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Effect of strontium ions on the early formation of biomimetic apatite on single crystalline rutile
Applied Surface Science 266 (2013) 199–204
Contents lists available at SciVerse ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Effect of strontium ions on the early formation of biomimetic apatite on single crystalline rutile Carl Lindahl a,b , Håkan Engqvist a,b , Wei Xia a,b,∗ a b
Applied Materials Science, Department of Engineering Sciences, Uppsala University, Uppsala, Sweden BIOMATCELL, VINN Excellence Center of Biomaterials and Cell Therapy, Gothenburg, Sweden
a r t i c l e
i n f o
Article history: Received 11 September 2012 Received in revised form 6 November 2012 Accepted 27 November 2012 Available online 17 December 2012 Keywords: Single crystalline rutile Apatite Strontium Ion adsorption
a b s t r a c t Single crystalline rutile is a good model to investigate the growth mechanism of hydroxyapatite on bioactive Ti surfaces. Previous studies have shown the difference on different crystalline rutile faces in the early stage and during the growth of HAp crystals from simulated body fluids. It is known that the biological apatite crystal is an ion-substituted apatite. Ion substitution will influence the HAp crystal growth and morphology. In the present study, the effect of strontium ions on the adsorption of Ca and phosphate ions on three different faces of single crystalline rutile substrates has been investigated. The ion adsorption is the crucial step in the nucleation of HAp crystals on specific surfaces. Single crystalline rutile surfaces ((1 1 0), (1 0 0) and (0 0 1)) were soaked in phosphate buffer solutions containing calcium and strontium ions for different time periods. The results showed that the adsorption of Sr, Ca and P is faster on the (1 1 0) surface than on the (1 0 0) and (0 0 1) surfaces. Almost same amount of Sr ion was adsorbed on the surfaces compared to the adsorption of Ca ion. Strontium ion influenced the biological apatite formation in the early stage in this study. © 2012 Elsevier B.V. All rights reserved.
1. Introduction To achieve a chemical bonding at the interface between material and bone is important to reduce the risk of the implant failure. Hydroxyapatite (HAp), the mineral phase of bone, is widely used as an implant coating for this purpose [1,2] but also bioactive surfaces can result in a chemical bonding to bone in vivo. Bioactivity of an implant surface is described as the ability to form HAp on the surface in vivo (or in simulated body fluids in vitro). The mechanism of biological apatite growth on bioactive surfaces is not fully understood [3], and to understand how bone-like apatite nucleates is important for the understanding of the mechanism of the bonding between bone and materials [3,4]. The biomineralization method based on SBF (simulated body fluid) soaking process mimic’s the bone mineral growth on bioactive surfaces [5]. If HAp forms on the surface of a material after immersing in SBF or modified SBF (mimicking the ionic content of blood plasma) the surface is considered to form a chemical bonding with bone in vivo. Crystalline titanium dioxide has previously been shown to be bioactive, due to its negative surface charge and the presence of hydroxyl groups on the surfaces [6]. The formation of hydroxyl groups occurs when H2 O is
∗ Corresponding author at: Applied Materials Science, Department of Engineering Sciences, Uppsala University, Uppsala, Sweden. Tel.: +46 18 471 3065. E-mail address: [email protected] (W. Xia). 0169-4332/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsusc.2012.11.147
adsorbed and dissociated on the surface [4]. The hydroxyl groups then induce hydroxyapatite formation nucleation and growth on its surface. The first step in the formation of hydroxyapatite is the adsorption of calcium and phosphate ions. This has proven to be a crucial step in the nucleation of apatite from simulated body fluids (SBF) were the hydroxyl groups are involved in the binding of ions like Ca and P from the body fluid [3,4,7]. From the previous studies it has been shown that the mechanism of hydroxyapatite formation in the first step involves adsorption of Ca2+ ions on the negatively charged substrate which is then followed by binding of P ions to the adsorbed Ca2+ ions forming the HAp layer. This knowledge also gives some insight in the process how bone formation occurs in vivo. However, the bone mineral is not a stochiometric HAp but a multisubstituted HAp containing traces of ions like Sr2+ , Mg2+ , F− , Si4+ in the HAp lattice [9–11]. These ions not only play an important role in the process of bone formation, but also influence the growth of HAp crystals, such as crystal size, crystallinity, and composition. One ion of interest in the bone formation process is strontium (Sr). Strontium is a trace element in bone and has been shown to incorporate into HAp crystals [8] due to its similar ion radius with Ca [9]. When strontium is incorporated into biological apatite it has also been shown that Sr2+ not only competes with Ca2+ on the binding sites, but also inhibits the first step in the mineralization of bone [10]. To evaluate how strontium effects the formation of apatite Olivera et al. have evaluated how Sr effects the Ca and P
