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A new simplified calcifying solution to synthesize calcium phosphate coatings
Surface & Coatings Technology 232 (2013) 13–21
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A new simplified calcifying solution to synthesize calcium phosphate coatings Barbara Bracci, Silvia Panzavolta ⁎, Adriana Bigi Department of Chemistry “G. Ciamician”, University of Bologna, Via Selmi 2, 40126 Bologna, Italy
a r t i c l e
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Article history: Received 19 February 2013 Accepted in revised form 20 April 2013 Available online 4 May 2013 Keywords: Biomimetic Coatings Calcium phosphates Phosphate buffer
a b s t r a c t In this work we set up new experimental conditions to deposit biomimetic coatings of different calcium phosphates onto titanium (Ti) substrates using over-simplified, economically convenient, slightly supersaturated solutions. The new supersaturated solutions, CaPPs, contain just calcium chloride and phosphate buffer, without addition of further salts and organic buffers usually employed for biomimetic coatings. The product precipitated at pH 7.2 and 37 °C was constituted of spherical aggregates of poorly crystalline hydroxyapatite (HA), similar to that obtained using HEPES as buffer system. Reduction of starting pH, which was varied from 7.2 to 6.6, promoted the precipitation of the kinetically favored phase, octacalcium phosphate (OCP), together with HA. Furthermore, OCP could be obtained as a single phase by increasing the Ca/P molar ratio of the calcifying solution from 1/1 to 2/1. Temperature reduction from 37 °C to 25 °C promoted the co-precipitation of calcium monohydrogen phosphate dihydrate (DCPD) together with HA and OCP, in agreement with the solubility isotherms of the different calcium phosphates. Variation of the experimental conditions was utilized to synthesize the coatings of spherical aggregates of poorly crystalline HA, petal-like crystalline OCP, as well as coatings containing both HA and OCP, onto titanium substrates in a few hours. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Short and long-term performance of dental and orthopedic implants is related to early osteointegration [1–3]. Calcium phosphate coating of metallic implants is generally accepted to be advantageous for clinical success. In fact, it allows retaining the excellent mechanical properties of titanium (Ti) and its alloys, widely employed in this field, while providing the surface of the implant with the bioactivity of calcium phosphates. A variety of physical techniques are commercially applied to this purpose [4–7]. However, physically deposited coatings exhibit several inherent drawbacks that can be overcome by wet-chemical deposition techniques [8]. In particular, the biomimetic approach involves deposition from slightly supersaturated solution at physiological, or nearly physiological, values of pH and temperature. The method, which leads to the deposition of a poorly crystalline hydroxyapatite (HA) phase similar to the inorganic phase of bone, is economic and can be applied to complex-shaped materials [9,10]. Moreover, it can be used to co-precipitate ions, drugs, macromolecules, proteins and growth factors [11–17]. The first formulation of a calcifying solution, known as simulated body fluid (SBF), was developed by Kokubo et al. [18] in 1990. Since then, a number of compositions have been proposed in order to accelerate the biomimetic deposition of poorly crystalline HA on metallic substrates. Most methods rely on buffer systems, such as TRIS (tris-hydroxymethyl-aminomethane)-HCl or HEPES (2-(4-(2-hydroxyethyl)-1-piperazinyl)ethane sulphonic acid)-NaOH ⁎ Corresponding author. Tel.: +39 051 2099566; fax: +39 051 2099456. E-mail address: [email protected] (S. Panzavolta). 0257-8972/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.surfcoat.2013.04.046
for the control of pH. Lactic acid/sodium lactate was also employed as buffer system [19]. We previously developed a slightly supersaturated calcium phosphate solution (CaP), characterized by a very simple composition and buffered with HEPES, which can deposit coatings of poorly crystalline HA on metallic substrates in a few hours [20]. In this work, we explored the possibility to further simplify the composition of the CaP solution, in order to reduce the coating cost without provoking significant lengthening of the deposition time. To this aim, we removed HEPES buffer and carbonate ions from the composition of the calcifying solution, and employed a phosphate buffer solution as source of phosphate ions. Furthermore, we investigated the influence of concentration, pH and temperature on the composition of the inorganic deposits. Selected conditions were used to modulate the crystalline phases deposited on Ti substrates from hydroxyapatite to octacalcium phosphate (OCP). 2. Materials and methods 2.1. Preparation of the reference CaP calcifying solution The reference CaP calcifying solution was prepared as previously reported [20]. Briefly, the reagent grade chemical CaCl2·2H2O was dissolved in double distilled water and buffered at pH 7.2 with HEPES (Ca solution). The reagent grade chemicals Na3PO4·12H2O, and NaHCO3 were dissolved in double distilled water and buffered at pH 7.2 with HEPES (P solution). CaP supersaturated calcifying solution was freshly prepared by mixing equal volumes of the Ca and the P solutions at 37 °C. The ionic concentration of the CaP solution is reported in Table 1 [20].
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Table 1 Composition and starting pH of the different calcifying solutions. Solution
Ca/P (molar ratio)
[Ca2+] (mM)
CaP CaPPC
1/1 1/1
2.5 2.5
CaPP1
1/1
2.5
CaPP2
1/1
5
CaPP3
2/1
a b
5
[CO32−] (mM)
Buffer solution
pH
2.5a 2.5b
18 18
Hepes
2.5b
–
7.2 7.2 7.0 7.2 7.0 6.8 6.6 7.0 6.8 6.6 6.8
[phosphate] (mM)
5b
2.5b
–
–
PO43−
Concentration of ions for CaP solution. Concentration of H2PO4−/HPO42− for CaPP solutions.
detector. CuKα radiation was used (40 mA, 40 kV). The 2θ range was from 3.8° to 11° and from 24° to 41° with a step size of 0.083° and a time/step of 1000 s/step. Morphological investigation of the coatings was performed using a Philips XL-20 scanning electron microscope operating at 15 kV. The samples were sputter-coated with gold before examination. For the observation of cross-sectional microstructure, the specimens, embedded into Methylmetacrylate (Merck Schuchardt, Hohenbrunn, Germany), were cross-sectioned with a Leica SP 1600 diamond saw (Leica, Milano, Italy). The specimens were polished, and sputter-coated with gold before SEM investigation. Energy dispersive X-ray spectrometry (EDX) analyses were performed in order to evaluate the Ca/P ratio of the deposits. 3. Results 3.1. Powder samples
2.2. Preparation of the new CaPP calcifying solutions The new calcifying solutions, CaPPs, were prepared using 0.1 M phosphate buffer (PBS) solutions at different pH values as sources of phosphate ions without addition of the HEPES solution. The 0.1 M phosphate buffer solutions at pH values 7.2, 7.0, 6.8, and 6.6, were prepared by mixing proper amounts of 0.2 M NaH2PO4 and 0.2 M Na2HPO4 solutions. The reagent grade chemical CaCl2·2H2O was dissolved in double distilled water (Ca solution). The P solution was prepared using the proper amounts of phosphate buffer solutions, in order to obtain the required phosphate concentration. NaHCO3, when used, was added to the P solution. CaPP solutions were freshly prepared by mixing appropriate volumes of the Ca and the P solutions at 37 °C, or at 25 °C, as reported in Table 1.
