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Effect of calcium ions on transformation brushite to hydroxyapatite in aqueous solutions
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Colloids and Surfaces A: Physicochem. Eng. Aspects 316 (2008) 104–109
Effect of calcium ions on transformation brushite to hydroxyapatite in aqueous solutions ˇ R. Stulajterov´ a ∗ , L’. Medveck´y Institute of Materials Research, Slovak Academy of Sciences, Watsonova 47, 043 53 Koˇsice, Slovakia Received 13 April 2007; received in revised form 17 August 2007; accepted 23 August 2007 Available online 30 August 2007
Abstract The conversion of brushite into hydroxyapatite (HAP) by hydrolysis has been studied in alkaline solutions at pH 10.8 and temperature of 39 ◦ C with and without addition of calcium ions. Morphology of particles was analyzed by SEM. It was shown that the precipitation of hydroxyapatite crucial depends on temperature and pH. The stoichiometric hydroxyapatite as thermodynamically the most stable form of calcium phosphates was obtained in the solution without addition of Ca2+ ions after long-time hydrolysis only. In pure aqueous solution at applied pH, the surface nucleation of HAP and gradual transformation of brushite by dissolution–precipitation mechanism were confirmed. The addition of Ca2+ ions into solution causes the acceleration of brushite conversion to calcium deficient HAP forms. © 2007 Elsevier B.V. All rights reserved. Keywords: Brushite; Calcium ions; Transformation; Hydrolysis; Hydroxyapatite
1. Introduction Acidic calcium phosphates – dicalcium phosphate anhydrous (CaHPO4 , monetite), dicalcium phosphate dihydrate (CaHPO4 ·2H2 O, brushite) – are thermodynamically unstable under pH values greater than 6–7 and undergo transformation into more stable calcium phosphates (e.g., hydroxyapatite). Brushite can be applied as a coating for orthopedic implants [1]. It was found that brushite is transformed into hydroxyapatite at room temperature in deionized water or in Hank’s type solution with or without calcium and magnesium ions. The complete conversion of monetite to hydroxyapatite has been found after 4 h from immersion in alkaline solutions. The transformation occured by dissolution–precipitation mechanism [2]. The effect of NaOH concentration in hydrolyzing solution on mechanism of brushite transformation was investigated at 25 ◦ C [3]. The precipitation process of brushite, preparing by reaction calcium hydroxide suspension and an orthophosphoric acid solution was found to be divided into several stages as a function of pH and reagents’ concentrations [4]. Different conditions during brushite hydrolysis (pH 6 to 14, temperatures
∗
Corresponding author. ˇ E-mail address: [email protected] (R. Stulajterov´ a).
0927-7757/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2007.08.036
between 60 and 140 ◦ C) have been investigated to determine their influence on size and morphology of HAP particles [5]. Results have shown that pH value is a significant parameter in altering the HAP particle morphology. The formation of elongated particles was observed at pH 9 and 120 ◦ C. The rate of brushite conversion to hydroxyapatite in the presence of calcium ions and protein-free aqueous body fluid (Hank’s balanced salt solution, HBSS) was investigated electrolytically from monocalcium phosphate solution at various concentration of KCl in solution (as a supporting electrolyte) [6]. Modified brushite deposit demonstrated a faster transformation to HAP. The modified form of brushite with potassium substituting for calcium at specific sites demonstrated accelerated transformation to HAP when exposed to nonproteinaceous Hank’s balanced aqueous salt solutions (HBSS) [7]. In the presence of a protein-free environment, transformation is faster in buffered medium than in nonbuffered medium. The presence bovine serum albumin (BSA) in either buffered or nonbuffered medium retards the transformation in comparison to the corresponding BSA-free medium. Tas and Bhaduri [8] studied chemical synthesis procedure for the manufacture of Na- and K-doped dicalcium phosphate dihydrate powder and their transformation into apatitic calcium phosphates by immersing them in simulated body fluid (SBF) solution [9]. One of the factors influencing the conversion of DCPD to HAP is its incongruent dissolution.
ˇ R. Stulajterov´ a, L’. Medveck´y / Colloids and Surfaces A: Physicochem. Eng. Aspects 316 (2008) 104–109
The hydrolysis of DCPD should also be expected to depend on the reaction temperature. HAP has been shown to exhibit retrograde solubility [10] while DCPD has been shown to exhibit a maximum in its solubility product at about 25 ◦ C [11]. The concentration gradient between the surfaces of the dissolving DCPD crystals and the bulk solution will be reduced and the overgrowth by HAP will occur at a lower extent of dissolution [12]. Incongruent dissolution of either CaHPO4 or CaHPO4 ·2H2 O results in the formation of hydroxyapatite during the dissolution process [13]. Kanzaki et al. [14] confirm by atomic force microscopy that the precipitation of hydroxyapatite occurred after the dissolution of DCPD and DCPD acts as a simple heterogenous growth center for hydroxyapatite. In our work we are studied of the kinetics of brushite conversion to calcium phosphate (hydroxyapatite-like phase) in aqueous solutions at pH equals 10.8 at temperature 39 ◦ C and the effect of calcium on above kinetics.
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2.3. Characterization
2. Experimental procedures
The pH of solutions were measured and adjusted using the pH-meter (WTW, Inolab 720) with combined glass electrode (SenTix 41). The degrees of brushite conversion were determined from Ca/P ratio in products at given hydrolysis times so as from chemical analysis of composition of solutions. The concentration of phosphorus in amonium solution was determined by the calorimetry as P–Mo–V complex (Hitachi 1100 U). The Ca2+ ions content in samples was determined by complexometric titration (EDTA, Ca2+ ion selective electrode was used to determine of equivalence point). The phase composition of powder samples was analyzed using the X-ray diffraction analysis (Philips, X’Pert Pro, Cu K␣ radiation), infrared spectroscopy (Specord M80, 1 mg sample + 400 mg KBr) and thermogravimetric analysis (Mettler 2000C). Morphology of calcium phosphate particles was observed by scanning electron microscopy (SEM, Tesla BS 340) equipped with EDX analyzer (LINK ISIS, Oxford Instruments) after deposition of gold on sample surfaces.