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concentrations in SBF solution during the formation of biomimetic HAp and SrHA [11]. It was shown that there was an initial drop in the concentration of Ca and P ions in the SBF solution during HAp formation. The drop in the Ca and P concentration was delayed when Sr was added to the solution [11]. This showed that Sr could delay the formation of calcium phosphates. In addition a decrease in the crystal size was observed when adding increasing amounts of Sr to the solution [11]. Also Xia et al. reported that Sr could dramatically alter the morphology of biomimetic apatite, changing from a plate-like to a sphere-like morphology [12]. The incorporation of strontium in apatite could also increase the solubility compared to non-substituted apatite [13]. It has been reported that the adsorption of ions of Ca and phosphate is faster on the (0 0 1) and (1 0 0) surfaces than on the surface (1 1 0) of single crystalline rutile [14,15]. The single crystals were immersed in phosphate buffer solutions containing Ca and Mg ions for soaking times up to 4 weeks. It was observed that the formation of hydroxyapatite crystals was different on different faces of rutile [14,15]. Because it has been shown that Sr2+ affects both the formation of bone in vivo and also the formation of hydroxyapatite the role of strontium in the initial formation of strontium substituted hydroxyapatite is important to understand. It is also interesting to study the competition between Ca and Sr in the initial phase of hydroxyapatite formation. It can help us understand the formation of biological apatite at the interface between implant surfaces and bone. In this study, single crystalline rutile in different crystal faces, (0 0 1), (1 0 0), and (1 1 0) is used as the well controlled surfaces. The objective is to experimentally investigate if strontium has an effect on the adsorption of calcium and phosphate on the different crystal faces after soaking into strontium modified simulated body fluid from 1 h to 1 week. This is the first step in understanding the competition of the adsorption of substituted ion with calcium during the early nucleation of apatite on a well-controlled surface. 2. Materials and methods Dulbecco’s phosphate buffer saline (DPBS) solution (Dulbecco’s (Aldrich)) was used as soaking medium [15]. Sr(NO3 )2 was used as a strontium source and was added to the PBS (0.6 mM of strontium). This strontium concentration has previously been used in the study by Xia et al. to prepare strontium substituted apatite coatings on titanium substrates [12]. The size of single crystalline rutile was 5 mm × 5 mm × 0.5 mm. Before and between each experiment, the single crystalline rutile was ultrasonically cleaned in 75 mL of 0.5 M HCl for 30 min, then washed in 75 mL of MilliQ water for 30 min. Afterwards, the rutile plates were polished using 1 m diamond spray and DP lubricant blue. Each rutile plate was polished for 2.5 min, and washed by deionized water, and then the process was repeated once. After polishing, the rutile plates were washed in EtOH for 10 min and then in MilliQ water for 10 min. Then the rutile plates were treated by UV ozone for 1 h and then transferred directly to a Peri-dish filled with MilliQ water. The rutile plates were then soaked into PBS containing 0.6 mM of Sr (Sr-PBS) at 37 ◦ C and incubated for 1 h, 24 h and 1 week. The volume PBS/rutile plate was fixed at 10 mL/crystal. After soaking, the rutile plates were taken out and washed in MilliQ water gently using tweezers for 20 s and thereafter dried in LAF hood prior to analysis. 3. Characterisation The morphology of the surfaces was analyzed using a field emission scanning electron microscopy (FE-SEM, Zeiss 1550). An Inlens
Table 1 Binding energies (eV) obtained from XPS of single crystalline rutile in different crystal faces. Crystal faces
Time
O1s
Ca2p
Sr3p
(0 0 1)
0 1h 24 h 1 week 0 1h 24 h 1 week 0 1h 24 h 1 week
528.87 529.35 529.25 530.83 529.30 529.73 530.44 530.87 529.39 530.11 530.83 530.78
– 346.54 346.30 346.25 – 347.01 347.59 347.01 – 347.54 347.30 346.87
– 284.32 284.02 283.78 – 284.55 285.21 284.55 – 284.97 284.79 284.20
(1 0 0)
(1 1 0)