3.1.1. Characterization of the precipitates obtained at 37 °C The new calcifying solutions containing just CaCl2 as source of calcium ions, and NaH2PO4/Na2HPO4 buffer as source of phosphate ions, allowed to obtain fast precipitation of calcium phosphates and
2.3. Powder samples In order to better evaluate the effect of the different preparation methods on the inorganic phase precipitated from the calcifying solution, preliminary data were obtained on the powders precipitated in the absence of Ti substrates: after mixing the P solution with the Ca solution the temperature was kept constant either at 37 °C or 25 °C for 6 h, then the precipitate deposited on the bottom of the flask was filtered, washed with double distilled water and air-dried at 37 °C overnight. XRD measurements were performed using a PANalytical powder diffractometer equipped with a fast X'celerator detector. CuKα radiation was used (40 mA, 40 kV). The 2θ range was from 3 to 50° with a step size of 0.033° and time/step of 10 s/step. Morphological investigation was performed using a Philips XL-20 scanning electron microscope operating at 15 kV. The samples were sputter-coated with gold before examination. 2.4. Substrate coatings Ti disks Grade 2 (diameter 15 mm; thickness 0.5 mm, NextMaterials, Milano, Italy) were cleaned by means of soaking in an acidic mixture, containing 30% HNO3 and 3% HF in distilled water (10 ml/2 disks), then rinsed with distilled water and dried at 37 °C overnight, prior to use. Coating of the Ti substrates was performed by immersion of the disk-shaped samples in the calcifying solutions at 37 °C for 6 h. In some experiment, the calcifying solution was refreshed after 3 h. Coated samples were accurately rinsed in double distilled water, and dried at 37 °C overnight. XRD measurements were performed on the coatings using a PANalytical powder diffractometer equipped with a fast X'celerator
Fig. 1. Powder X-ray diffraction patterns of the powder samples obtained at 37 °C.
B. Bracci et al. / Surface & Coatings Technology 232 (2013) 13–21 Table 2 Crystalline phase composition of the products synthesized under different conditions. Sample
pH
37 °C
25 °C
CaPP1 CaPP1 CaPP1 CaPP1 CaPP2 CaPP2 CaPP2 CaPP2 CaPP3
7.2 7.0 6.8 6.6 7.2 7.0 6.8 6.6 6.8
HA HA HA/OCP n.d. HA/OCP HA/OCP HA/OCP HA/OCP OCP
HA/OCP HA/OCP HA/OCP n.d. HA/OCP HA/OCP/DCPD HA/OCP/DCPD HA/OCP/DCPD OCP/DCPD
n.d.: not detectable.
to modulate the composition of the coating by variation of the experimental conditions. The XRD patterns of the powders obtained at pH 7.2 and 7.0 from the calcifying solutions containing carbonate (CaPPC), revealed the presence of poorly crystalline HA, very similar to the one obtained from the reference CaP solution (Fig. 1). The product obtained using
Fig. 2. Powder X-ray diffraction patterns of the powder samples obtained at 37 °C. The main reflections of OCP are indicated with (*).
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a starting pH 7.0 (Fig. 1c) displayed slightly broader X-ray diffraction peaks than the material precipitated from the pH 7.2 solution (Fig. 1b), indicating a lower degree of crystallinity. The presence of NaHCO3 prevented the possibility to prepare solution at pH values lower than 7.0, therefore further investigations were performed on solutions not including NaHCO3 in their composition. The XRD patterns of the precipitates obtained in absence of NaHCO3 (CaPP1) still showed the presence of poorly crystalline hydroxyapatite as unique phase at pH 7.2 and 7.0 (Table 2, Fig. 2a,b). However, on comparing the XRD patterns reported in Fig. 2 with those shown in Fig. 1, it is evident that the products precipitated in the absence of carbonate display a higher degree of crystallinity than those obtained in the presence of carbonate, in agreement with the reduced broadening of the diffraction peaks. The amount of precipitate was reduced at pH 6.8, when OCP appeared as a second phase (Fig. 2c). No precipitate was appreciable at pH 6.6. Electron microscope images showed that the sample obtained from CaP at pH 7.2 is constituted of almost spherical aggregates with mean diameter of about 2–4 μm (Fig. 3a) while the mean dimensions
Fig. 3. SEM micrographs of the powder samples obtained at 37 °C. (a) CaP pH 7.2; (b) CaPP1 pH 7.2; and (c) CaPP1 pH 6.8. Bar = 5 μm.
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Fig. 4. Powder X-ray diffraction pattern and SEM micrograph of the powder sample CaPP3 obtained at 37 °C. Bar = 6 μm.
of the spherical aggregates decreased to 1 μm in the sample obtained from CaPP1 at pH 7.2 (Fig. 3b). Powders obtained from CaPP1 at pH 6.8 presented a disordered petal-like morphology (Fig. 3c). In order to increase the amount of precipitation, the concentrations of the Ca and P solutions were doubled (CaPP2), which provoked the onset of precipitation just after mixing the Ca and P solutions. The XRD patterns of CaPP2 powder revealed the presence of both HA and OCP in the explored pH range 7.2–6.6. The crystalline phases identified by means of XRD analysis are reported in Table 2. Further modifications of composition allowed to verify that a Ca/P molar ratio of 2/1 (CaPP3) at pH 6.8 promoted the precipitation of pure crystalline OCP, with the characteristic petal-like morphology (Fig. 4). The absence of the diffraction peak at 10.8°/2θ confirms the absence of HA. 3.1.2. Characterization of the precipitates obtained at 25 °C As it occurred at 37 °C, the syntheses performed at 25 °C from CaPP1 yielded no precipitation at pH 6.6. In the range of pH 7.2–6.8 the precipitates contained always OCP, together with HA (Table 2). Doubling the concentration of Ca and P solutions (CaPP2) still gave the precipitation of a mixed OCP/HA phase. However, the 11.68°/2θ reflection indicated the presence of a third phase, CaHPO4·2H2O (DCPD), in the pH range 7.0–6.6. The relative amount of DCPD increased on decreasing pH (Fig. 5, Table 2). Similarly, the precipitate obtained from the CaPP3 solution at pH 6.8 contained DCPD together with poorly crystalline HA/OCP (Table 2). 3.2. Substrate coatings On the basis of the results obtained for the powder samples, different conditions were tested to coat Ti substrates. Table 3 reports the results of the coating experiments performed at 37 °C using CaPP1 at pH 7.2 and at pH 7.0, and CaPP3 at pH 6.8. For comparison, some coating experiments were performed using the reference CaP solution containing carbonate and buffered with HEPES at pH 7.2 and 7.0, as well as at pH 6.8. In this last condition, the Ca/P molar ratio of the CaP solution was 2/1 (this allowed direct comparison with CaPP3). The SEM micrographs of the coated Ti substrates are reported in Fig. 6. All the TiCaP samples were completely covered by inorganic
Fig. 5. Powder X-ray diffraction patterns of the powder samples obtained at 25 °C. The main reflections of OCP are indicated with (*). The main reflections of DCPD are indicated with (°).
