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Dissolution studies of hydroxyapatite and glass-reinforced hydroxyapatite ceramics
Materials Characterization 50 (2003) 197 – 202
Dissolution studies of hydroxyapatite and glass-reinforced hydroxyapatite ceramics A.C. Queiroz a,b,c,1, J.D. Santos a,b,1, F.J. Monteiro a,b,1, M.H. Prado da Silva d,* a
Laborato´rio de Biomateriais, Instituto de Engenharia Biome´dica (INEB), Rua do Campo Alegre 823, 4150-180 Porto, Portugal b Departamento de Engenharia Metalu´rgica e de Materiais, Faculdade de Engenharia da Universidade do Porto, Rua Roberto Frias, 4200-466 Porto, Portugal c Escola Superior de Tecnologia e Gesta˜o, Ap 574, 4901 Viana do Castelo Codex, Portugal d DEMP – UFC, Campus PICI, Bl. 714, Fortaleza, CE 60455-760, Brazil Received 10 January 2003; accepted 30 March 2003
Abstract In the continuous agitation assays, glass-reinforced hydroxyapatite (GR-HA) was shown to form a calcium phosphate (CaP) layer, but hydroxyapatite (HA) only formed dispersed precipitates. The formation of this layer was first detected on the GR-HA with a 7.5% glass addition (7.5 GR-HA) after only 3 days of immersion in simulated body fluid (SBF). The time required for layer formation decreased as the amount of glass added to the HA increased. The dissolution rate of the materials followed a similar pattern, i.e. the dissolution rate for GR-HA was higher than for HA, and increased with the addition of glass. The immersion of 7.5 GR-HA in water showed almost linear dissolution kinetics over the immersion periods (3, 7, 15, 30 and 60 days). The concentration of calcium ions in solution and the scanning electron microscopy (SEM) analysis of the 7.5 GR-HA specimens immersed in water and in SBF revealed a clear competition between the material dissolution and the precipitation of a CaP phase. Fourier transformed infrared spectroscopy with alternated total reflectance (FTIR-ATR) analysis indicated that the CaP phase that formed during longer immersion times (30 and 60 days) could be a carbonate-substituted CaP precipitate. As expected from previous work, the GR-HA behavior in terms of its in vitro bioactivity is higher than HA because a homogeneous CaP layer is formed and the precipitation occurs faster. From the dissolution test and in accordance with the chemical composition of the samples, GR-HA was more soluble than HA. D 2003 Elsevier Inc. All rights reserved. Keywords: Dissolution; Hydroxyapatite; Glass-reinforced hydroxyapatite
1. Introduction
* Correspondence author. Tel.: +55-85-288-9635; fax: +55-85288-9636. E-mail address: [email protected] (M.H. Prado da Silva). 1 Tel.: +351-22-6074900; fax: +351-22-6094567. 1044-5803/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1044-5803(03)00092-5
Hydroxyapatite (HA) is a well-known material that is extensively used in biomaterials [1]. It is commonly used as a coating layer for hip prostheses, as well as for dental implants [2] and several other types of medical applications because of its reasonable mechanical behavior under low-load conditions and excellent biocompatibility. Newly developed glass-
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reinforced hydroxyapatite (GR-HA) [3,4] has recently been characterized in terms of mechanical properties, which proves to be better than HA and in vitro and in vivo performance, both as coatings [5,6] and as a dense material [7,8]. The formation of an apatite layer in vitro is an important step towards the bioactivity of biomaterials. This layer is formed not only in vivo but also in vitro when these materials are immersed in solutions simulating human plasma, namely, Kokubo et al.’s [9,10] simulated body fluid (SBF). GR-HA ceramics may contain a number of crystalline phases, such as HA, h- and a-TCP [tricalcium phosphate, Ca3(PO4)2], depending on the content of CaO – P2O5 glass added and the applied sintering conditions. Both h-TCP and a-TCP are known to biodegrade faster than HA [11,12], and therefore, GRHA composites may provide several advantages over commercial HA in some applications, namely, those requiring a high dissolution rate. Although some dissolution studies have already been performed to characterize the dissolution behavior of HA and GR-HA [13 –15], in this work, a flowthrough dissolution system and a continuous agitation test were used in order to predict their dissolution rates in vivo and also to evaluate their in vitro bioactivity both in SBF and dissolution in deionised water. These materials are also being studied for use as drug carriers for controlled delivery [16]. Thus, the dissolution rate of these materials is of utmost importance because it can be one of the controlling mechanisms for drug release. The flow-through system has been extensively used to study the drug release kinetics and the dissolution rate of materials in different environments [17].