2.1. DCPD preparation
3. Results and discussion
DCPD was prepared by the precipitation of calcium ions (4 g CaCO3 (analytical grade) was dissolved in 13% HNO3 (analytical grade)) by 67.4 mL of 3.4% H3 PO4 (analytical grade). CO2 was ultrasonically released from solution. H3 PO4 was slowly dropped to Ca(NO3 )2 solution after pH adjustment to value 3 with 6.25% NH3 (aq) (analytical grade). The rotation speed of stirrer was kept at 500 rpm and pH was maintained at 4 during precipitation. Obtained DCPD was filtered over the glass filter and washed with distilled water (V = 500 mL) and ethanol (50 mL) and dried at 70 ◦ C for 2 h.
3.1. The effect of calcium addition on reactions of brushite in aqueous solutions
2.2. Transformation of brushite in aqueous solutions Hydrolysis of brushite was carried out in NH3 (aq) solution—initial pH 10.8 without the presence of Ca2+ ions or with addition of 0.5 g Ca2+ (CaCl2 ·2H2 O solution). Total volume of solution during conversion was 120 mL. Before adding of brushite (1.2648 g), the reaction solution pH was adjusted to value 10.8 by adding 25% NH3 (aq) whereas temperature during experiments was kept at 39 ±1 ◦ C (close to physiological temperature). The reaction was carried out in closed glass vessel with stirrer (500 rpm). The kinetics of brushite transformation was determined from the changes in Ca/P ratio in final products of hydrolysis and changes in composition of solution after immersion of brushite at various times from reaction start (1, 2, 4, 6, 24 and 48 h). The resulting products (at given times from start of hydrolysis) were filtered, washed with distilled water and dried at 70 ◦ C for 2 h. The solutions after brushite conversion at selected reaction times were filtered over the membrane filter (Millipore, polyvinylidene fluoride, <0.2 m) for separation and trapping of very fine particles that could be affect on chemical analysis of solutions. Simultaneously, the pH solutions during hydrolysis was measured.
The changes of pH in solutions during brushite hydrolysis at temperature of 39 ◦ C and starting pH 10.8 are shown in Fig. 1. During the time of conversion of brushite in ammonium solution without Ca2+ addition (liquid/solid ratio = 100), a gradual decrease of pH with conversion time was observed whereas the pH value reached ∼6.6 (Fig. 1, curve 1) after 24 h. The gradual decrease of pH value in the solution containing of calcium ions (concentration of Ca2+ equals 0.5 g/120 mL solution) with time of DCPD conversion (liquid/solid ratio = 100) is shown in Fig. 1, curve 3. The pH of solution decreased slowly from starting value of 10.8 to 10.5 during first 2 h with following the rapid decrease to ≈9 and gradually down to ≈8 after 24 h. From the comparison of dependences of pH on time in Fig. 1, curves 1 and 2 and curves 3 and 4 results that the final pH of solution after 24 h conversion
Fig. 1. The changes of pH solution and Ca/P ratio in final products during brushite hydrolysis at temperature of 39 ◦ C and initial pH 10.8 without addition of calcium ions, curves 1 and 2 (- - -) and with addition of calcium ions (0.5 g/120 mL), curves 3 and 4 (—).
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Fig. 2. DSC and TG analysis of reaction products in the solution without addition of calcium ions: (a) after 2 h, (b) after 24 h and with addition 0.5 g of calcium ions/120 mL solution, (c) after 2 h and (d) after 24 h.
in the solution of Ca2+ ions is shifted to basic region, which confirms the fact that no phosphoric acid is released during brushite transformation (only a very low phosphate concentration has observed in solution) to reaction solution, respectively, as a consequence of high supersaturation in solution considering HAP after adding of the calcium ions. Calcium ions interact with released phosphate ions at surfaces of DCPD particles and hydroxyl ions are consumed in the same time. After 24 h from start of reaction, Ca/P ratio achieved approximately value of 1.5 that corresponds to one in calcium deficient hydroxyapatite. The same ratio was determined by the chemical analysis of product after transformation for 48 h. It is evident
that the Ca/P ratio at applied conditions is increased only slowly and the value equals 1.67 which represents the stoichiometric HAP, is achieved after long-time hydrolysis. For such a slow shift in Ca/P ratio in brushite hydrolysis product, the acidic pH of solution are responsible. The system achieves of conditions of the quasi-chemical equilibrium and the rate of phosphate release into solution is markedly decelerated, whereas a thermodynamic equilibrium and overall a system stability are not attained. The rate of reaction is increased again after pH solution adjustment in direction to basic region which causes the rise of supersaturation solution considering HAP. The hydrolysis rate was significantly increased approximately after 10 h from immersion of brushite
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Fig. 3. IR spectra of the products after DCPD hydrolysis in solutions: (a) without addition of Ca2+ after 24 h, (b) with addition of 0.5 g Ca2+ /120 mL solution after 24 h, (c) after 2 h and (d) after 4 h.