detector was used with an acceleration voltage of 5 kV. Energy Dispersive Spectroscopy (EDS) was performed using an acceleration voltage of 10 kV. The chemical compositions of the samples were analyzed using X-ray photoelectron spectroscopy (XPS, Physical Electronics Quantum 2000) using Al K␣ X-ray source. Since TiO2 and calcium phosphates are insulators, the electron and ion gun neutralizer were used. Atomic force microscopy (PSIA XE150 SPM/AFM) was used to analyze the surfaces roughness before and after immersion in PBS containing strontium using a non-contact mode. Samples for SEM analysis were coated with Au/Pd for 30 s using a sputter coater. Samples for XPS and AFM analyses was not treated before analysis. 4. Results After 1 h soaking none or only a few particles could be observed on the surfaces, see Fig 1. After 24 h soaking there was a clear increase in the number of particles on the surfaces compared to 1 h soaking. After 1 week soaking the number of particles on the surfaces increased even more, see Fig. 1. EDS mappings of the surfaces after one week showed Ca, P, Mg and Sr signals, see Fig. 2. This indicates that these particles were strontium doped calcium phosphate. The bonding energies of O1s, Ca2p, and Sr3p, which were obtained by XPS analysis of the different crystal faces after soaking at different soaking times, are summarized in Table 1. There was a shift in the bonding energies for all of elements with the increase of soaking time. The O1s peak detected on the clean surfaces was ascribed to the Ti O bond [16]. After soaking in Sr-PBS the O1s peak became broader, and showed an asymmetry. The O1s peak after analysis of the (1 1 0) surface soaked in Sr-PBS for 1 week was fitted using the peak deconvolution method, and could be decomposed into two components, see Fig. 3. Because the O1s peak had similar binding energy for all of the directions one direction was chosen for analysis. In addition the samples soaked for 1 week show the strongest O1s peak. The first component at 530.2 eV corresponded to the O linked to Ti as in Ti O which is from the substrate. The second at 531.6 eV was attributed to PO4 3− and OH− which corresponded to the adsorption of phosphates and hydroxyl groups from the PBS. There were an increase of Ca, P and Sr concentrations on the surfaces with immersion times, see Fig. 4. No significant difference was observed in the adsorption of Ca, P and Sr on the three directions within 1 h. After 24 h, the relative intensities for all of Ca, P and Sr were higher at the (1 1 0) face than the (1 0 0) and (0 0 1) faces. After 1 week of soaking, the adsorption at the (1 1 0) and (1 0 0) faces were higher than that at (0 0 1) face. The results indicated that the adsorption of Ca, phosphate and Sr ions at (1 1 0) face were faster up to 24 h on the (1 1 0) face, and then faster adsorption on both (1 1 0) and (1 0 0) faces appeared up to one week. The Ca/Sr ratios at (0 0 1) and (1 1 0) faces after 1 h soaking were higher than that after 24 h and 1 week soaking, see Fig. 5. The Ca/Sr
C. Lindahl et al. / Applied Surface Science 266 (2013) 199–204
Fig. 1. SEM images of the (0 0 1), (1 0 0) and (1 1 0) surfaces after soaking in Sr-PBS at 37 ◦ C for 1 h, 24 h and 1 week.
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Fig. 2. EDS mapping of deposits observed on the (001), (1 0 0) and (1 1 0) surfaces after soaking in Sr-PBS at 37 ◦ C for 1 week.
ratios at (0 0 1) and (1 1 0) faces after 1 h soaking were 1.2. However, the adsorption of Ca and Sr were almost similar after 24 h of soaking. This showed that the early adsorption of Ca at (0 0 1) and (1 1 0) faces was quicker than Sr, however, the adsorption of Sr increased with increase of soaking time. The (Ca + Sr)/P ratio in all cases was far below the stoichiometric Ca/P ratio for hydroxyapatite. The initial (Ca + Sr)/P ratio at 1 h for (0 0 1) and (1 1 0) faces was a little higher than the ratio at 24 h and 1 week. The surface roughness of different crystal faces (0 0 1), (1 0 0) and (1 1 0) were evaluated before and after immersion in Sr-PBS at 37 ◦ C, see Fig. 6. The surface roughness increased with immersion time in Sr-PBS for all crystal faces as expected. The surface roughness was found to be higher on the (1 1 0) face than the (0 0 1) and (1 0 0) face after 24 h. After 1 weeks immersion the surface roughness was higher for the (1 1 0) face and (1 0 0) face than the (0 0 1) face, see Fig. 6.
O1s
O1s Peak 1 Peak 2
2
1
5. Discussion
540
538
536
534
532
530
528
526
Binding energy (eV) Fig. 3. XPS peak fitting of O1s obtained from XPS analysis of (1 1 0) surface after soaking in Sr-PBS for 1 week; 1: Oxides, 2: PO4 , OH.
In this study, strontium has been added into the phosphate buffer solution to study its influence on the adsorption of Ca and P in the early stage of hydroxyapatite formation. Because Sr ion has ˚ compared to Ca ionic radius (0.99 A), ˚ a similar ionic radius (1.13 A) thus Ca could be easily replaced by Sr in the HA lattice. Is there a
C. Lindahl et al. / Applied Surface Science 266 (2013) 199–204
Calcium adsorpon (A) 1h
Ra(nm)
at%
9 8 7 6 5 4 3 2 1 0
24h 1week
[001]
[100]
[110]
20 18 16 14 12 10 8 6 4 2 0
203
clean 1h 24h 1week
[001]
[100]
[110]
Crystal direcon 30 25
(B) 1h 24h 1week
20
at%
Fig. 6. Surface roughness for the different crystal directions (0 0 1), (1 0 0) and (1 1 0) before and after immersion into Sr-PBS at 37 ◦ C for 1 h, 24 h, 1 weeks soaking.
Phosphate adsorpon
15 10 5 0
[001]
[100]
[110]
Crystal direcon 9 8
Stronum adsorpon (C)
7
at%
6
1h
5
24h
4
1week
3 2 1 0
[001]
[100]
[110]
Crystal direcon Fig. 4. Atomic % of (A) Calcium, (B) Phosphate and (C) Strontium for different crystal surfaces and immersion times.