2. Materials and methods 2.1. Materials preparation The materials used in this study were commercial HA (from Plasma Biotal, ref. P120 powder) and GRHA granules. A glass of the P2O5 – CaO system (75 P2O5, 15 CaO, 10 CaF2 mol%) was prepared using reagent grade chemicals, heated for 1 h at 1350 jC and quenched in water. The glass was dried for 24 h in an oven at 100 jC, ball milled and sieved until a
particle size < 75 Am was achieved. HA was also sieved to approximately the same particle size. GR-HA was obtained with 4.0 and 7.5 wt.% glass addition to HA powder (4.0 and 7.5 GR-HA, respectively). The mixture was wet milled for 8 h with methanol as a suspension medium, dried for 24 h in an oven at 100 jC and sieved to a particle size < 75 Am in order to obtain a homogeneous free-flowing mixed powder. HA and GR-HA were uniaxially pressed to cylindrical samples at 288 MPa. The green compacts were sintered for 1 h at 1200 jC with a heating rate of 4 Cj/min followed by natural cooling inside the furnace. Samples were milled and sieved in order to obtain 250 –850 Am granules. Coulter’s laser particle size analyzer was used for granulometric control [16]. 2.2. Dissolution studies All of the dissolution tests were conducted in triplicate, both in deionised water and in SBF. The SBF was prepared according to Kokubo’s [18] formulation, which is a buffered (pH of 7.4 at 25 jC) solution with an ionic concentration close to that found in the human blood plasma (Table 1—chemical composition of formulated SBF). Continuous agitation assays were performed by immersing 0.5 g of the previously prepared granules in 5 ml of the two testing solutions using 20-ml capacity polypropylene flasks, thermostatised in an orbital shaker (37 F 0.5 jC) and continuously stirred at 120 rpm (2 Hz). The material-to-solution ratio, temperature, buffer pH and agitation were selected according to ISO/FDIS 10993-14 [19]. After immersion periods of 3, 7, 15, 30 and 60 days, the samples
Table 1 Reagent concentrations for SBF preparation Reagents
SBF, mass (g)
NaCl NaHCO3 KCl KH2PO43H2O MgCl26H2O Na2SO4 CaCl2 Adjustment pH 7.4 Tris HCl (1 M)
8.003 0.353 0.224 0.228 0.305 0.071 0.278 Needed Needed
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were centrifuged at 4000 rpm for 5 min, and after decanting the supernatant, the granules were carefully washed with monodistilled deionised water. Both supernatant and granules were reserved for posterior analysis using several analysis techniques. The flow-through dissolution assays were performed in a dissolution system (Sotax CE6) consisting of a water-heated bath at 37 F 0.2 jC with four cells with controlled volume, a four-channel peristaltic pump (Gilson Minipulse3) and a fraction collector. For each sample, 0.5 g was placed in a cell and the testing solution was passed through the samples at a constant rate of 10 ml/min and collected at the same rate. The total time of these assays was 30 min, with samples collected every minute, as these were the testing conditions used with the same materials to evaluate their drug-releasing capability [16]. Atomic absorption spectrophotometry (AAS) was used to determine the calcium concentration in the supernatants. The fluoride concentration was evaluated with an F ion selective electrode. The granules were individually analysed by scanning electron microscopy (SEM) at 15 KeV, energy-dispersive X-ray analysis (EDS) and Fourier transformed infrared spectroscopy with alternated total reflectance (FTIR-ATR) using the split-pea accessory to characterize the precipitates formed on the samples’ surface.
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Fig. 2. SEM micrographs of 7.5 GR-HA after (a) 0, (b) 3 and (c) 60 days of immersion in water.
face. This probably occurred because of the high flow rate, which may have removed any calcium phosphate (CaP) formation on the surface and a short immersion period (only 30 min) when compared to the immersion periods used in the continuous agitation assays. The fluoride concentration was not analysed because there was no difference in the calcium concentration and the fluoride concentration in the granules is much lower.
3. Results and discussion When analysing the results obtained from the flowthrough dissolution assays, the concentration of calcium on the collected solution samples was similar to the initial calcium concentration both for HA and GRHA samples. SEM analysis also revealed no deposition of newly formed precipitates on materials’ sur-
Fig. 1. Continuous dissolution assay in water for (x) HA, (.) 4.0 GR-HA and (x) 7.5 GR-HA.