into solution as it is shown in Fig. 1. The more probable reason for such a course of conversion is high nucleation rate of calcium deficient hydroxyapatite [15]. This process provides the formation of high number of nucleus for crystallization at the surface of DCPD particles. Brushite solubility markedly decreased by the creation of compact HAP layer at the surface of brushite particles because the diffusion of phosphates into reactive solution is reduced resp. it is not possible exchange of ions between reacting brushite and surrounding solution. Following increase in the transformation rate can be observed after crystallization of small HAP particles by dissolution–precipitation process (confirmed by the XRD analysis, not shown) which free the DCPD particle surfaces for continuation of the reaction. Recrystallized coarser particles of calcium deficient HAP represent nucleation seeds for growth of the hydroxyapatite particles and this evidence provides suitable conditions for fast DCPD conversion despite the nucleation rate fell down by decreasing of solution pH. The analysis of DSC and TG curves (Fig. 2a) verify above results, where any significant changes in mass losses or magnitudes, respectively, area ratios of thermal endo-effects at 160 and 200 ◦ C corresponding to transformation of DCPD to DCPA and effect at 440 ◦ C characterizing CaHPO4 conversion to Ca2 P2 O7 in comparison with ones in pure brushite were not observed after 2 h from hydrolysis start. In Fig. 2b, no presence of the brushite (no endo-effects on the calorimetric curve at 160 and 200 ◦ C) and monetite (moreover not found effect at 440 ◦ C) are visible on curves after 24 h DCPD conversion. The observed summary mass loss is possible to assign to physically bonded water on fine particles of the apatitic phase. IR spectrum of DCPD hydrolysis product after 24 h is shown in Fig. 3a and b. Antisymmetric (ν3 ) and symmetric (ν1 ) P–O stretching vibrations of PO4 group at
1050, 1100 and 962 cm−1 , O–P–O bending (ν4 ) vibrations of PO4 group at 565 and 603 cm−1 , ν2 and ν3 modes of CO3 2− at wavenumbers of 870 and 1400–1500 cm−1 , vibrational mode at 630 cm−1 of OH hydroxyapatite group and band at 1650 cm−1 from vibration modes of adsorbed water can be found in spectrum. No peaks representing DCPD or DCPA are visible in spectrum. Primary brushite plate-like particles had average size approximately 40 m and a smooth surface (Fig. 4a). After 24 and 48 h (Fig. 4b and c) from beginning of hydrolysis, the plate-like morphology of the brushite particles was maintained, however, newly crystallized 1–5 m particles tightly bonded on the surface of origin particles can be observed in figure. The stronger agglomerates with size up to 50 m are formed probably as the result of overgrowing of primary brushite particles during transformation and recrystallization to the calcium deficient HAP. Present results are in accordance with accounts in Ref. [13], where the formation of the calcium deficient HAP systems during brushite hydrolysis was assumed on the basis of the analysis of the phase diagram of CaO–P2 O5 –H2 O system. The changes in phase composition of product during conversion are shown in Fig. 5. After 2 h from brushite immersion into the solution of calcium ions, besides initial DCPD phase (JCPDS 09-0077), peaks correspond to formed hydroxyapatite phase (JCPDS 24-0033) were present and no additional phase were observed in product. In the spectrum, well observable angle shift in DCPD peaks in direction to higher diffraction angles which is consequence of shortening of interplanar distances whereas the locations of lines from reflections of planes of the hydroxyapatite phase are not changed. It is evident that new-formed particles are very fine which confirm large half peak widths in XRD record. The significant change in prod-
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Fig. 4. Morphologies of products of transformation: (a) origin plate-like brushite particles, (b) in solution without addition of Ca2+ after 24 h of hydrolysis, (c) in solution without addition of Ca2+ after 48 h of hydrolysis, (d) in solution with addition of 0.5 g Ca2+ after 24 h of hydrolysis.
uct composition was observed after 4 h of conversion, where the brushite peaks were not found in spectrum and lines corresponding almost amorphous particles of HAP are possible to see only. The rise in crystallinity of the HAP particles with another prolongation of transformation was verified from the increase of intensity of peaks and decrease of half peak width in XRD spectrum. Considerable increasing of Ca/P ratio was observed after 4 h of conversion (Fig. 1). The reason this effect is rapid rise in the calcium content in sample (Fig. 6) by the interacting of
calcium ions in solution with DCPD—the Ca/P ratio in product after 24 h was 1.64 which is ratio approaching to stoichiometric HAP. The results of XRD diffraction analysis coincide with ones of the IR analysis of spectra samples after 2 and 4 h transformation (Fig. 3c and d), where the spectrum of the product of 2 h hydrolysis represents the spectrum almost pure DCPD whereas the peaks corresponding to vibrations of the phosphate group are shifted about 5–10 cm−1 in the direction to shorter wavelengths. This indicates strengthening of bonds in brushite.
Fig. 5. X-ray diffraction patterns of calcium phosphate powders: (a) HAP, (b) pure DCPD, (c) in solution with addition of 0.5 g Ca2+ /120 mL solution after 2 h, (d) after 4 h and (e) after 48 h.
Fig. 6. Dependence of calcium and phosphorus contents in products on hydrolysis time.