Table 3 Crystalline phases deposited onto Ti substrates under the different conditions. Sample
pH
Crystalline phases
TiCaP TiCaP TiCaP (Ca/P = 2/1) TiCaPP1 TiCaPP1 TiCaPP3
7.2 7.0 6.8 7.2 7.0 6.8
HA HA HA HA OCP/HA OCP
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deposits in the shape of almost spherical aggregates, as in the image reported in Fig. 6a and b for TiCaP at pH 7.2. TiCaPP1 at pH 7.2 showed just a few aggregates (Fig. 6c, d) while the deposits precipitated at pH 7.0 exhibited the presence of a first homogeneous layer
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and of a second deposition made of globular aggregates (Fig. 6e, f), smaller than those obtained for the analogous TiCaP sample. At variance, TiCaPP3 at pH 6.8 was completely different: the substrate was coated by a first layer of typical OCP petal-like lamellae, with
Fig. 6. SEM micrographs of the surface of the etched Ti substrates after soaking for 6 h in the calcifying solutions: (a, b) TiCaP pH 7.2; (c, d) TiCaPP1 pH 7.2; (e, f) TiCaPP1 pH 7.0; and (g, h) TiCaPP3 pH 6.8. Bars: (a, c, e, g) = 5 μm; and (b, d, f, h) = 2 μm.
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dimensions of a few microns, and by a second deposition constituted of spherical aggregates, 2 to 4 μm wide (Fig. 6g, h). In order to increase the amount of inorganic phase deposited on the Ti substrates, further experiments were performed in the same conditions, but refreshing the calcifying solutions after 3 h, and maintaining the total experimental time at 6 h. Accordingly, SEM micrographs, reported in Fig. 7a–c, display an increase of deposition. SEM images of the cross-sections (Fig. 7d–f) show that the coatings deposited from CaPP1 at pH 7.2 and pH 7.0 were about 2 μm thick (Fig. 7d, e), while TiCaPP3 at pH 6.8 could reach a thickness up to 8 μm (Fig. 7f). In comparison the thickness of the coating deposited from the reference CaP solutions was around 5 μm. Ca/P average molar ratio of the different coatings was evaluated through EDX analysis. TiCaPP3 at pH 6.8 exhibited a Ca/P average molar ratio of 1.20, whereas the values obtained for TiCaPP1 at pH 7.0 and 7.2 were 1.47 and 1.54, respectively. A Ca/P average molar ratio of 1.57 was obtained for the coatings deposited from the CaP reference solutions. The XRD patterns recorded on the different coatings show that soaking
in the HEPES buffered CaP solutions provoked the deposition of poorly crystalline HA, whatever the starting pH, as reported in Table 3. On the contrary, soaking in the new CaPP solutions provided the deposition of poorly crystalline HA just at the highest pH value, whereas it yielded an OCP/HA mixed phase at pH 7.0. Only pure OCP was deposited at pH 6.8 from CaPP3 (Fig. 8). During coating experiments, pH values of CaP solution did not exhibit significant variations. At variance, pH values of the CaPP solutions decreased with time and remained almost constant after the onset of precipitation. Fig. 9 reports the pH variation of the calcifying solutions with time for CaPP1 at pH 7.2 and CaPP3 pH 6.8. pH decrease and precipitation occurred at different times depending on the starting pH of the CaPP solutions. The comparison between the values obtained with and without Ti substrates in solution showed that the presence of substrate generally accelerated precipitation. In fact, precipitation onset from CaPP1 pH 7.2, CaPP1 pH 7.0, and CaPP3 pH 6.8, occurred after about 15, 30, and 50 min respectively on Ti substrates, and after 30, 45 and 210 min in the absence of substrates.
Fig. 7. SEM micrographs of the surface and relative sections of the etched Ti substrates after soaking for 6 h with refreshment of the calcifying solutions after 3 h: (a, d) TiCaPP1 pH 7.2; (b, e) TiCaPP1 pH 7.0; and (c, f) TiCaPP3 pH 6.8. Bars = (a, b, c) = 5 μm; and (d, e, f) = 2 μm.
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4. Discussion The results of this paper indicate that CaPP solutions, containing just calcium chloride and phosphate buffer, can be used to obtain fast calcium phosphate deposition on metallic substrates. This strategy is a very economic route, since CaPP composition is even simpler than that of CaP, which contains also carbonate and the quite expensive HEPES buffer [20], but it still provides deposition of the inorganic phase in just a few hours. A further advantage of this method is that variation of the experimental conditions, namely starting pH and calcium and phosphate concentration, allows depositing different crystalline phases. 4.1. Powder samples
Fig. 8. Powder X-ray diffraction patterns of the coatings deposited on Ti substrates after soaking for 6 h with refreshment of the calcifying solutions after 3 h. The main reflection of OCP is indicated with (*).