Fig. 3. SEM micrographs after 60 days of immersion in SBF of (a) HA, (b) 4.0 GR-HA and (c) 7.5 GR-HA.
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Fig. 4. Continuous dissolution assay in SBF for (x) HA, (.) 4.0 GR-HA and (x) 7.5 GR-HA.
In contrast to the results obtained from the flowthrough dissolution assays, continuous agitation assays indicated a clear dissolution of 7.5 GR-HA when immersed in water (Fig. 1). SEM/EDS observation of these samples showed the presence of a CaP precipitate after 3 days of immersion. This precipitate tended to organize and form a CaP layer after 60 days of immersion (Fig. 2). This precipitation was because of the previous dissolution of the material because the medium used was calcium-free (monodistilled deionised water). For the 3-day immersion period, the calcium concentration in solution was higher for HA than for GR-HA. This phenomenon was probably because of a dissolution/precipitation mechanism occurring at the GR-HA surface but that did not occur to such an extent with HA. In fact, abundant CaP formation occurred at the surface of 7.5 GR-HA,
which led to a decrease in calcium concentration. Regarding the HA and 4.0 GR-HA behavior, this pattern was maintained for all immersion times, although for the longer immersion periods, dispersed precipitates probably formed on the HA surface. For 7.5 GR-HA, the dissolution is much higher than for the two other tested materials, so the amount of calcium in solution after 30 days is already higher than for HA. This effect is explained by the presence of a significant content of highly soluble phases, like h-TCP, as shown previously [16]. So what firstly was thought to be a strange result, as it would be expected that the calcium concentration in the water assays should be higher for the GR-HA materials, can now be explained as a competition of dissolution of the material and the precipitation of newly formed CaP on its surface. When immersed in SBF, all samples were characterized by the presence of CaP precipitates, yet there is a clear difference between the tested samples even after 60 days of immersion. This is illustrated by the SEM analysis (Fig. 3) in which only a few dispersed precipitates are present in the HA. The amount of precipitation increased for 4.0 GR-HA and a homogeneous layer was formed for 7.5 GR-HA. All precipitates formed are CaP, as evidenced by EDS analysis. For each material, the amount of calcium present in the solution varied, indicating the occurrence of
Fig. 5. FTIR-ATR analysis of 7.5 GR-HA granules, ‘‘as is,’’ and after 3, 30 and 60 days of immersion.
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dissolution – precipitation phenomena. It appeared that initially, there was significant dissolution of the material and, as CaP precipitates were formed, dissolution of the material, together with the dissolution of some possible amorphous precipitates, occurred. This was clearer for the 7.5 GR-HA, although it also seemed to be present, at a smaller scale, for 4.0 GRHA. In the immersed HA, the amount of material dissolution seemed to be stabilized and similar to the pattern observed in water (Fig. 4), and only few dispersed precipitates were observed. In these assays, the fluoride concentration was measured although no fluoride appeared to be present in an amount above the selective electrode’s detection limit ( < 10 6 mol/l F ). When comparing the calcium concentration results obtained from the water and SBF continuous agitation assays, there is a clear difference in the amount of calcium dissolved for the GR-HA ceramics. This fact is probably because of the nature of the solutions used. For the test in water (calcium-free medium), the calcium from the dissolved GR-HA material is used in the formation of the CaP precipitates. Therefore, the calcium concentration is theoretically higher than the value given from the calcium concentration in the solution. When using SBF, there is an initial amount of calcium present in the solution. This calcium can be used for the formation of the CaP precipitates almost immediately after immersion. For this reason, the calcium concentration in the supernatant that is attributed to material dissolution obtained in these assays is higher and probably more realistic than the values given for the water assays. From all the granules tested, only those of 7.5 GRHA were analysed by FTIR-ATR because this material was the only one forming a continuous surface layer. FTIR results are shown in Fig. 5. After 3 days of immersion, CaP precipitates could be analysed and there was no significant difference compared to the untreated granules. For the longer immersion period samples, the appearance of the carbonate bands (1454, 1417 and 880 cm 1) indicates the presence of carbonate-substituted CaP precipitates because the phosphate bands are still present. Moreover, for the 60-day immersion sample, the bands characteristic of OH could also be distinguished (3330 and 2974 cm 1). As the CaP layer formed in the 7.5 GR-HA after 30 days of immersion is a carbonated one, it can be
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concluded that its origin is the SBF as NaHCO3 is used in its preparation.