ˇ R. Stulajterov´ a, L’. Medveck´y / Colloids and Surfaces A: Physicochem. Eng. Aspects 316 (2008) 104–109
On the other hand, both the significantly changes in peak intensities of DCPD at 1650, 1230, 980, 870 and 800 cm−1 and in the ratio of intensities of the bands at 560 and 540 cm−1 were observed in the IR spectrum of sample after 4 h of the reaction. Completely modified is shape of bands at 1050 and 1100 cm−1 and the new peaks appearances in vicinity of the wavelengths at 960 and 605 cm−1 —all above bands are characteristic for the hydroxyapatite spectrum after hydrolysis for 24 h. An presence of the pure HAP after conversion of brushite in calcium solution for 24 h without any residue of brushite or monetite is evident from the analysis of TG and DSC curves (Fig. 2d), where no thermal effects, which would be possible assign to decomposition of DCPD or DCPA, were found and the summary mass loss represents the amount of physical bonded water. The changes in the shape of the TG curves and large modification of the shape of DSC curves were observed 2 h after transformation (Fig. 2c). The changes in ratios of the thermal endo-effects are occured at 160 and 200 ◦ C in comparison with effects which were found in the pure brushite—the lower mass loss and magnitude of thermal effect at 200 ◦ C were observed and maximum of endo-effect is moved to 165 ◦ C. From above result that fraction of the chemically bonded water in DCPD is decreased which induces the rise of compactness of the crystal lattice. The fall of the content of chemically bonded water was responsible for the existence of maximum on dependences of the content of calcium and phosphorus in products of reaction on the transformation time (in Fig. 6) because of increasing of the sample densities. This fact is possible to support from comparison of the volumes of crystalline cell of brushite (JCPDS 09-0077) and monetite (JCPDS 09-0080), where both the decrease in cell volume from 493 to ˚ 3 and the increase in the density of monetite about 30% 309 A were found after elimination of the chemically bonded water. In DSC record, a new endo-effect with maximum at temperature of 375 ◦ C was found that is not related to any change in mass. Its presence can be connected with some transformation of the calcium phosphate phases or immediate interaction between residual-modified DCPD and created hydroxyapatite phase. The morphology of hydroxyapatite particles after conversion of brushite for 24 h is shown in Fig. 4d. Similarly as in the case of brushite transformation in solution without addition of calcium ions, plate-like agglomerates of size around 30 m composed from fine particles (<1 m) are visible in figure. From analysis above results it can be stated the conversion of DCPD in solution of calcium ions with initial pH 10.8 does not carry out by the simple dissolving of DCPD and following gradual precipitation of a new hydroxyapatite phase on the surface of brushite particles but there is transformation in volume of DCPD crystal lattice whereas the diffusion of calcium ions into brushite particles is probably one of important effects. 4. Conclusion The results of experimental works showed that the calcium deficient types of HAP are formed by the transformation of brushite in aqueous solution with initial pH 10.8 at temperature
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of 39 ◦ C whereas the stoichiometric hydroxyapatite as thermodynamically the most stable form of calcium phosphates was obtained in the solution without addition of Ca2+ ions after long-time hydrolysis only. In pure aqueous solution at applied pH, the surface nucleation of HAP and gradual transformation of brushite by dissolution–precipitation mechanism were confirmed. The addition of Ca2+ ions into solution causes the acceleration of brushite conversion to calcium deficient HAP forms. The stoichiometric hydroxyapatite is created after 24 h from the immersion of brushite to the reactive solution. As it was shown, the transformation in this case is carried out inside of particles of powder brushite too. Acknowledgements This study was carried out as a project supported by the Slovak Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences, Project No. 2/5143/25. References [1] M. Kumar, H. Dasarathy, C. Riley, Electrodeposition of brushite coatings and their transformation to hydroxyapatite in aqueous solutions, J. Biomed. Mater. Res. 45 (4) (1999) 302–310. [2] M.H. Prado Da Silva, J.H.C. Lima, G.A. Soares, C.N. Elias, M.C. Deandrade, S.M. Best, I.R. Gibson, Transformation of monetite to hydroxyapatite in bioactive coatings on titanium, Surf. Coat. Technol. 137 (2001) 270–276. [3] K. Furutaka, H. Monma, T. Okura, S. Takahashi, Characteristic reaction processes in the system brushite–NaOH solution, J. Eur. Ceram. Soc. 26 (2006) 543–547. [4] A. Ferreira, C. Oliveira, F. Rocha, The different phases in the precipitation of dicalcium phosphate dihydrate, J. Cryst. Growth 252 (2003) 599–611. [5] J.B. Liu, X. Ye, H. Wang, M. Zhu, B. Wang, H. Yan, The influence of pH and temperature on the morphology of hydroxyapatite synthesized by hydrothermal method, Ceram. Int. 29 (2003) 629–633. [6] M. Kumar, J. Xie, K. Chittur, C. Riley, Transformation of modified brushite to hydroxyapatite in aqueous solution: effects of potassium substitution, Biomaterials 20 (1999) 1389–1399. [7] J. Xie, C. Riley, K. Chittur, Effect of albumin on brushite transformation to hydroxyapatite, J. Biomed. Mater. Res. 57 (3) (2001) 357–365. [8] A.C. Tas, S.B. Bhaduri, Chemical processing of CaHPO4 ·2H2 O: its conversion to hydroxyapatite, J. Am. Ceram. Soc. 87 (12) (2004) 2195–2200. [9] A.C. Tas, Synthesis of biomimetic Ca–hydroxyapatite powders at 37 ◦ C in synthetic body fluids, Biomaterials 21 (2000) 1429–1438. [10] K.S. Tenhuisen, P.W. Brown, The kinetics of calcium deficient and stoichiometric hydroxyapatite formation from CaHPO4 ·2H2 O and Ca4 (PO4 )2 O, J. Mater. Sci.: Mater. Med. 7 (1996) 309–316. [11] E.C. Moreno, T.M. Gregory, W.E. Brown, Solubility of CaHPO4 ·2H2 O and formation of ion pairs in the system Ca(OH)2 –H3 PO4 –H2 O at 37.5 ◦ C, J. Res. NBS 70A (1996) 545–552. [12] M.T. Fulmer, P.W. Brown, Hydrolysis of dicalcium phosphate dihydrate to hydroxyapatite, J. Mater. Sci.: Mater. Med. 9 (1998) 197–202. [13] P.W. Brown, Phase relationship in the ternary system CaO–P2 O5 –H2 O at 25 ◦ C, J. Am. Ceram. Soc. 25 (1992) 17–22. [14] N. Kanzaki, K. Onuma, G. Treboux, A. Ito, Dissolution kinetics of dicalcium phosphate dihydrate under pseudophysiological conditions, J. Cryst. Growth 235 (2002) 465–470. [15] X. Lu, Y. Leng, Theoretical analysis of calcium phosphate precipitation in simulated body fluid, Biomaterials 26 (2005) 1097–1108.