The results obtained on the powders deposited at 37 °C in the absence of metallic substrates showed that CaPP1, which has the same calcium and phosphate concentration as CaP, gave rise to precipitation of a poorly crystalline apatitic phase at pH 7.2 and 7.0, whereas pH 6.8 promoted the formation of octacalcium phosphate together with apatite. The apatite precipitated from CaPP1 was constituted of spherical aggregates with smaller mean dimensions than those precipitated from CaP, and it displayed more defined XRD peaks in agreement with a greater degree of crystallinity, most likely because of the absence of NaHCO3 in CaPP composition [21]. The precipitation of OCP at pH 6.8, is not surprising since, although HA is thermodynamically more stable than OCP, the difference between the relative stability of the two phases reduces as pH decreases [22]. Moreover, OCP precipitation is kinetically favored, and it was shown that in solutions with ionic composition similar to that of human blood plasma the difference between the nucleation rates of OCP and HA increases on decreasing pH [23]. The decrease of the amount of precipitate on decreasing pH is in agreement with the solubility isotherms of calcium phosphates (Fig. 10a) [24,25]. Doubling the concentration of the Ca and P solutions (CaPP2) yielded the precipitation of OCP, together with poorly crystalline HA, even at pH 7.2 in agreement with the higher level of relative supersaturation that favors the thermodynamically less stable phase. However, HA was present, together with OCP, in the powders precipitated from CaPP2 solutions in the whole range of explored pH, 7.2–6.6. At variance, OCP could be obtained as a single phase at pH 6.8 from CaPP3 (Ca/P molar ratio of 2/1). Lowering the temperature down to 25 °C provoked the precipitation of OCP together with poorly crystalline HA even at pH 7.2 from both CaPP1 and CaPP2. Temperature decrease from 37 °C to 25 °C provokes a modest increase of the solubility of both HA and OCP [22], and it promotes the kinetically favored phase, namely OCP. The nucleation rate (J) depends on the temperature according to: J ¼ k expð−ΔGcrit =kTÞ
Fig. 9. Time dependent pH values of the calcifying solutions CaPP1 pH 7.2 in the presence ( ) and in absence (Δ) of Ti substrates, and CaPP3 pH 6.8 in the presence ( ) and in absence (◊) of Ti substrates.
where T is the absolute temperature, k is the Boltzmann's constant, and k is a preexponential factor [22]. It follows that lower temperature generally results in slower nucleation rate. However, the higher the value of ΔGcrit, the greater the decrease of nucleation rate. Thus, the decrease of temperature promotes the kinetically favored phase. Moreover, CaPP2 solutions at 25 °C yielded the precipitation of a third crystalline phase, CaHPO4·2H2O (DCPD), in the range of pH 7.0–6.6. The precipitation of DCPD can be explained on the basis of the solubility isotherms of calcium phosphates [24–26]. The DCPD/OCP singular point, that is the point where the two isotherms cross and the solution is saturated with respect to both salt, is at pH 5.0 at 37 °C and at pH 6.4 at 25 °C [24]. Accordingly, at 25 °C DCPD precipitates at higher values of pH than at 37 °C. The solubility isotherms of HA, OCP and DCPD at 37 °C and at 25 °C are reported in Fig. 10a and b.
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which started after 45 min in the absence of substrate and after 30 min on Ti substrates. The different rates of nucleation justify the different compositions of the materials deposited onto Ti substrates, OCP and poorly crystalline HA, and in the absence of substrates, just poorly crystalline HA. The different conditions utilized in TiCaPP3, namely the increase of the Ca/P molar ratio of the calcifying solution to 2/1 and the value 6.8 of starting pH, enhanced the differences between the rate of nucleation on Ti substrates and those observed in the absence of substrates. In fact, the first deposits on the metallic substrates were appreciable after about 45 min from immersion in the CaPP3 solution, whereas much longer time was required for precipitation, as well as for pH decrease, in the absence of substrate (Fig. 9). TiCaPP3 coating was constituted of crystalline OCP. Refreshment of the calcifying solutions after 3 h immersion increased deposition without modifying coating compositions (Figs. 7, 8). The thickness of the coatings obtained from CaPP1 at pH 7.2 and at pH 7.0 was smaller (about 2 μm) than that deposited from the reference CaP solution (about 5 μm); however, a thickness of 2 μm is within the range of biomimetic coatings [8]. TiCaPP3, which was constituted just of crystalline OCP, exhibited a greater thickness of about 8 μm, most likely because of the different morphologies of the coating. The different values of Ca/P molar ratio obtained through EDX analysis are in agreement with the different crystalline phases deposited in the different conditions. The possibility to use the CaPP solutions to deposit on the substrates the selected inorganic phase, HA, HA/OCP or OCP, is of great interest in view of the different properties of OCP and HA. As a matter of fact, OCP is considered the precursor phase of the inorganic phase of bone, due to its structural similarity with HA. Its solubility is greater than that of the thermodynamically more stable HA, and it easily hydrolyzes to poorly crystalline HA [27]. Moreover, OCP exhibits high osteoconductive characteristics and a speed of resorption that far exceeds that of HA [28–30]. Therefore, OCP coatings have a high potential to improve osseointegration [30–32], and the possibility to control the coating mineral phase should imply modulation of resorption rate. 5. Conclusions The newly developed method provides a very simple, economically convenient route to realize fast deposition of calcium phosphates on Ti substrates. Variations of pH, Ca/P molar ratio and/or temperature can be utilized to modulate the crystalline phase composition of the precipitates. In particular, the conditions to synthesize coatings constituted alternatively of HA, OCP, or HA/OCP have been optimized.
Fig. 10. Solubility phase diagrams of some calcium phosphates at (a) 37 °C and (b) 25 °C. Diagrams have been modified from the original ones, cited in Reference [24,25] with permissions.
4.2. Substrate coatings Coatings were deposited at 37 °C, with the aim to obtain complete coverage of the substrates with poorly crystalline HA and/or OCP. The TiCaPP1 coating obtained when the starting pH was 7.2 was constituted of poorly crystalline HA, similar to that obtained from the reference CaP solution, although the amount of material deposited from CaPP1 was less abundant. The onset of precipitation occurred about 15 min after mixing the Ca and P solutions, and it was accompanied by a decrease of pH. Precipitation was quite fast in these conditions and just a small part of the deposits were laid down onto the Ti substrates (Fig. 6c, d). In agreement with the accelerating role of the foreign surface, powder precipitation, as well as pH decrease, occurred later in the absence of Ti substrate: the first precipitates were observed at about 30 min from mixing the Ca and P solution, when pH fell to about 6.6. Reduction of the starting pH of the CaPP1 solution to 7.0 retarded nucleation,
Abbreviations CaP calcium phosphate solution CaPP calcium phosphate buffer solution SBF simulated body fluid HA hydroxyapatite OCP octacalcium phosphate DCPD calcium monohydrogen phosphate dihydrate XRD X-ray diffraction SEM Scanning electron microscopy EDX Energy-dispersive X-ray spectroscopy PBS Phosphate-buffered saline TiCaP Ti coated by calcium phosphate solution TiCaPP Ti coated by calcium phosphate buffer solution.
Acknowledgements This research was carried out with the financial support of MIUR. The authors thank NextMaterials s.r.l. (Milano, Italy) for providing the titanium substrates.
