simulated body fluid, the alkali titanate layer is again hydrated to transform into TiO2 hydrogel via
The ESEM technique was used to observe the evolution of the surface appearance of a single sample in the SBF solution. The ESEM is an extremely useful tool as it enables the sample to be observed in wet state, and it is therefore not necessary to handle the sample and thus interrupt the apatite deposition reaction. When using a conventional scanning electron microscope, the sample tends to dry out as a result of the vacuum in the microscope’s chamber. Moreover, due to the low electric conductivity of the hydrogel layer, it must be covered with a gold conductive layer to prevent electrostatic charging. These factors mean that the sample could not be reused. Therefore, it could not be submerged again in SBF to allow the apatite precipitation process to continue. Fig. 3 shows how the first apatite nuclei appeared after 3 days. Fig. 4 shows that once the first nuclei had appeared, the growth of the apatite layer was very rapid. In
Fig. 4. Apatitic surface.
F.J. Gil et al. / Materials Science and Engineering C 22 (2002) 53–60
57
Fig. 5. New nucleation on the apatite structure.
this figure, the surface completely coated with the apatite can be observed. In Fig. 5, the new nucleation of apatite crystals on the apatite layer can be observed. It is possible to design the thickness of the apatite layer controlling the time of the treatment in SBF. Therefore, it is clear that the stage that controls the kinetics of the process is precisely
the nucleation stage. The nature of this deposited layer on the surface of the hydrogel is characterised by X-ray diffraction, as shown in Fig. 6. The spectrum shows a number of peaks at values around 25 – 34 and 46– 50 corresponding to the apatite; the titanate peaks were gradually disappearing.
Fig. 6. X-ray diffraction of the apatite layer.
58
F.J. Gil et al. / Materials Science and Engineering C 22 (2002) 53–60
Table 2 Mean cell count and osteocalcine concentration values F standard deviation
Negative control Positive control Bioactive cp Ti
Cell count (n/0.01 cm2)
Osteocalcine concentration (ng/ml)
23.18 F 2.80 21.00 F 2.52 29.67 F 0.93
1.87 F 0.05 2.59 F 0.12 4.39 F 0.31
The main disadvantage of the convenctional methods (plasma spray) is related to the high temperature used. Due to the low thermal properties of titanium alloys, local heating of the surface is produced, thus changing the structure and the mechanical properties of the surface material. Another main problem concerns the control of the chemical composition and crystallinity of the apatite coating as well as its physical and mechanical properties during and after deposition [19 – 21]. Hydroxyapatite is thermically unstable during cooling, thus producing amorphous calcium phosphate phases. The presence of amorphous phases in the coating goes against the stability of the coating at long term after implantation. As it is known, amorphous calcium phosphates and even phases as tetra-
calcium and tricalcium phosphates, which are also formed during cooling, dissolve faster than hydroxyapatite, thus leading to complete mechanical disintegration of the coating and loosening of the implant fixation [21 – 23]. It has also been reported that adhesion of the coating to the implant is very limited, leading to early decohesion of the coating even after short implantation. This is related to the absence of any chemical link between the titanium surface and the hydroxyapatite coating. This is precisely the reason used by the new methods to improve adhesion by modifying the surface of the metal with different chemical agents so that apatite crystals could be deposited chemically. As an example, commercially pure titanium can be bioactive by attacking its oxide layer so that a gel of sodium titanate is produced, which is able to induce nucleation of apatite crystals. The human osteoblast cultures revealed that bioactive titanium surfaces had better cell response than positive and negative controls ( p-value < 0.05). The apatite layer has a significant effect on the adhesion (cell count) and differentiation (osteocalcine concentration) of osteoblast-like cells (Table 2). In Fig. 7, an osteoblast on the bioactive surface can be observed.
Fig. 7. Human osteoblast on the bioactive surface.
F.J. Gil et al. / Materials Science and Engineering C 22 (2002) 53–60
59
Fig. 8. Colony of bacilli and cocci appeared after 4 days of immersion in SBF.
One of the main drawbacks of this method was that the samples covered in apatite were susceptible to contamination by bacteria. SBF at 37 C is an ideal medium for bacterial growth unless very strict measures are taken to ensure aseptic conditions (autoclave, UV radiation, etc.). The ESEM permits easy analysis and determination of this contamination and the stage of the process at which it takes place. For example, Fig. 8 shows a colony of bacilli and cocci, which appeared after 4 days of immersion in SBF. The experiments performed on titanium samples revealed that it is after the third day of immersion in SBF, coinciding with the appearance of the apatite, that the sample is most susceptible to contamination. The fact that we can observe the deposition process in situ enables us, for instance, to study the effect of the incorporation of antibiotics such as vancomycin or gentamycin, and to see the influence of antibiotic concentration on bacteria removal. Acknowledgements This work was supported by CICYT project MAT-980415 and by Klockner Impants. The authors are specially grateful to Mercedes Rolda´n Chesa.
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