Colloids and Surfaces A: Physicochem. Eng. Aspects 316 (2008) 104–109
Effect of calcium ions on transformation brushite to hydroxyapatite in aqueous solutions ˇ R. Stulajterov´ a ∗ , L’. Medveck´y Institute of Materials Research, Slovak Academy of Sciences, Watsonova 47, 043 53 Koˇsice, Slovakia Received 13 April 2007; received in revised form 17 August 2007; accepted 23 August 2007 Available online 30 August 2007
Abstract The conversion of brushite into hydroxyapatite (HAP) by hydrolysis has been studied in alkaline solutions at pH 10.8 and temperature of 39 ◦ C with and without addition of calcium ions. Morphology of particles was analyzed by SEM. It was shown that the precipitation of hydroxyapatite crucial depends on temperature and pH. The stoichiometric hydroxyapatite as thermodynamically the most stable form of calcium phosphates was obtained in the solution without addition of Ca2+ ions after long-time hydrolysis only. In pure aqueous solution at applied pH, the surface nucleation of HAP and gradual transformation of brushite by dissolution–precipitation mechanism were confirmed. The addition of Ca2+ ions into solution causes the acceleration of brushite conversion to calcium deficient HAP forms. © 2007 Elsevier B.V. All rights reserved. Keywords: Brushite; Calcium ions; Transformation; Hydrolysis; Hydroxyapatite
1. Introduction Acidic calcium phosphates – dicalcium phosphate anhydrous (CaHPO4 , monetite), dicalcium phosphate dihydrate (CaHPO4 ·2H2 O, brushite) – are thermodynamically unstable under pH values greater than 6–7 and undergo transformation into more stable calcium phosphates (e.g., hydroxyapatite). Brushite can be applied as a coating for orthopedic implants [1]. It was found that brushite is transformed into hydroxyapatite at room temperature in deionized water or in Hank’s type solution with or without calcium and magnesium ions. The complete conversion of monetite to hydroxyapatite has been found after 4 h from immersion in alkaline solutions. The transformation occured by dissolution–precipitation mechanism [2]. The effect of NaOH concentration in hydrolyzing solution on mechanism of brushite transformation was investigated at 25 ◦ C [3]. The precipitation process of brushite, preparing by reaction calcium hydroxide suspension and an orthophosphoric acid solution was found to be divided into several stages as a function of pH and reagents’ concentrations [4]. Different conditions during brushite hydrolysis (pH 6 to 14, temperatures
∗
Corresponding author. ˇ E-mail address: [email protected] (R. Stulajterov´ a).
0927-7757/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2007.08.036
between 60 and 140 ◦ C) have been investigated to determine their influence on size and morphology of HAP particles [5]. Results have shown that pH value is a significant parameter in altering the HAP particle morphology. The formation of elongated particles was observed at pH 9 and 120 ◦ C. The rate of brushite conversion to hydroxyapatite in the presence of calcium ions and protein-free aqueous body fluid (Hank’s balanced salt solution, HBSS) was investigated electrolytically from monocalcium phosphate solution at various concentration of KCl in solution (as a supporting electrolyte) [6]. Modified brushite deposit demonstrated a faster transformation to HAP. The modified form of brushite with potassium substituting for calcium at specific sites demonstrated accelerated transformation to HAP when exposed to nonproteinaceous Hank’s balanced aqueous salt solutions (HBSS) [7]. In the presence of a protein-free environment, transformation is faster in buffered medium than in nonbuffered medium. The presence bovine serum albumin (BSA) in either buffered or nonbuffered medium retards the transformation in comparison to the corresponding BSA-free medium. Tas and Bhaduri [8] studied chemical synthesis procedure for the manufacture of Na- and K-doped dicalcium phosphate dihydrate powder and their transformation into apatitic calcium phosphates by immersing them in simulated body fluid (SBF) solution [9]. One of the factors influencing the conversion of DCPD to HAP is its incongruent dissolution.
ˇ R. Stulajterov´ a, L’. Medveck´y / Colloids and Surfaces A: Physicochem. Eng. Aspects 316 (2008) 104–109
The hydrolysis of DCPD should also be expected to depend on the reaction temperature. HAP has been shown to exhibit retrograde solubility [10] while DCPD has been shown to exhibit a maximum in its solubility product at about 25 ◦ C [11]. The concentration gradient between the surfaces of the dissolving DCPD crystals and the bulk solution will be reduced and the overgrowth by HAP will occur at a lower extent of dissolution [12]. Incongruent dissolution of either CaHPO4 or CaHPO4 ·2H2 O results in the formation of hydroxyapatite during the dissolution process [13]. Kanzaki et al. [14] confirm by atomic force microscopy that the precipitation of hydroxyapatite occurred after the dissolution of DCPD and DCPD acts as a simple heterogenous growth center for hydroxyapatite. In our work we are studied of the kinetics of brushite conversion to calcium phosphate (hydroxyapatite-like phase) in aqueous solutions at pH equals 10.8 at temperature 39 ◦ C and the effect of calcium on above kinetics.
105
2.3. Characterization
2. Experimental procedures
The pH of solutions were measured and adjusted using the pH-meter (WTW, Inolab 720) with combined glass electrode (SenTix 41). The degrees of brushite conversion were determined from Ca/P ratio in products at given hydrolysis times so as from chemical analysis of composition of solutions. The concentration of phosphorus in amonium solution was determined by the calorimetry as P–Mo–V complex (Hitachi 1100 U). The Ca2+ ions content in samples was determined by complexometric titration (EDTA, Ca2+ ion selective electrode was used to determine of equivalence point). The phase composition of powder samples was analyzed using the X-ray diffraction analysis (Philips, X’Pert Pro, Cu K␣ radiation), infrared spectroscopy (Specord M80, 1 mg sample + 400 mg KBr) and thermogravimetric analysis (Mettler 2000C). Morphology of calcium phosphate particles was observed by scanning electron microscopy (SEM, Tesla BS 340) equipped with EDX analyzer (LINK ISIS, Oxford Instruments) after deposition of gold on sample surfaces.