4. Conclusion The continuous agitation assays indicated the presence of competing mechanisms between dissolution and precipitation that became evident because of the differences obtained when using calcium-free and SBF solutions. For these tests, it was also noted that 7.5 GR-HA is more soluble than 4.0 GR-HA, which is more soluble than HA. The ability to form a CaP layer is by far greater in the 7.5 GR-HA than in the other tested materials, although after 60 days the precipitates forming in the 4.0 GR-HA were already covering most of its surface.
Acknowledgements The authors would like to acknowledge the help provided by Instituto Geolo´gico e Mineiro and its director Prof. Machado Leite.
References [1] Barinov SM, Baschenko YV. Application of ceramic composites as implants: result and problem. In: Ravaglioli A, Krajewski A, editors. Bioceramics and the human body. United Kingdom: Elsevier; 1992. p. 206 – 10. [2] Nishihara K. Studies on peri-root tissue formation around new type artificial root made of dense hydroxyapatite. Clin Mater 1993;12:159 – 67. [3] Lopes MA, Monteiro FJ, Santos JD, Serro AP, Saramago B. Hydrophobicity, surface tensions, and zeta potential measurements of glass-reinforced hydroxyapatite composites. J Biomed Mater Res 1999;45:370 – 5. [4] Santos JD, Silva PL, Knowles JC, Talal S, Monteiro FJ. Reinforcement of hydroxyapatite by adding P2O5 – CaO glasses with Na2O, K2O, MgO. J Mater Sci Mater Med 1996;7: 187 – 9. [5] Ferraz MP, Knowles JC, Olsen I, Monteiro FJ, Santos JD. Flow cytometry analysis of effects of glass on response of osteosarcoma cells to plasma-sprayed hydroxyapatite/CaO – P2O5 coatings. J Biomed Mater Res 1999;47:603 – 11. [6] Ferraz MP, Monteiro FJ, Santos JD. CaO – P2O5 glass hydroxyapatite double-layer plasma-sprayed coating: in vitro bioactivity evaluation. J Biomed Mater Res 1999;45:376 – 83. [7] Lopes MA, Santos JD, Monteiro FJ, Ohtsuki C, Osaka A, Kaneko S, et al. Push-out testing and histological evaluation
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A.C. Queiroz et al. / Materials Characterization 50 (2003) 197–202 of glass reinforced hydroxyapatite composites implanted in the tibia of rabbits. J Biomed Mater Res 2001;54:463 – 9. Lopes MA, Knowles JC, Kuru L, Santos JD, Monteiro FJ, Olsen I. Flow cytometry for assessing biocompatibility. J Biomed Mater Res 1998;41:649 – 56. Kokubo T, Ito S, Huang ZT, Hayashi T, Sakka S. Ca, P-rich layer formed on high-strength bioactive glass-ceramic A – W. J Biomed Mater Res 1990;24:331 – 43. Kokubo T, Kushitani H, Kitsugi T, Yamamuro T. Solution able to reproduce in vivo surface-structure changes of bioactive glass-ceramic A – W. J Biomed Mater Res 1990;24:721 – 34. Klein CPAT, Wolke JGC, de Blieck-Hogervorst JMA, de Groot K. Features of calcium phosphate plasma-sprayed coatings: an in vitro study. J Biomed Mater Res 1994;28:961 – 7. Klein CPAT, Wolke JGC, de Blieck-Hogervorst JMA, de Groot K. Calcium phosphate plasma-sprayed coatings and their stability: an in vivo study. J Biomed Mater Res 1994;28: 909 – 17. Kokubo T, Kushitani H, Ohtsuki C, Sakka S, Yamamuro T. Chemical reaction of bioactive glass and glass-ceramics with a simulated body fluid. J Mater Sci Mater Med 1992;3: 79 – 83.