Ca/Sr
1.6 1.4
1h
1.2
24h
1
1 week
0.8 0.6 0.4 0.2 0
[001]
[100]
[110]
crystal direcon
(Ca+Sr)/P
0.7
6. Conclusions
1h 24h 1 week
0.6 0.5 0.4 0.3 0.2 0.1 0
[001]
[100]
natural selection between Sr and Ca during HAp formation? How the competition performs in this process, especially in the early stage of HA formation, is not clear. Our results showed that the adsorption of Ca and Sr was almost the same after 24 h soaking. Within 1 h, the adsorption of Ca was higher than Sr, showing that the adsorption of Ca was quicker than Sr. SEM analysis of the surfaces after 1 h soaking show no or very few particles on the surface. This indicates that mostly adsorption of ions had occurred within 1 h soaking in Sr-PBS. Then the adsorption of Sr increased more quickly, and the ratio of Ca/Sr was close to one after 24 h. Analysis of the surfaces after 24 h also shows formation of calcium phosphate containing strontium according to SEM and EDS analysis. Previous studies has shown that the adsorption of Ca and P ions on the (0 0 1) and (1 0 0) surface is faster than on the (1 1 0) surface. In the present study the adsorption of Ca and P ions on different crystal faces was changed when Sr was added to the solution. The adsorption of ions was faster on (1 1 0) surface up to 24 h. It seems that the addition of Sr in the solution affects not only the adsorption of Ca, but also other ions adsorption on different crystal faces, and further influence the formation of biological apatite. The low (Ca + Sr)/P ratio indicates that there is only amorphous apatite even after the substrates were soaked for 1 week in the buffer solution. The substitution of Sr could be close to 50% according to results in the early stage of HAp formation. Studies by Uchida et al. have shown that the bone bonding of titanium oxides was due to the presence of hydroxyl groups on its surface that induced apatite growth [17]. The peak fitting of O1s suggested that the hydroxyl groups appeared within 1 h soaking. A downshift in binding energy for the O1s peak when comparing a clean (1 1 0) surface with a (1 1 0) surface immersed in PBS has also previously has been shown [15]. When Ca or Sr is bonded to the hydroxyl groups, Ca O and Sr O bonds are formed according to theoretical investigations [4,18]. The negatively charged Ti OH groups incorporate positively charged calcium ions in the fluid. The strontium, also positively charged, can be adsorbed on the surfaces in the same way as Ca ion by interacting with Ti OH groups on the surface. So there is competition between Ca and Sr ions adsorption.
[110]
crystal direcon Fig. 5. Ca/Sr and (Ca + Sr)/P ratios at different crystal faces for varied immersion times in Sr-PBS.
The results in this study show that Sr ions affected the adsorption of Ca ions in the initial stages of strontium apatite formation on single crystalline rutile. Within one hour’s soaking the adsorption of Ca ions was quicker than Sr ions. However, after 1 h, the adsorption of Sr ions was increased. After 24 h soaking the adsorption of Ca and Sr ions was almost equal on the different crystal faces. Furthermore, the results also showed that the adsorption of Sr, Ca and P is faster on the (1 1 0) surface than on the (0 0 1) and (1 0 0) surfaces up to 24 h soaking. After one week’s soaking, the adsorption of ions was faster on the (1 1 0) and (1 0 0) faces than on (0 0 1) face. The results could be used to explain how a multi-substituted
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hydroxyapatite starts to form on bioactive surfaces in a biological surrounding. References [1] M. Vallet-Regí, Ceramics for medical applications, Journal of Chemical Society, Dalton Transactions (2001) 97–108. [2] T. Kokubo, Recent progress in glass-based materials for biomedical applications, Journal of the Ceramic Society Japan 99 (1991) 965–973. [3] H. Takadama, H. Kim, F. Miyaji, T. Kokubo, T. Nakamura, Mechanism of apatite formation induced by silanol groups – TEM observation, Journal of the Ceramic Society Japan 108 (2000) 118–121. [4] M. Svetina, L.C. Ciacchi, O. Sbaizero, S. Meriani, A.D. Vita, Deposition of calcium ions on rutile (1 1 0): a first-principles investigation, Acta Materialia 49 (2001) 2169–2177. [5] T. Kokubo, H. Takadama, How useful is SBF in predicting in vivo bone bioactivity, Biomaterials 27 (2006) 2907–2915. [6] P. Li, C. Ohtsuki, T. Kukobo, The role of hydrated silica, titania and alumina in inducing apatite on implants, Journal of Biomedical Materials Research 28 (1994) 7–15. [7] T. Miyazaki, H.M. Kim, T. Kokubo, C. Ohtsuki, H. Kato, T. Nakamura, Mechanism of bonelike apatite formation on bioactive tantalum metal in a simulated body fluid, Biomaterials 23 (2002) 827–832. [8] E. Landi, A. Tampieri, G. Celotti, S. Sprio, M. Sandri, G. Logroscino, Sr-substituted hydroxyapatites for osteoporotic bone replacement, Acta Biomaterialia 3 (2007) 961–969. [9] Z.Y. Li, W.M. Lam, C. Yang, B. Xu, G.X. Ni, S.A. Abbah, K.M.C. Cheung, K.D.K. Luk, W.W. Lu, Chemical composition, crystal size and lattice structural changes after incorporation of strontium into biomimetic apatite, Biomaterials 28 (2007) 1452–1460.