2.1. DCPD preparation
3. Results and discussion
DCPD was prepared by the precipitation of calcium ions (4 g CaCO3 (analytical grade) was dissolved in 13% HNO3 (analytical grade)) by 67.4 mL of 3.4% H3 PO4 (analytical grade). CO2 was ultrasonically released from solution. H3 PO4 was slowly dropped to Ca(NO3 )2 solution after pH adjustment to value 3 with 6.25% NH3 (aq) (analytical grade). The rotation speed of stirrer was kept at 500 rpm and pH was maintained at 4 during precipitation. Obtained DCPD was filtered over the glass filter and washed with distilled water (V = 500 mL) and ethanol (50 mL) and dried at 70 ◦ C for 2 h.
3.1. The effect of calcium addition on reactions of brushite in aqueous solutions
2.2. Transformation of brushite in aqueous solutions Hydrolysis of brushite was carried out in NH3 (aq) solution—initial pH 10.8 without the presence of Ca2+ ions or with addition of 0.5 g Ca2+ (CaCl2 ·2H2 O solution). Total volume of solution during conversion was 120 mL. Before adding of brushite (1.2648 g), the reaction solution pH was adjusted to value 10.8 by adding 25% NH3 (aq) whereas temperature during experiments was kept at 39 ±1 ◦ C (close to physiological temperature). The reaction was carried out in closed glass vessel with stirrer (500 rpm). The kinetics of brushite transformation was determined from the changes in Ca/P ratio in final products of hydrolysis and changes in composition of solution after immersion of brushite at various times from reaction start (1, 2, 4, 6, 24 and 48 h). The resulting products (at given times from start of hydrolysis) were filtered, washed with distilled water and dried at 70 ◦ C for 2 h. The solutions after brushite conversion at selected reaction times were filtered over the membrane filter (Millipore, polyvinylidene fluoride, <0.2 m) for separation and trapping of very fine particles that could be affect on chemical analysis of solutions. Simultaneously, the pH solutions during hydrolysis was measured.
The changes of pH in solutions during brushite hydrolysis at temperature of 39 ◦ C and starting pH 10.8 are shown in Fig. 1. During the time of conversion of brushite in ammonium solution without Ca2+ addition (liquid/solid ratio = 100), a gradual decrease of pH with conversion time was observed whereas the pH value reached ∼6.6 (Fig. 1, curve 1) after 24 h. The gradual decrease of pH value in the solution containing of calcium ions (concentration of Ca2+ equals 0.5 g/120 mL solution) with time of DCPD conversion (liquid/solid ratio = 100) is shown in Fig. 1, curve 3. The pH of solution decreased slowly from starting value of 10.8 to 10.5 during first 2 h with following the rapid decrease to ≈9 and gradually down to ≈8 after 24 h. From the comparison of dependences of pH on time in Fig. 1, curves 1 and 2 and curves 3 and 4 results that the final pH of solution after 24 h conversion
Fig. 1. The changes of pH solution and Ca/P ratio in final products during brushite hydrolysis at temperature of 39 ◦ C and initial pH 10.8 without addition of calcium ions, curves 1 and 2 (- - -) and with addition of calcium ions (0.5 g/120 mL), curves 3 and 4 (—).
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Fig. 2. DSC and TG analysis of reaction products in the solution without addition of calcium ions: (a) after 2 h, (b) after 24 h and with addition 0.5 g of calcium ions/120 mL solution, (c) after 2 h and (d) after 24 h.
in the solution of Ca2+ ions is shifted to basic region, which confirms the fact that no phosphoric acid is released during brushite transformation (only a very low phosphate concentration has observed in solution) to reaction solution, respectively, as a consequence of high supersaturation in solution considering HAP after adding of the calcium ions. Calcium ions interact with released phosphate ions at surfaces of DCPD particles and hydroxyl ions are consumed in the same time. After 24 h from start of reaction, Ca/P ratio achieved approximately value of 1.5 that corresponds to one in calcium deficient hydroxyapatite. The same ratio was determined by the chemical analysis of product after transformation for 48 h. It is evident
that the Ca/P ratio at applied conditions is increased only slowly and the value equals 1.67 which represents the stoichiometric HAP, is achieved after long-time hydrolysis. For such a slow shift in Ca/P ratio in brushite hydrolysis product, the acidic pH of solution are responsible. The system achieves of conditions of the quasi-chemical equilibrium and the rate of phosphate release into solution is markedly decelerated, whereas a thermodynamic equilibrium and overall a system stability are not attained. The rate of reaction is increased again after pH solution adjustment in direction to basic region which causes the rise of supersaturation solution considering HAP. The hydrolysis rate was significantly increased approximately after 10 h from immersion of brushite
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Fig. 3. IR spectra of the products after DCPD hydrolysis in solutions: (a) without addition of Ca2+ after 24 h, (b) with addition of 0.5 g Ca2+ /120 mL solution after 24 h, (c) after 2 h and (d) after 4 h.