[14] Zhang H, Li S, Yan Y. Dissolution behaviour of hydroxyapatite powder in hydrothermal solution. Ceram Int 2001; 27:451 – 4. [15] LeGeros RZ, LeGeros JP. Calcium phosphate biomaterials in medical applications. In: Kokubo T, Nakamura T, Miyaji F, editors. Bioceramics, vol. 9. Otsu, Japan: Elsevier; 1996. p. 7 – 10. [16] Queiroz AC, Santos JD, Monteiro FJ, Gibson IR, Knowles JC. Adsorption and release studies of sodium ampicillin from hydroxyapatite and glass-reinforced hydroxyapatite composites. Biomaterials 2001;22:1393 – 400. [17] Langenbucher F, Benz D, Ku¨rth W, Mo¨ller H, Otz M. Standardized flow-cell method as an alternative to existing pharmacopoeial dissolution testing. Pharm Ind 1989;51(11): 1276 – 81. [18] Kokubo T. In: Yamamuro T, Hench LL, Wilson J, editors. CRC handbook of bioactive ceramics, vol. 1. Boca Raton (FL): CRC Press; 1990. p. 41. [19] International Standard ISO/FDIS 10993-14, Biological evaluation of medical devices: Part 14. Identification and quantification of degradation products from ceramics; 2001.
Dissolution studies of hydroxyapatite and glass-reinforced hydroxyapatite ceramics A.C. Queiroz a,b,c,1, J.D. Santos a,b,1, F.J. Monteiro a,b,1, M.H. Prado da Silva d,* a
Laborato´rio de Biomateriais, Instituto de Engenharia Biome´dica (INEB), Rua do Campo Alegre 823, 4150-180 Porto, Portugal b Departamento de Engenharia Metalu´rgica e de Materiais, Faculdade de Engenharia da Universidade do Porto, Rua Roberto Frias, 4200-466 Porto, Portugal c Escola Superior de Tecnologia e Gesta˜o, Ap 574, 4901 Viana do Castelo Codex, Portugal d DEMP – UFC, Campus PICI, Bl. 714, Fortaleza, CE 60455-760, Brazil Received 10 January 2003; accepted 30 March 2003
Abstract In the continuous agitation assays, glass-reinforced hydroxyapatite (GR-HA) was shown to form a calcium phosphate (CaP) layer, but hydroxyapatite (HA) only formed dispersed precipitates. The formation of this layer was first detected on the GR-HA with a 7.5% glass addition (7.5 GR-HA) after only 3 days of immersion in simulated body fluid (SBF). The time required for layer formation decreased as the amount of glass added to the HA increased. The dissolution rate of the materials followed a similar pattern, i.e. the dissolution rate for GR-HA was higher than for HA, and increased with the addition of glass. The immersion of 7.5 GR-HA in water showed almost linear dissolution kinetics over the immersion periods (3, 7, 15, 30 and 60 days). The concentration of calcium ions in solution and the scanning electron microscopy (SEM) analysis of the 7.5 GR-HA specimens immersed in water and in SBF revealed a clear competition between the material dissolution and the precipitation of a CaP phase. Fourier transformed infrared spectroscopy with alternated total reflectance (FTIR-ATR) analysis indicated that the CaP phase that formed during longer immersion times (30 and 60 days) could be a carbonate-substituted CaP precipitate. As expected from previous work, the GR-HA behavior in terms of its in vitro bioactivity is higher than HA because a homogeneous CaP layer is formed and the precipitation occurs faster. From the dissolution test and in accordance with the chemical composition of the samples, GR-HA was more soluble than HA. D 2003 Elsevier Inc. All rights reserved. Keywords: Dissolution; Hydroxyapatite; Glass-reinforced hydroxyapatite
1. Introduction
* Correspondence author. Tel.: +55-85-288-9635; fax: +55-85288-9636. E-mail address: [email protected] (M.H. Prado da Silva). 1 Tel.: +351-22-6074900; fax: +351-22-6094567. 1044-5803/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/S1044-5803(03)00092-5
Hydroxyapatite (HA) is a well-known material that is extensively used in biomaterials [1]. It is commonly used as a coating layer for hip prostheses, as well as for dental implants [2] and several other types of medical applications because of its reasonable mechanical behavior under low-load conditions and excellent biocompatibility. Newly developed glass-
198
A.C. Queiroz et al. / Materials Characterization 50 (2003) 197–202
reinforced hydroxyapatite (GR-HA) [3,4] has recently been characterized in terms of mechanical properties, which proves to be better than HA and in vitro and in vivo performance, both as coatings [5,6] and as a dense material [7,8]. The formation of an apatite layer in vitro is an important step towards the bioactivity of biomaterials. This layer is formed not only in vivo but also in vitro when these materials are immersed in solutions simulating human plasma, namely, Kokubo et al.’s [9,10] simulated body fluid (SBF). GR-HA ceramics may contain a number of crystalline phases, such as HA, h- and a-TCP [tricalcium phosphate, Ca3(PO4)2], depending on the content of CaO – P2O5 glass added and the applied sintering conditions. Both h-TCP and a-TCP are known to biodegrade faster than HA [11,12], and therefore, GRHA composites may provide several advantages over commercial HA in some applications, namely, those requiring a high dissolution rate. Although some dissolution studies have already been performed to characterize the dissolution behavior of HA and GR-HA [13 –15], in this work, a flowthrough dissolution system and a continuous agitation test were used in order to predict their dissolution rates in vivo and also to evaluate their in vitro bioactivity both in SBF and dissolution in deionised water. These materials are also being studied for use as drug carriers for controlled delivery [16]. Thus, the dissolution rate of these materials is of utmost importance because it can be one of the controlling mechanisms for drug release. The flow-through system has been extensively used to study the drug release kinetics and the dissolution rate of materials in different environments [17].