[10] J.F. Staub, E. Foos, B. Courtin, R. Jochemsen, A.M. Perault-Staub, A nonlinear compartmental model of Sr metabolism. II. Its physiological relevance for Ca metabolism, American Journal of Physiology - Regulatory, Integrative and Comparative Physiology 284 (2003) R835–R852. [11] A. Oliveira, R. Reis, P. Li, Strontium-substituted apatite coating grown on Ti6Al4V substrate through biomimetic synthesis, Journal of Biomedical Materials Research Part B: Applied Biomaterials 83B (2007) 258–265. [12] W. Xia, C. Lindahl, J. Lausmaa, P. Borchardt, A. Ballo, P. Thomsen, H. Engqvist, Biomineralized strontium-substituted apatite/titanium dioxide coating on titanium surfaces, Acta Biomaterialia 6 (2010) 1591–1600. [13] H.B. Pan, Z.Y. Li, W.M. Lam, J.C. Wong, B.W. Darvell, K.D.K. Luk, Solubility of strontium substituted apatite by solid titration, Acta Biomaterialia 5 (2009) 1678–1685. [14] F. Lindberg, J. Heinrichs, F. Ericson, P. Thomsen, H. Engqvist, Hydroxylapatite growth on single-crystal rutile substrates, Biomaterials 29 (2008) 3317–3323. [15] C. Lindahl, P. Borchardt, J. Lausmaa, W. Xia, H. Engqvist, Studies of early growth mechanisms of hydroxyapatite on single crystalline rutile: a model system for bioactive surfaces, Journal of Materials Science: Materials in Medicine 21 (2010) 2743–2749. [16] S. Kaciulis, G. Mattogno, L. Pandolfi, M. Cavalli, G. Gnappi, A. Montenero, XPS study of apatite-based coatings prepared by sol–gel technique, Applied Surface Science 151 (1999) 1–5. [17] M. Uchida, H. Kim, T. Kokubo, S. Fujibashi, T. Nakamura, Structural dependence of apatite formation on titania gels in a simulated body fluid, Journal of Biomedical Materials Research 64A (2003) 164–170. [18] V. Kohli, Z. Zhang, C. Park, P. Fenter, Rb+ and Sr2+ Adsorption at the TiO2 (1 1 0)-electrolyte interface observed with resonant anomalous X-ray reflectivity, Langmuir 26 (2010) 950–958.
Contents lists available at SciVerse ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Effect of strontium ions on the early formation of biomimetic apatite on single crystalline rutile Carl Lindahl a,b , Håkan Engqvist a,b , Wei Xia a,b,∗ a b
Applied Materials Science, Department of Engineering Sciences, Uppsala University, Uppsala, Sweden BIOMATCELL, VINN Excellence Center of Biomaterials and Cell Therapy, Gothenburg, Sweden
a r t i c l e
i n f o
Article history: Received 11 September 2012 Received in revised form 6 November 2012 Accepted 27 November 2012 Available online 17 December 2012 Keywords: Single crystalline rutile Apatite Strontium Ion adsorption
a b s t r a c t Single crystalline rutile is a good model to investigate the growth mechanism of hydroxyapatite on bioactive Ti surfaces. Previous studies have shown the difference on different crystalline rutile faces in the early stage and during the growth of HAp crystals from simulated body fluids. It is known that the biological apatite crystal is an ion-substituted apatite. Ion substitution will influence the HAp crystal growth and morphology. In the present study, the effect of strontium ions on the adsorption of Ca and phosphate ions on three different faces of single crystalline rutile substrates has been investigated. The ion adsorption is the crucial step in the nucleation of HAp crystals on specific surfaces. Single crystalline rutile surfaces ((1 1 0), (1 0 0) and (0 0 1)) were soaked in phosphate buffer solutions containing calcium and strontium ions for different time periods. The results showed that the adsorption of Sr, Ca and P is faster on the (1 1 0) surface than on the (1 0 0) and (0 0 1) surfaces. Almost same amount of Sr ion was adsorbed on the surfaces compared to the adsorption of Ca ion. Strontium ion influenced the biological apatite formation in the early stage in this study. © 2012 Elsevier B.V. All rights reserved.