into solution as it is shown in Fig. 1. The more probable reason for such a course of conversion is high nucleation rate of calcium deficient hydroxyapatite [15]. This process provides the formation of high number of nucleus for crystallization at the surface of DCPD particles. Brushite solubility markedly decreased by the creation of compact HAP layer at the surface of brushite particles because the diffusion of phosphates into reactive solution is reduced resp. it is not possible exchange of ions between reacting brushite and surrounding solution. Following increase in the transformation rate can be observed after crystallization of small HAP particles by dissolution–precipitation process (confirmed by the XRD analysis, not shown) which free the DCPD particle surfaces for continuation of the reaction. Recrystallized coarser particles of calcium deficient HAP represent nucleation seeds for growth of the hydroxyapatite particles and this evidence provides suitable conditions for fast DCPD conversion despite the nucleation rate fell down by decreasing of solution pH. The analysis of DSC and TG curves (Fig. 2a) verify above results, where any significant changes in mass losses or magnitudes, respectively, area ratios of thermal endo-effects at 160 and 200 ◦ C corresponding to transformation of DCPD to DCPA and effect at 440 ◦ C characterizing CaHPO4 conversion to Ca2 P2 O7 in comparison with ones in pure brushite were not observed after 2 h from hydrolysis start. In Fig. 2b, no presence of the brushite (no endo-effects on the calorimetric curve at 160 and 200 ◦ C) and monetite (moreover not found effect at 440 ◦ C) are visible on curves after 24 h DCPD conversion. The observed summary mass loss is possible to assign to physically bonded water on fine particles of the apatitic phase. IR spectrum of DCPD hydrolysis product after 24 h is shown in Fig. 3a and b. Antisymmetric (ν3 ) and symmetric (ν1 ) P–O stretching vibrations of PO4 group at
1050, 1100 and 962 cm−1 , O–P–O bending (ν4 ) vibrations of PO4 group at 565 and 603 cm−1 , ν2 and ν3 modes of CO3 2− at wavenumbers of 870 and 1400–1500 cm−1 , vibrational mode at 630 cm−1 of OH hydroxyapatite group and band at 1650 cm−1 from vibration modes of adsorbed water can be found in spectrum. No peaks representing DCPD or DCPA are visible in spectrum. Primary brushite plate-like particles had average size approximately 40 m and a smooth surface (Fig. 4a). After 24 and 48 h (Fig. 4b and c) from beginning of hydrolysis, the plate-like morphology of the brushite particles was maintained, however, newly crystallized 1–5 m particles tightly bonded on the surface of origin particles can be observed in figure. The stronger agglomerates with size up to 50 m are formed probably as the result of overgrowing of primary brushite particles during transformation and recrystallization to the calcium deficient HAP. Present results are in accordance with accounts in Ref. [13], where the formation of the calcium deficient HAP systems during brushite hydrolysis was assumed on the basis of the analysis of the phase diagram of CaO–P2 O5 –H2 O system. The changes in phase composition of product during conversion are shown in Fig. 5. After 2 h from brushite immersion into the solution of calcium ions, besides initial DCPD phase (JCPDS 09-0077), peaks correspond to formed hydroxyapatite phase (JCPDS 24-0033) were present and no additional phase were observed in product. In the spectrum, well observable angle shift in DCPD peaks in direction to higher diffraction angles which is consequence of shortening of interplanar distances whereas the locations of lines from reflections of planes of the hydroxyapatite phase are not changed. It is evident that new-formed particles are very fine which confirm large half peak widths in XRD record. The significant change in prod-
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Fig. 4. Morphologies of products of transformation: (a) origin plate-like brushite particles, (b) in solution without addition of Ca2+ after 24 h of hydrolysis, (c) in solution without addition of Ca2+ after 48 h of hydrolysis, (d) in solution with addition of 0.5 g Ca2+ after 24 h of hydrolysis.
uct composition was observed after 4 h of conversion, where the brushite peaks were not found in spectrum and lines corresponding almost amorphous particles of HAP are possible to see only. The rise in crystallinity of the HAP particles with another prolongation of transformation was verified from the increase of intensity of peaks and decrease of half peak width in XRD spectrum. Considerable increasing of Ca/P ratio was observed after 4 h of conversion (Fig. 1). The reason this effect is rapid rise in the calcium content in sample (Fig. 6) by the interacting of
calcium ions in solution with DCPD—the Ca/P ratio in product after 24 h was 1.64 which is ratio approaching to stoichiometric HAP. The results of XRD diffraction analysis coincide with ones of the IR analysis of spectra samples after 2 and 4 h transformation (Fig. 3c and d), where the spectrum of the product of 2 h hydrolysis represents the spectrum almost pure DCPD whereas the peaks corresponding to vibrations of the phosphate group are shifted about 5–10 cm−1 in the direction to shorter wavelengths. This indicates strengthening of bonds in brushite.
Fig. 5. X-ray diffraction patterns of calcium phosphate powders: (a) HAP, (b) pure DCPD, (c) in solution with addition of 0.5 g Ca2+ /120 mL solution after 2 h, (d) after 4 h and (e) after 48 h.
Fig. 6. Dependence of calcium and phosphorus contents in products on hydrolysis time.