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Surface & Coatings Technology journal homepage: www.elsevier.com/locate/surfcoat
A new simplified calcifying solution to synthesize calcium phosphate coatings Barbara Bracci, Silvia Panzavolta ⁎, Adriana Bigi Department of Chemistry “G. Ciamician”, University of Bologna, Via Selmi 2, 40126 Bologna, Italy
a r t i c l e
i n f o
Article history: Received 19 February 2013 Accepted in revised form 20 April 2013 Available online 4 May 2013 Keywords: Biomimetic Coatings Calcium phosphates Phosphate buffer
a b s t r a c t In this work we set up new experimental conditions to deposit biomimetic coatings of different calcium phosphates onto titanium (Ti) substrates using over-simplified, economically convenient, slightly supersaturated solutions. The new supersaturated solutions, CaPPs, contain just calcium chloride and phosphate buffer, without addition of further salts and organic buffers usually employed for biomimetic coatings. The product precipitated at pH 7.2 and 37 °C was constituted of spherical aggregates of poorly crystalline hydroxyapatite (HA), similar to that obtained using HEPES as buffer system. Reduction of starting pH, which was varied from 7.2 to 6.6, promoted the precipitation of the kinetically favored phase, octacalcium phosphate (OCP), together with HA. Furthermore, OCP could be obtained as a single phase by increasing the Ca/P molar ratio of the calcifying solution from 1/1 to 2/1. Temperature reduction from 37 °C to 25 °C promoted the co-precipitation of calcium monohydrogen phosphate dihydrate (DCPD) together with HA and OCP, in agreement with the solubility isotherms of the different calcium phosphates. Variation of the experimental conditions was utilized to synthesize the coatings of spherical aggregates of poorly crystalline HA, petal-like crystalline OCP, as well as coatings containing both HA and OCP, onto titanium substrates in a few hours. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Short and long-term performance of dental and orthopedic implants is related to early osteointegration [1–3]. Calcium phosphate coating of metallic implants is generally accepted to be advantageous for clinical success. In fact, it allows retaining the excellent mechanical properties of titanium (Ti) and its alloys, widely employed in this field, while providing the surface of the implant with the bioactivity of calcium phosphates. A variety of physical techniques are commercially applied to this purpose [4–7]. However, physically deposited coatings exhibit several inherent drawbacks that can be overcome by wet-chemical deposition techniques [8]. In particular, the biomimetic approach involves deposition from slightly supersaturated solution at physiological, or nearly physiological, values of pH and temperature. The method, which leads to the deposition of a poorly crystalline hydroxyapatite (HA) phase similar to the inorganic phase of bone, is economic and can be applied to complex-shaped materials [9,10]. Moreover, it can be used to co-precipitate ions, drugs, macromolecules, proteins and growth factors [11–17]. The first formulation of a calcifying solution, known as simulated body fluid (SBF), was developed by Kokubo et al. [18] in 1990. Since then, a number of compositions have been proposed in order to accelerate the biomimetic deposition of poorly crystalline HA on metallic substrates. Most methods rely on buffer systems, such as TRIS (tris-hydroxymethyl-aminomethane)-HCl or HEPES (2-(4-(2-hydroxyethyl)-1-piperazinyl)ethane sulphonic acid)-NaOH ⁎ Corresponding author. Tel.: +39 051 2099566; fax: +39 051 2099456. E-mail address: [email protected] (S. Panzavolta). 0257-8972/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.surfcoat.2013.04.046
for the control of pH. Lactic acid/sodium lactate was also employed as buffer system [19]. We previously developed a slightly supersaturated calcium phosphate solution (CaP), characterized by a very simple composition and buffered with HEPES, which can deposit coatings of poorly crystalline HA on metallic substrates in a few hours [20]. In this work, we explored the possibility to further simplify the composition of the CaP solution, in order to reduce the coating cost without provoking significant lengthening of the deposition time. To this aim, we removed HEPES buffer and carbonate ions from the composition of the calcifying solution, and employed a phosphate buffer solution as source of phosphate ions. Furthermore, we investigated the influence of concentration, pH and temperature on the composition of the inorganic deposits. Selected conditions were used to modulate the crystalline phases deposited on Ti substrates from hydroxyapatite to octacalcium phosphate (OCP). 2. Materials and methods 2.1. Preparation of the reference CaP calcifying solution The reference CaP calcifying solution was prepared as previously reported [20]. Briefly, the reagent grade chemical CaCl2·2H2O was dissolved in double distilled water and buffered at pH 7.2 with HEPES (Ca solution). The reagent grade chemicals Na3PO4·12H2O, and NaHCO3 were dissolved in double distilled water and buffered at pH 7.2 with HEPES (P solution). CaP supersaturated calcifying solution was freshly prepared by mixing equal volumes of the Ca and the P solutions at 37 °C. The ionic concentration of the CaP solution is reported in Table 1 [20].
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Table 1 Composition and starting pH of the different calcifying solutions. Solution
Ca/P (molar ratio)
[Ca2+] (mM)
CaP CaPPC
1/1 1/1
2.5 2.5
CaPP1
1/1
2.5
CaPP2
1/1
5
CaPP3
2/1
a b
5
[CO32−] (mM)
Buffer solution
pH
2.5a 2.5b
18 18
Hepes
2.5b
–
7.2 7.2 7.0 7.2 7.0 6.8 6.6 7.0 6.8 6.6 6.8
[phosphate] (mM)
5b
2.5b
–
–
PO43−
Concentration of ions for CaP solution. Concentration of H2PO4−/HPO42− for CaPP solutions.
detector. CuKα radiation was used (40 mA, 40 kV). The 2θ range was from 3.8° to 11° and from 24° to 41° with a step size of 0.083° and a time/step of 1000 s/step. Morphological investigation of the coatings was performed using a Philips XL-20 scanning electron microscope operating at 15 kV. The samples were sputter-coated with gold before examination. For the observation of cross-sectional microstructure, the specimens, embedded into Methylmetacrylate (Merck Schuchardt, Hohenbrunn, Germany), were cross-sectioned with a Leica SP 1600 diamond saw (Leica, Milano, Italy). The specimens were polished, and sputter-coated with gold before SEM investigation. Energy dispersive X-ray spectrometry (EDX) analyses were performed in order to evaluate the Ca/P ratio of the deposits. 3. Results 3.1. Powder samples
2.2. Preparation of the new CaPP calcifying solutions The new calcifying solutions, CaPPs, were prepared using 0.1 M phosphate buffer (PBS) solutions at different pH values as sources of phosphate ions without addition of the HEPES solution. The 0.1 M phosphate buffer solutions at pH values 7.2, 7.0, 6.8, and 6.6, were prepared by mixing proper amounts of 0.2 M NaH2PO4 and 0.2 M Na2HPO4 solutions. The reagent grade chemical CaCl2·2H2O was dissolved in double distilled water (Ca solution). The P solution was prepared using the proper amounts of phosphate buffer solutions, in order to obtain the required phosphate concentration. NaHCO3, when used, was added to the P solution. CaPP solutions were freshly prepared by mixing appropriate volumes of the Ca and the P solutions at 37 °C, or at 25 °C, as reported in Table 1.