1. Introduction To achieve a chemical bonding at the interface between material and bone is important to reduce the risk of the implant failure. Hydroxyapatite (HAp), the mineral phase of bone, is widely used as an implant coating for this purpose [1,2] but also bioactive surfaces can result in a chemical bonding to bone in vivo. Bioactivity of an implant surface is described as the ability to form HAp on the surface in vivo (or in simulated body fluids in vitro). The mechanism of biological apatite growth on bioactive surfaces is not fully understood [3], and to understand how bone-like apatite nucleates is important for the understanding of the mechanism of the bonding between bone and materials [3,4]. The biomineralization method based on SBF (simulated body fluid) soaking process mimic’s the bone mineral growth on bioactive surfaces [5]. If HAp forms on the surface of a material after immersing in SBF or modified SBF (mimicking the ionic content of blood plasma) the surface is considered to form a chemical bonding with bone in vivo. Crystalline titanium dioxide has previously been shown to be bioactive, due to its negative surface charge and the presence of hydroxyl groups on the surfaces [6]. The formation of hydroxyl groups occurs when H2 O is
∗ Corresponding author at: Applied Materials Science, Department of Engineering Sciences, Uppsala University, Uppsala, Sweden. Tel.: +46 18 471 3065. E-mail address: [email protected] (W. Xia). 0169-4332/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.apsusc.2012.11.147
adsorbed and dissociated on the surface [4]. The hydroxyl groups then induce hydroxyapatite formation nucleation and growth on its surface. The first step in the formation of hydroxyapatite is the adsorption of calcium and phosphate ions. This has proven to be a crucial step in the nucleation of apatite from simulated body fluids (SBF) were the hydroxyl groups are involved in the binding of ions like Ca and P from the body fluid [3,4,7]. From the previous studies it has been shown that the mechanism of hydroxyapatite formation in the first step involves adsorption of Ca2+ ions on the negatively charged substrate which is then followed by binding of P ions to the adsorbed Ca2+ ions forming the HAp layer. This knowledge also gives some insight in the process how bone formation occurs in vivo. However, the bone mineral is not a stochiometric HAp but a multisubstituted HAp containing traces of ions like Sr2+ , Mg2+ , F− , Si4+ in the HAp lattice [9–11]. These ions not only play an important role in the process of bone formation, but also influence the growth of HAp crystals, such as crystal size, crystallinity, and composition. One ion of interest in the bone formation process is strontium (Sr). Strontium is a trace element in bone and has been shown to incorporate into HAp crystals [8] due to its similar ion radius with Ca [9]. When strontium is incorporated into biological apatite it has also been shown that Sr2+ not only competes with Ca2+ on the binding sites, but also inhibits the first step in the mineralization of bone [10]. To evaluate how strontium effects the formation of apatite Olivera et al. have evaluated how Sr effects the Ca and P
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concentrations in SBF solution during the formation of biomimetic HAp and SrHA [11]. It was shown that there was an initial drop in the concentration of Ca and P ions in the SBF solution during HAp formation. The drop in the Ca and P concentration was delayed when Sr was added to the solution [11]. This showed that Sr could delay the formation of calcium phosphates. In addition a decrease in the crystal size was observed when adding increasing amounts of Sr to the solution [11]. Also Xia et al. reported that Sr could dramatically alter the morphology of biomimetic apatite, changing from a plate-like to a sphere-like morphology [12]. The incorporation of strontium in apatite could also increase the solubility compared to non-substituted apatite [13]. It has been reported that the adsorption of ions of Ca and phosphate is faster on the (0 0 1) and (1 0 0) surfaces than on the surface (1 1 0) of single crystalline rutile [14,15]. The single crystals were immersed in phosphate buffer solutions containing Ca and Mg ions for soaking times up to 4 weeks. It was observed that the formation of hydroxyapatite crystals was different on different faces of rutile [14,15]. Because it has been shown that Sr2+ affects both the formation of bone in vivo and also the formation of hydroxyapatite the role of strontium in the initial formation of strontium substituted hydroxyapatite is important to understand. It is also interesting to study the competition between Ca and Sr in the initial phase of hydroxyapatite formation. It can help us understand the formation of biological apatite at the interface between implant surfaces and bone. In this study, single crystalline rutile in different crystal faces, (0 0 1), (1 0 0), and (1 1 0) is used as the well controlled surfaces. The objective is to experimentally investigate if strontium has an effect on the adsorption of calcium and phosphate on the different crystal faces after soaking into strontium modified simulated body fluid from 1 h to 1 week. This is the first step in understanding the competition of the adsorption of substituted ion with calcium during the early nucleation of apatite on a well-controlled surface. 