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On the other hand, both the significantly changes in peak intensities of DCPD at 1650, 1230, 980, 870 and 800 cm−1 and in the ratio of intensities of the bands at 560 and 540 cm−1 were observed in the IR spectrum of sample after 4 h of the reaction. Completely modified is shape of bands at 1050 and 1100 cm−1 and the new peaks appearances in vicinity of the wavelengths at 960 and 605 cm−1 —all above bands are characteristic for the hydroxyapatite spectrum after hydrolysis for 24 h. An presence of the pure HAP after conversion of brushite in calcium solution for 24 h without any residue of brushite or monetite is evident from the analysis of TG and DSC curves (Fig. 2d), where no thermal effects, which would be possible assign to decomposition of DCPD or DCPA, were found and the summary mass loss represents the amount of physical bonded water. The changes in the shape of the TG curves and large modification of the shape of DSC curves were observed 2 h after transformation (Fig. 2c). The changes in ratios of the thermal endo-effects are occured at 160 and 200 ◦ C in comparison with effects which were found in the pure brushite—the lower mass loss and magnitude of thermal effect at 200 ◦ C were observed and maximum of endo-effect is moved to 165 ◦ C. From above result that fraction of the chemically bonded water in DCPD is decreased which induces the rise of compactness of the crystal lattice. The fall of the content of chemically bonded water was responsible for the existence of maximum on dependences of the content of calcium and phosphorus in products of reaction on the transformation time (in Fig. 6) because of increasing of the sample densities. This fact is possible to support from comparison of the volumes of crystalline cell of brushite (JCPDS 09-0077) and monetite (JCPDS 09-0080), where both the decrease in cell volume from 493 to ˚ 3 and the increase in the density of monetite about 30% 309 A were found after elimination of the chemically bonded water. In DSC record, a new endo-effect with maximum at temperature of 375 ◦ C was found that is not related to any change in mass. Its presence can be connected with some transformation of the calcium phosphate phases or immediate interaction between residual-modified DCPD and created hydroxyapatite phase. The morphology of hydroxyapatite particles after conversion of brushite for 24 h is shown in Fig. 4d. Similarly as in the case of brushite transformation in solution without addition of calcium ions, plate-like agglomerates of size around 30 m composed from fine particles (<1 m) are visible in figure. From analysis above results it can be stated the conversion of DCPD in solution of calcium ions with initial pH 10.8 does not carry out by the simple dissolving of DCPD and following gradual precipitation of a new hydroxyapatite phase on the surface of brushite particles but there is transformation in volume of DCPD crystal lattice whereas the diffusion of calcium ions into brushite particles is probably one of important effects. 4. Conclusion The results of experimental works showed that the calcium deficient types of HAP are formed by the transformation of brushite in aqueous solution with initial pH 10.8 at temperature
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of 39 ◦ C whereas the stoichiometric hydroxyapatite as thermodynamically the most stable form of calcium phosphates was obtained in the solution without addition of Ca2+ ions after long-time hydrolysis only. In pure aqueous solution at applied pH, the surface nucleation of HAP and gradual transformation of brushite by dissolution–precipitation mechanism were confirmed. The addition of Ca2+ ions into solution causes the acceleration of brushite conversion to calcium deficient HAP forms. The stoichiometric hydroxyapatite is created after 24 h from the immersion of brushite to the reactive solution. As it was shown, the transformation in this case is carried out inside of particles of powder brushite too. Acknowledgements This study was carried out as a project supported by the Slovak Grant Agency of the Ministry of Education of the Slovak Republic and the Slovak Academy of Sciences, Project No. 2/5143/25. References [1] M. Kumar, H. Dasarathy, C. Riley, Electrodeposition of brushite coatings and their transformation to hydroxyapatite in aqueous solutions, J. Biomed. Mater. Res. 45 (4) (1999) 302–310. [2] M.H. Prado Da Silva, J.H.C. Lima, G.A. Soares, C.N. Elias, M.C. Deandrade, S.M. Best, I.R. Gibson, Transformation of monetite to hydroxyapatite in bioactive coatings on titanium, Surf. Coat. Technol. 137 (2001) 270–276. [3] K. Furutaka, H. Monma, T. Okura, S. Takahashi, Characteristic reaction processes in the system brushite–NaOH solution, J. Eur. Ceram. Soc. 26 (2006) 543–547. [4] A. Ferreira, C. Oliveira, F. Rocha, The different phases in the precipitation of dicalcium phosphate dihydrate, J. Cryst. Growth 252 (2003) 599–611. [5] J.B. Liu, X. Ye, H. Wang, M. Zhu, B. Wang, H. Yan, The influence of pH and temperature on the morphology of hydroxyapatite synthesized by hydrothermal method, Ceram. Int. 29 (2003) 629–633. [6] M. Kumar, J. Xie, K. Chittur, C. Riley, Transformation of modified brushite to hydroxyapatite in aqueous solution: effects of potassium substitution, Biomaterials 20 (1999) 1389–1399. [7] J. Xie, C. Riley, K. Chittur, Effect of albumin on brushite transformation to hydroxyapatite, J. Biomed. Mater. Res. 57 (3) (2001) 357–365. [8] A.C. Tas, S.B. Bhaduri, Chemical processing of CaHPO4 ·2H2 O: its conversion to hydroxyapatite, J. Am. Ceram. Soc. 87 (12) (2004) 2195–2200. [9] A.C. Tas, Synthesis of biomimetic Ca–hydroxyapatite powders at 37 ◦ C in synthetic body fluids, Biomaterials 21 (2000) 1429–1438. [10] K.S. Tenhuisen, P.W. Brown, The kinetics of calcium deficient and stoichiometric hydroxyapatite formation from CaHPO4 ·2H2 O and Ca4 (PO4 )2 O, J. Mater. Sci.: Mater. Med. 7 (1996) 309–316. [11] E.C. Moreno, T.M. Gregory, W.E. Brown, Solubility of CaHPO4 ·2H2 O and formation of ion pairs in the system Ca(OH)2 –H3 PO4 –H2 O at 37.5 ◦ C, J. Res. NBS 70A (1996) 545–552. [12] M.T. Fulmer, P.W. Brown, Hydrolysis of dicalcium phosphate dihydrate to hydroxyapatite, J. Mater. Sci.: Mater. Med. 9 (1998) 197–202. [13] P.W. Brown, Phase relationship in the ternary system CaO–P2 O5 –H2 O at 25 ◦ C, J. Am. Ceram. Soc. 25 (1992) 17–22. [14] N. Kanzaki, K. Onuma, G. Treboux, A. Ito, Dissolution kinetics of dicalcium phosphate dihydrate under pseudophysiological conditions, J. Cryst. Growth 235 (2002) 465–470. [15] X. Lu, Y. Leng, Theoretical analysis of calcium phosphate precipitation in simulated body fluid, Biomaterials 26 (2005) 1097–1108.