3.1.1. Characterization of the precipitates obtained at 37 °C The new calcifying solutions containing just CaCl2 as source of calcium ions, and NaH2PO4/Na2HPO4 buffer as source of phosphate ions, allowed to obtain fast precipitation of calcium phosphates and
2.3. Powder samples In order to better evaluate the effect of the different preparation methods on the inorganic phase precipitated from the calcifying solution, preliminary data were obtained on the powders precipitated in the absence of Ti substrates: after mixing the P solution with the Ca solution the temperature was kept constant either at 37 °C or 25 °C for 6 h, then the precipitate deposited on the bottom of the flask was filtered, washed with double distilled water and air-dried at 37 °C overnight. XRD measurements were performed using a PANalytical powder diffractometer equipped with a fast X'celerator detector. CuKα radiation was used (40 mA, 40 kV). The 2θ range was from 3 to 50° with a step size of 0.033° and time/step of 10 s/step. Morphological investigation was performed using a Philips XL-20 scanning electron microscope operating at 15 kV. The samples were sputter-coated with gold before examination. 2.4. Substrate coatings Ti disks Grade 2 (diameter 15 mm; thickness 0.5 mm, NextMaterials, Milano, Italy) were cleaned by means of soaking in an acidic mixture, containing 30% HNO3 and 3% HF in distilled water (10 ml/2 disks), then rinsed with distilled water and dried at 37 °C overnight, prior to use. Coating of the Ti substrates was performed by immersion of the disk-shaped samples in the calcifying solutions at 37 °C for 6 h. In some experiment, the calcifying solution was refreshed after 3 h. Coated samples were accurately rinsed in double distilled water, and dried at 37 °C overnight. XRD measurements were performed on the coatings using a PANalytical powder diffractometer equipped with a fast X'celerator
Fig. 1. Powder X-ray diffraction patterns of the powder samples obtained at 37 °C.
B. Bracci et al. / Surface & Coatings Technology 232 (2013) 13–21 Table 2 Crystalline phase composition of the products synthesized under different conditions. Sample
pH
37 °C
25 °C
CaPP1 CaPP1 CaPP1 CaPP1 CaPP2 CaPP2 CaPP2 CaPP2 CaPP3
7.2 7.0 6.8 6.6 7.2 7.0 6.8 6.6 6.8
HA HA HA/OCP n.d. HA/OCP HA/OCP HA/OCP HA/OCP OCP
HA/OCP HA/OCP HA/OCP n.d. HA/OCP HA/OCP/DCPD HA/OCP/DCPD HA/OCP/DCPD OCP/DCPD
n.d.: not detectable.
to modulate the composition of the coating by variation of the experimental conditions. The XRD patterns of the powders obtained at pH 7.2 and 7.0 from the calcifying solutions containing carbonate (CaPPC), revealed the presence of poorly crystalline HA, very similar to the one obtained from the reference CaP solution (Fig. 1). The product obtained using
Fig. 2. Powder X-ray diffraction patterns of the powder samples obtained at 37 °C. The main reflections of OCP are indicated with (*).
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a starting pH 7.0 (Fig. 1c) displayed slightly broader X-ray diffraction peaks than the material precipitated from the pH 7.2 solution (Fig. 1b), indicating a lower degree of crystallinity. The presence of NaHCO3 prevented the possibility to prepare solution at pH values lower than 7.0, therefore further investigations were performed on solutions not including NaHCO3 in their composition. The XRD patterns of the precipitates obtained in absence of NaHCO3 (CaPP1) still showed the presence of poorly crystalline hydroxyapatite as unique phase at pH 7.2 and 7.0 (Table 2, Fig. 2a,b). However, on comparing the XRD patterns reported in Fig. 2 with those shown in Fig. 1, it is evident that the products precipitated in the absence of carbonate display a higher degree of crystallinity than those obtained in the presence of carbonate, in agreement with the reduced broadening of the diffraction peaks. The amount of precipitate was reduced at pH 6.8, when OCP appeared as a second phase (Fig. 2c). No precipitate was appreciable at pH 6.6. Electron microscope images showed that the sample obtained from CaP at pH 7.2 is constituted of almost spherical aggregates with mean diameter of about 2–4 μm (Fig. 3a) while the mean dimensions
Fig. 3. SEM micrographs of the powder samples obtained at 37 °C. (a) CaP pH 7.2; (b) CaPP1 pH 7.2; and (c) CaPP1 pH 6.8. Bar = 5 μm.
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Fig. 4. Powder X-ray diffraction pattern and SEM micrograph of the powder sample CaPP3 obtained at 37 °C. Bar = 6 μm.
of the spherical aggregates decreased to 1 μm in the sample obtained from CaPP1 at pH 7.2 (Fig. 3b). Powders obtained from CaPP1 at pH 6.8 presented a disordered petal-like morphology (Fig. 3c). In order to increase the amount of precipitation, the concentrations of the Ca and P solutions were doubled (CaPP2), which provoked the onset of precipitation just after mixing the Ca and P solutions. The XRD patterns of CaPP2 powder revealed the presence of both HA and OCP in the explored pH range 7.2–6.6. The crystalline phases identified by means of XRD analysis are reported in Table 2. Further modifications of composition allowed to verify that a Ca/P molar ratio of 2/1 (CaPP3) at pH 6.8 promoted the precipitation of pure crystalline OCP, with the characteristic petal-like morphology (Fig. 4). The absence of the diffraction peak at 10.8°/2θ confirms the absence of HA. 3.1.2. Characterization of the precipitates obtained at 25 °C As it occurred at 37 °C, the syntheses performed at 25 °C from CaPP1 yielded no precipitation at pH 6.6. In the range of pH 7.2–6.8 the precipitates contained always OCP, together with HA (Table 2). Doubling the concentration of Ca and P solutions (CaPP2) still gave the precipitation of a mixed OCP/HA phase. However, the 11.68°/2θ reflection indicated the presence of a third phase, CaHPO4·2H2O (DCPD), in the pH range 7.0–6.6. The relative amount of DCPD increased on decreasing pH (Fig. 5, Table 2). Similarly, the precipitate obtained from the CaPP3 solution at pH 6.8 contained DCPD together with poorly crystalline HA/OCP (Table 2). 3.2. Substrate coatings On the basis of the results obtained for the powder samples, different conditions were tested to coat Ti substrates. Table 3 reports the results of the coating experiments performed at 37 °C using CaPP1 at pH 7.2 and at pH 7.0, and CaPP3 at pH 6.8. For comparison, some coating experiments were performed using the reference CaP solution containing carbonate and buffered with HEPES at pH 7.2 and 7.0, as well as at pH 6.8. In this last condition, the Ca/P molar ratio of the CaP solution was 2/1 (this allowed direct comparison with CaPP3). The SEM micrographs of the coated Ti substrates are reported in Fig. 6. All the TiCaP samples were completely covered by inorganic
Fig. 5. Powder X-ray diffraction patterns of the powder samples obtained at 25 °C. The main reflections of OCP are indicated with (*). The main reflections of DCPD are indicated with (°).