2. Materials and methods 2.1. Materials preparation The materials used in this study were commercial HA (from Plasma Biotal, ref. P120 powder) and GRHA granules. A glass of the P2O5 – CaO system (75 P2O5, 15 CaO, 10 CaF2 mol%) was prepared using reagent grade chemicals, heated for 1 h at 1350 jC and quenched in water. The glass was dried for 24 h in an oven at 100 jC, ball milled and sieved until a
particle size < 75 Am was achieved. HA was also sieved to approximately the same particle size. GR-HA was obtained with 4.0 and 7.5 wt.% glass addition to HA powder (4.0 and 7.5 GR-HA, respectively). The mixture was wet milled for 8 h with methanol as a suspension medium, dried for 24 h in an oven at 100 jC and sieved to a particle size < 75 Am in order to obtain a homogeneous free-flowing mixed powder. HA and GR-HA were uniaxially pressed to cylindrical samples at 288 MPa. The green compacts were sintered for 1 h at 1200 jC with a heating rate of 4 Cj/min followed by natural cooling inside the furnace. Samples were milled and sieved in order to obtain 250 –850 Am granules. Coulter’s laser particle size analyzer was used for granulometric control [16]. 2.2. Dissolution studies All of the dissolution tests were conducted in triplicate, both in deionised water and in SBF. The SBF was prepared according to Kokubo’s [18] formulation, which is a buffered (pH of 7.4 at 25 jC) solution with an ionic concentration close to that found in the human blood plasma (Table 1—chemical composition of formulated SBF). Continuous agitation assays were performed by immersing 0.5 g of the previously prepared granules in 5 ml of the two testing solutions using 20-ml capacity polypropylene flasks, thermostatised in an orbital shaker (37 F 0.5 jC) and continuously stirred at 120 rpm (2 Hz). The material-to-solution ratio, temperature, buffer pH and agitation were selected according to ISO/FDIS 10993-14 [19]. After immersion periods of 3, 7, 15, 30 and 60 days, the samples
Table 1 Reagent concentrations for SBF preparation Reagents
SBF, mass (g)
NaCl NaHCO3 KCl KH2PO43H2O MgCl26H2O Na2SO4 CaCl2 Adjustment pH 7.4 Tris HCl (1 M)
8.003 0.353 0.224 0.228 0.305 0.071 0.278 Needed Needed
A.C. Queiroz et al. / Materials Characterization 50 (2003) 197–202
were centrifuged at 4000 rpm for 5 min, and after decanting the supernatant, the granules were carefully washed with monodistilled deionised water. Both supernatant and granules were reserved for posterior analysis using several analysis techniques. The flow-through dissolution assays were performed in a dissolution system (Sotax CE6) consisting of a water-heated bath at 37 F 0.2 jC with four cells with controlled volume, a four-channel peristaltic pump (Gilson Minipulse3) and a fraction collector. For each sample, 0.5 g was placed in a cell and the testing solution was passed through the samples at a constant rate of 10 ml/min and collected at the same rate. The total time of these assays was 30 min, with samples collected every minute, as these were the testing conditions used with the same materials to evaluate their drug-releasing capability [16]. Atomic absorption spectrophotometry (AAS) was used to determine the calcium concentration in the supernatants. The fluoride concentration was evaluated with an F ion selective electrode. The granules were individually analysed by scanning electron microscopy (SEM) at 15 KeV, energy-dispersive X-ray analysis (EDS) and Fourier transformed infrared spectroscopy with alternated total reflectance (FTIR-ATR) using the split-pea accessory to characterize the precipitates formed on the samples’ surface.