2. Materials and methods Dulbecco’s phosphate buffer saline (DPBS) solution (Dulbecco’s (Aldrich)) was used as soaking medium [15]. Sr(NO3 )2 was used as a strontium source and was added to the PBS (0.6 mM of strontium). This strontium concentration has previously been used in the study by Xia et al. to prepare strontium substituted apatite coatings on titanium substrates [12]. The size of single crystalline rutile was 5 mm × 5 mm × 0.5 mm. Before and between each experiment, the single crystalline rutile was ultrasonically cleaned in 75 mL of 0.5 M HCl for 30 min, then washed in 75 mL of MilliQ water for 30 min. Afterwards, the rutile plates were polished using 1 m diamond spray and DP lubricant blue. Each rutile plate was polished for 2.5 min, and washed by deionized water, and then the process was repeated once. After polishing, the rutile plates were washed in EtOH for 10 min and then in MilliQ water for 10 min. Then the rutile plates were treated by UV ozone for 1 h and then transferred directly to a Peri-dish filled with MilliQ water. The rutile plates were then soaked into PBS containing 0.6 mM of Sr (Sr-PBS) at 37 ◦ C and incubated for 1 h, 24 h and 1 week. The volume PBS/rutile plate was fixed at 10 mL/crystal. After soaking, the rutile plates were taken out and washed in MilliQ water gently using tweezers for 20 s and thereafter dried in LAF hood prior to analysis. 3. Characterisation The morphology of the surfaces was analyzed using a field emission scanning electron microscopy (FE-SEM, Zeiss 1550). An Inlens
Table 1 Binding energies (eV) obtained from XPS of single crystalline rutile in different crystal faces. Crystal faces
Time
O1s
Ca2p
Sr3p
(0 0 1)
0 1h 24 h 1 week 0 1h 24 h 1 week 0 1h 24 h 1 week
528.87 529.35 529.25 530.83 529.30 529.73 530.44 530.87 529.39 530.11 530.83 530.78
– 346.54 346.30 346.25 – 347.01 347.59 347.01 – 347.54 347.30 346.87
– 284.32 284.02 283.78 – 284.55 285.21 284.55 – 284.97 284.79 284.20
(1 0 0)
(1 1 0)
detector was used with an acceleration voltage of 5 kV. Energy Dispersive Spectroscopy (EDS) was performed using an acceleration voltage of 10 kV. The chemical compositions of the samples were analyzed using X-ray photoelectron spectroscopy (XPS, Physical Electronics Quantum 2000) using Al K␣ X-ray source. Since TiO2 and calcium phosphates are insulators, the electron and ion gun neutralizer were used. Atomic force microscopy (PSIA XE150 SPM/AFM) was used to analyze the surfaces roughness before and after immersion in PBS containing strontium using a non-contact mode. Samples for SEM analysis were coated with Au/Pd for 30 s using a sputter coater. Samples for XPS and AFM analyses was not treated before analysis. 4. Results After 1 h soaking none or only a few particles could be observed on the surfaces, see Fig 1. After 24 h soaking there was a clear increase in the number of particles on the surfaces compared to 1 h soaking. After 1 week soaking the number of particles on the surfaces increased even more, see Fig. 1. EDS mappings of the surfaces after one week showed Ca, P, Mg and Sr signals, see Fig. 2. This indicates that these particles were strontium doped calcium phosphate. The bonding energies of O1s, Ca2p, and Sr3p, which were obtained by XPS analysis of the different crystal faces after soaking at different soaking times, are summarized in Table 1. There was a shift in the bonding energies for all of elements with the increase of soaking time. The O1s peak detected on the clean surfaces was ascribed to the Ti O bond [16]. After soaking in Sr-PBS the O1s peak became broader, and showed an asymmetry. The O1s peak after analysis of the (1 1 0) surface soaked in Sr-PBS for 1 week was fitted using the peak deconvolution method, and could be decomposed into two components, see Fig. 3. Because the O1s peak had similar binding energy for all of the directions one direction was chosen for analysis. In addition the samples soaked for 1 week show the strongest O1s peak. The first component at 530.2 eV corresponded to the O linked to Ti as in Ti O which is from the substrate. The second at 531.6 eV was attributed to PO4 3− and OH− which corresponded to the adsorption of phosphates and hydroxyl groups from the PBS. There were an increase of Ca, P and Sr concentrations on the surfaces with immersion times, see Fig. 4. No significant difference was observed in the adsorption of Ca, P and Sr on the three directions within 1 h. After 24 h, the relative intensities for all of Ca, P and Sr were higher at the (1 1 0) face than the (1 0 0) and (0 0 1) faces. After 1 week of soaking, the adsorption at the (1 1 0) and (1 0 0) faces were higher than that at (0 0 1) face. The results indicated that the adsorption of Ca, phosphate and Sr ions at (1 1 0) face were faster up to 24 h on the (1 1 0) face, and then faster adsorption on both (1 1 0) and (1 0 0) faces appeared up to one week. The Ca/Sr ratios at (0 0 1) and (1 1 0) faces after 1 h soaking were higher than that after 24 h and 1 week soaking, see Fig. 5. The Ca/Sr
C. Lindahl et al. / Applied Surface Science 266 (2013) 199–204
Fig. 1. SEM images of the (0 0 1), (1 0 0) and (1 1 0) surfaces after soaking in Sr-PBS at 37 ◦ C for 1 h, 24 h and 1 week.
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Fig. 2. EDS mapping of deposits observed on the (001), (1 0 0) and (1 1 0) surfaces after soaking in Sr-PBS at 37 ◦ C for 1 week.
ratios at (0 0 1) and (1 1 0) faces after 1 h soaking were 1.2. However, the adsorption of Ca and Sr were almost similar after 24 h of soaking. This showed that the early adsorption of Ca at (0 0 1) and (1 1 0) faces was quicker than Sr, however, the adsorption of Sr increased with increase of soaking time. The (Ca + Sr)/P ratio in all cases was far below the stoichiometric Ca/P ratio for hydroxyapatite. The initial (Ca + Sr)/P ratio at 1 h for (0 0 1) and (1 1 0) faces was a little higher than the ratio at 24 h and 1 week. The surface roughness of different crystal faces (0 0 1), (1 0 0) and (1 1 0) were evaluated before and after immersion in Sr-PBS at 37 ◦ C, see Fig. 6. The surface roughness increased with immersion time in Sr-PBS for all crystal faces as expected. The surface roughness was found to be higher on the (1 1 0) face than the (0 0 1) and (1 0 0) face after 24 h. After 1 weeks immersion the surface roughness was higher for the (1 1 0) face and (1 0 0) face than the (0 0 1) face, see Fig. 6.