Table 3 Crystalline phases deposited onto Ti substrates under the different conditions. Sample
pH
Crystalline phases
TiCaP TiCaP TiCaP (Ca/P = 2/1) TiCaPP1 TiCaPP1 TiCaPP3
7.2 7.0 6.8 7.2 7.0 6.8
HA HA HA HA OCP/HA OCP
B. Bracci et al. / Surface & Coatings Technology 232 (2013) 13–21
deposits in the shape of almost spherical aggregates, as in the image reported in Fig. 6a and b for TiCaP at pH 7.2. TiCaPP1 at pH 7.2 showed just a few aggregates (Fig. 6c, d) while the deposits precipitated at pH 7.0 exhibited the presence of a first homogeneous layer
17
and of a second deposition made of globular aggregates (Fig. 6e, f), smaller than those obtained for the analogous TiCaP sample. At variance, TiCaPP3 at pH 6.8 was completely different: the substrate was coated by a first layer of typical OCP petal-like lamellae, with
Fig. 6. SEM micrographs of the surface of the etched Ti substrates after soaking for 6 h in the calcifying solutions: (a, b) TiCaP pH 7.2; (c, d) TiCaPP1 pH 7.2; (e, f) TiCaPP1 pH 7.0; and (g, h) TiCaPP3 pH 6.8. Bars: (a, c, e, g) = 5 μm; and (b, d, f, h) = 2 μm.
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dimensions of a few microns, and by a second deposition constituted of spherical aggregates, 2 to 4 μm wide (Fig. 6g, h). In order to increase the amount of inorganic phase deposited on the Ti substrates, further experiments were performed in the same conditions, but refreshing the calcifying solutions after 3 h, and maintaining the total experimental time at 6 h. Accordingly, SEM micrographs, reported in Fig. 7a–c, display an increase of deposition. SEM images of the cross-sections (Fig. 7d–f) show that the coatings deposited from CaPP1 at pH 7.2 and pH 7.0 were about 2 μm thick (Fig. 7d, e), while TiCaPP3 at pH 6.8 could reach a thickness up to 8 μm (Fig. 7f). In comparison the thickness of the coating deposited from the reference CaP solutions was around 5 μm. Ca/P average molar ratio of the different coatings was evaluated through EDX analysis. TiCaPP3 at pH 6.8 exhibited a Ca/P average molar ratio of 1.20, whereas the values obtained for TiCaPP1 at pH 7.0 and 7.2 were 1.47 and 1.54, respectively. A Ca/P average molar ratio of 1.57 was obtained for the coatings deposited from the CaP reference solutions. The XRD patterns recorded on the different coatings show that soaking
in the HEPES buffered CaP solutions provoked the deposition of poorly crystalline HA, whatever the starting pH, as reported in Table 3. On the contrary, soaking in the new CaPP solutions provided the deposition of poorly crystalline HA just at the highest pH value, whereas it yielded an OCP/HA mixed phase at pH 7.0. Only pure OCP was deposited at pH 6.8 from CaPP3 (Fig. 8). During coating experiments, pH values of CaP solution did not exhibit significant variations. At variance, pH values of the CaPP solutions decreased with time and remained almost constant after the onset of precipitation. Fig. 9 reports the pH variation of the calcifying solutions with time for CaPP1 at pH 7.2 and CaPP3 pH 6.8. pH decrease and precipitation occurred at different times depending on the starting pH of the CaPP solutions. The comparison between the values obtained with and without Ti substrates in solution showed that the presence of substrate generally accelerated precipitation. In fact, precipitation onset from CaPP1 pH 7.2, CaPP1 pH 7.0, and CaPP3 pH 6.8, occurred after about 15, 30, and 50 min respectively on Ti substrates, and after 30, 45 and 210 min in the absence of substrates.
Fig. 7. SEM micrographs of the surface and relative sections of the etched Ti substrates after soaking for 6 h with refreshment of the calcifying solutions after 3 h: (a, d) TiCaPP1 pH 7.2; (b, e) TiCaPP1 pH 7.0; and (c, f) TiCaPP3 pH 6.8. Bars = (a, b, c) = 5 μm; and (d, e, f) = 2 μm.
B. Bracci et al. / Surface & Coatings Technology 232 (2013) 13–21
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4. Discussion The results of this paper indicate that CaPP solutions, containing just calcium chloride and phosphate buffer, can be used to obtain fast calcium phosphate deposition on metallic substrates. This strategy is a very economic route, since CaPP composition is even simpler than that of CaP, which contains also carbonate and the quite expensive HEPES buffer [20], but it still provides deposition of the inorganic phase in just a few hours. A further advantage of this method is that variation of the experimental conditions, namely starting pH and calcium and phosphate concentration, allows depositing different crystalline phases. 4.1. Powder samples
Fig. 8. Powder X-ray diffraction patterns of the coatings deposited on Ti substrates after soaking for 6 h with refreshment of the calcifying solutions after 3 h. The main reflection of OCP is indicated with (*).
The results obtained on the powders deposited at 37 °C in the absence of metallic substrates showed that CaPP1, which has the same calcium and phosphate concentration as CaP, gave rise to precipitation of a poorly crystalline apatitic phase at pH 7.2 and 7.0, whereas pH 6.8 promoted the formation of octacalcium phosphate together with apatite. The apatite precipitated from CaPP1 was constituted of spherical aggregates with smaller mean dimensions than those precipitated from CaP, and it displayed more defined XRD peaks in agreement with a greater degree of crystallinity, most likely because of the absence of NaHCO3 in CaPP composition [21]. The precipitation of OCP at pH 6.8, is not surprising since, although HA is thermodynamically more stable than OCP, the difference between the relative stability of the two phases reduces as pH decreases [22]. Moreover, OCP precipitation is kinetically favored, and it was shown that in solutions with ionic composition similar to that of human blood plasma the difference between the nucleation rates of OCP and HA increases on decreasing pH [23]. The decrease of the amount of precipitate on decreasing pH is in agreement with the solubility isotherms of calcium phosphates (Fig. 10a) [24,25]. Doubling the concentration of the Ca and P solutions (CaPP2) yielded the precipitation of OCP, together with poorly crystalline HA, even at pH 7.2 in agreement with the higher level of relative supersaturation that favors the thermodynamically less stable phase. However, HA was present, together with OCP, in the powders precipitated from CaPP2 solutions in the whole range of explored pH, 7.2–6.6. At variance, OCP could be obtained as a single phase at pH 6.8 from CaPP3 (Ca/P molar ratio of 2/1). Lowering the temperature down to 25 °C provoked the precipitation of OCP together with poorly crystalline HA even at pH 7.2 from both CaPP1 and CaPP2. Temperature decrease from 37 °C to 25 °C provokes a modest increase of the solubility of both HA and OCP [22], and it promotes the kinetically favored phase, namely OCP. The nucleation rate (J) depends on the temperature according to: J ¼ k expð−ΔGcrit =kTÞ
Fig. 9. Time dependent pH values of the calcifying solutions CaPP1 pH 7.2 in the presence ( ) and in absence (Δ) of Ti substrates, and CaPP3 pH 6.8 in the presence ( ) and in absence (◊) of Ti substrates.
where T is the absolute temperature, k is the Boltzmann's constant, and k is a preexponential factor [22]. It follows that lower temperature generally results in slower nucleation rate. However, the higher the value of ΔGcrit, the greater the decrease of nucleation rate. Thus, the decrease of temperature promotes the kinetically favored phase. Moreover, CaPP2 solutions at 25 °C yielded the precipitation of a third crystalline phase, CaHPO4·2H2O (DCPD), in the range of pH 7.0–6.6. The precipitation of DCPD can be explained on the basis of the solubility isotherms of calcium phosphates [24–26]. The DCPD/OCP singular point, that is the point where the two isotherms cross and the solution is saturated with respect to both salt, is at pH 5.0 at 37 °C and at pH 6.4 at 25 °C [24]. Accordingly, at 25 °C DCPD precipitates at higher values of pH than at 37 °C. The solubility isotherms of HA, OCP and DCPD at 37 °C and at 25 °C are reported in Fig. 10a and b.
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which started after 45 min in the absence of substrate and after 30 min on Ti substrates. The different rates of nucleation justify the different compositions of the materials deposited onto Ti substrates, OCP and poorly crystalline HA, and in the absence of substrates, just poorly crystalline HA. The different conditions utilized in TiCaPP3, namely the increase of the Ca/P molar ratio of the calcifying solution to 2/1 and the value 6.8 of starting pH, enhanced the differences between the rate of nucleation on Ti substrates and those observed in the absence of substrates. In fact, the first deposits on the metallic substrates were appreciable after about 45 min from immersion in the CaPP3 solution, whereas much longer time was required for precipitation, as well as for pH decrease, in the absence of substrate (Fig. 9). TiCaPP3 coating was constituted of crystalline OCP. Refreshment of the calcifying solutions after 3 h immersion increased deposition without modifying coating compositions (Figs. 7, 8). The thickness of the coatings obtained from CaPP1 at pH 7.2 and at pH 7.0 was smaller (about 2 μm) than that deposited from the reference CaP solution (about 5 μm); however, a thickness of 2 μm is within the range of biomimetic coatings [8]. TiCaPP3, which was constituted just of crystalline OCP, exhibited a greater thickness of about 8 μm, most likely because of the different morphologies of the coating. The different values of Ca/P molar ratio obtained through EDX analysis are in agreement with the different crystalline phases deposited in the different conditions. The possibility to use the CaPP solutions to deposit on the substrates the selected inorganic phase, HA, HA/OCP or OCP, is of great interest in view of the different properties of OCP and HA. As a matter of fact, OCP is considered the precursor phase of the inorganic phase of bone, due to its structural similarity with HA. Its solubility is greater than that of the thermodynamically more stable HA, and it easily hydrolyzes to poorly crystalline HA [27]. Moreover, OCP exhibits high osteoconductive characteristics and a speed of resorption that far exceeds that of HA [28–30]. Therefore, OCP coatings have a high potential to improve osseointegration [30–32], and the possibility to control the coating mineral phase should imply modulation of resorption rate. 5. Conclusions The newly developed method provides a very simple, economically convenient route to realize fast deposition of calcium phosphates on Ti substrates. Variations of pH, Ca/P molar ratio and/or temperature can be utilized to modulate the crystalline phase composition of the precipitates. In particular, the conditions to synthesize coatings constituted alternatively of HA, OCP, or HA/OCP have been optimized.
Fig. 10. Solubility phase diagrams of some calcium phosphates at (a) 37 °C and (b) 25 °C. Diagrams have been modified from the original ones, cited in Reference [24,25] with permissions.
4.2. Substrate coatings Coatings were deposited at 37 °C, with the aim to obtain complete coverage of the substrates with poorly crystalline HA and/or OCP. The TiCaPP1 coating obtained when the starting pH was 7.2 was constituted of poorly crystalline HA, similar to that obtained from the reference CaP solution, although the amount of material deposited from CaPP1 was less abundant. The onset of precipitation occurred about 15 min after mixing the Ca and P solutions, and it was accompanied by a decrease of pH. Precipitation was quite fast in these conditions and just a small part of the deposits were laid down onto the Ti substrates (Fig. 6c, d). In agreement with the accelerating role of the foreign surface, powder precipitation, as well as pH decrease, occurred later in the absence of Ti substrate: the first precipitates were observed at about 30 min from mixing the Ca and P solution, when pH fell to about 6.6. Reduction of the starting pH of the CaPP1 solution to 7.0 retarded nucleation,
Abbreviations CaP calcium phosphate solution CaPP calcium phosphate buffer solution SBF simulated body fluid HA hydroxyapatite OCP octacalcium phosphate DCPD calcium monohydrogen phosphate dihydrate XRD X-ray diffraction SEM Scanning electron microscopy EDX Energy-dispersive X-ray spectroscopy PBS Phosphate-buffered saline TiCaP Ti coated by calcium phosphate solution TiCaPP Ti coated by calcium phosphate buffer solution.
Acknowledgements This research was carried out with the financial support of MIUR. The authors thank NextMaterials s.r.l. (Milano, Italy) for providing the titanium substrates.
B. Bracci et al. / Surface & Coatings Technology 232 (2013) 13–21
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