O1s
O1s Peak 1 Peak 2
2
1
5. Discussion
540
538
536
534
532
530
528
526
Binding energy (eV) Fig. 3. XPS peak fitting of O1s obtained from XPS analysis of (1 1 0) surface after soaking in Sr-PBS for 1 week; 1: Oxides, 2: PO4 , OH.
In this study, strontium has been added into the phosphate buffer solution to study its influence on the adsorption of Ca and P in the early stage of hydroxyapatite formation. Because Sr ion has ˚ compared to Ca ionic radius (0.99 A), ˚ a similar ionic radius (1.13 A) thus Ca could be easily replaced by Sr in the HA lattice. Is there a
C. Lindahl et al. / Applied Surface Science 266 (2013) 199–204
Calcium adsorpon (A) 1h
Ra(nm)
at%
9 8 7 6 5 4 3 2 1 0
24h 1week
[001]
[100]
[110]
20 18 16 14 12 10 8 6 4 2 0
203
clean 1h 24h 1week
[001]
[100]
[110]
Crystal direcon 30 25
(B) 1h 24h 1week
20
at%
Fig. 6. Surface roughness for the different crystal directions (0 0 1), (1 0 0) and (1 1 0) before and after immersion into Sr-PBS at 37 ◦ C for 1 h, 24 h, 1 weeks soaking.
Phosphate adsorpon
15 10 5 0
[001]
[100]
[110]
Crystal direcon 9 8
Stronum adsorpon (C)
7
at%
6
1h
5
24h
4
1week
3 2 1 0
[001]
[100]
[110]
Crystal direcon Fig. 4. Atomic % of (A) Calcium, (B) Phosphate and (C) Strontium for different crystal surfaces and immersion times.
Ca/Sr
1.6 1.4
1h
1.2
24h
1
1 week
0.8 0.6 0.4 0.2 0
[001]
[100]
[110]
crystal direcon
(Ca+Sr)/P
0.7
6. Conclusions
1h 24h 1 week
0.6 0.5 0.4 0.3 0.2 0.1 0
[001]
[100]
natural selection between Sr and Ca during HAp formation? How the competition performs in this process, especially in the early stage of HA formation, is not clear. Our results showed that the adsorption of Ca and Sr was almost the same after 24 h soaking. Within 1 h, the adsorption of Ca was higher than Sr, showing that the adsorption of Ca was quicker than Sr. SEM analysis of the surfaces after 1 h soaking show no or very few particles on the surface. This indicates that mostly adsorption of ions had occurred within 1 h soaking in Sr-PBS. Then the adsorption of Sr increased more quickly, and the ratio of Ca/Sr was close to one after 24 h. Analysis of the surfaces after 24 h also shows formation of calcium phosphate containing strontium according to SEM and EDS analysis. Previous studies has shown that the adsorption of Ca and P ions on the (0 0 1) and (1 0 0) surface is faster than on the (1 1 0) surface. In the present study the adsorption of Ca and P ions on different crystal faces was changed when Sr was added to the solution. The adsorption of ions was faster on (1 1 0) surface up to 24 h. It seems that the addition of Sr in the solution affects not only the adsorption of Ca, but also other ions adsorption on different crystal faces, and further influence the formation of biological apatite. The low (Ca + Sr)/P ratio indicates that there is only amorphous apatite even after the substrates were soaked for 1 week in the buffer solution. The substitution of Sr could be close to 50% according to results in the early stage of HAp formation. Studies by Uchida et al. have shown that the bone bonding of titanium oxides was due to the presence of hydroxyl groups on its surface that induced apatite growth [17]. The peak fitting of O1s suggested that the hydroxyl groups appeared within 1 h soaking. A downshift in binding energy for the O1s peak when comparing a clean (1 1 0) surface with a (1 1 0) surface immersed in PBS has also previously has been shown [15]. When Ca or Sr is bonded to the hydroxyl groups, Ca O and Sr O bonds are formed according to theoretical investigations [4,18]. The negatively charged Ti OH groups incorporate positively charged calcium ions in the fluid. The strontium, also positively charged, can be adsorbed on the surfaces in the same way as Ca ion by interacting with Ti OH groups on the surface. So there is competition between Ca and Sr ions adsorption.
[110]
crystal direcon Fig. 5. Ca/Sr and (Ca + Sr)/P ratios at different crystal faces for varied immersion times in Sr-PBS.
The results in this study show that Sr ions affected the adsorption of Ca ions in the initial stages of strontium apatite formation on single crystalline rutile. Within one hour’s soaking the adsorption of Ca ions was quicker than Sr ions. However, after 1 h, the adsorption of Sr ions was increased. After 24 h soaking the adsorption of Ca and Sr ions was almost equal on the different crystal faces. Furthermore, the results also showed that the adsorption of Sr, Ca and P is faster on the (1 1 0) surface than on the (0 0 1) and (1 0 0) surfaces up to 24 h soaking. After one week’s soaking, the adsorption of ions was faster on the (1 1 0) and (1 0 0) faces than on (0 0 1) face. The results could be used to explain how a multi-substituted
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