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Fig. 2. SEM micrographs of 7.5 GR-HA after (a) 0, (b) 3 and (c) 60 days of immersion in water.
face. This probably occurred because of the high flow rate, which may have removed any calcium phosphate (CaP) formation on the surface and a short immersion period (only 30 min) when compared to the immersion periods used in the continuous agitation assays. The fluoride concentration was not analysed because there was no difference in the calcium concentration and the fluoride concentration in the granules is much lower.
3. Results and discussion When analysing the results obtained from the flowthrough dissolution assays, the concentration of calcium on the collected solution samples was similar to the initial calcium concentration both for HA and GRHA samples. SEM analysis also revealed no deposition of newly formed precipitates on materials’ sur-
Fig. 1. Continuous dissolution assay in water for (x) HA, (.) 4.0 GR-HA and (x) 7.5 GR-HA.
Fig. 3. SEM micrographs after 60 days of immersion in SBF of (a) HA, (b) 4.0 GR-HA and (c) 7.5 GR-HA.
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Fig. 4. Continuous dissolution assay in SBF for (x) HA, (.) 4.0 GR-HA and (x) 7.5 GR-HA.
In contrast to the results obtained from the flowthrough dissolution assays, continuous agitation assays indicated a clear dissolution of 7.5 GR-HA when immersed in water (Fig. 1). SEM/EDS observation of these samples showed the presence of a CaP precipitate after 3 days of immersion. This precipitate tended to organize and form a CaP layer after 60 days of immersion (Fig. 2). This precipitation was because of the previous dissolution of the material because the medium used was calcium-free (monodistilled deionised water). For the 3-day immersion period, the calcium concentration in solution was higher for HA than for GR-HA. This phenomenon was probably because of a dissolution/precipitation mechanism occurring at the GR-HA surface but that did not occur to such an extent with HA. In fact, abundant CaP formation occurred at the surface of 7.5 GR-HA,
which led to a decrease in calcium concentration. Regarding the HA and 4.0 GR-HA behavior, this pattern was maintained for all immersion times, although for the longer immersion periods, dispersed precipitates probably formed on the HA surface. For 7.5 GR-HA, the dissolution is much higher than for the two other tested materials, so the amount of calcium in solution after 30 days is already higher than for HA. This effect is explained by the presence of a significant content of highly soluble phases, like h-TCP, as shown previously [16]. So what firstly was thought to be a strange result, as it would be expected that the calcium concentration in the water assays should be higher for the GR-HA materials, can now be explained as a competition of dissolution of the material and the precipitation of newly formed CaP on its surface. When immersed in SBF, all samples were characterized by the presence of CaP precipitates, yet there is a clear difference between the tested samples even after 60 days of immersion. This is illustrated by the SEM analysis (Fig. 3) in which only a few dispersed precipitates are present in the HA. The amount of precipitation increased for 4.0 GR-HA and a homogeneous layer was formed for 7.5 GR-HA. All precipitates formed are CaP, as evidenced by EDS analysis. For each material, the amount of calcium present in the solution varied, indicating the occurrence of
Fig. 5. FTIR-ATR analysis of 7.5 GR-HA granules, ‘‘as is,’’ and after 3, 30 and 60 days of immersion.
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dissolution – precipitation phenomena. It appeared that initially, there was significant dissolution of the material and, as CaP precipitates were formed, dissolution of the material, together with the dissolution of some possible amorphous precipitates, occurred. This was clearer for the 7.5 GR-HA, although it also seemed to be present, at a smaller scale, for 4.0 GRHA. In the immersed HA, the amount of material dissolution seemed to be stabilized and similar to the pattern observed in water (Fig. 4), and only few dispersed precipitates were observed. In these assays, the fluoride concentration was measured although no fluoride appeared to be present in an amount above the selective electrode’s detection limit ( < 10 6 mol/l F ). When comparing the calcium concentration results obtained from the water and SBF continuous agitation assays, there is a clear difference in the amount of calcium dissolved for the GR-HA ceramics. This fact is probably because of the nature of the solutions used. For the test in water (calcium-free medium), the calcium from the dissolved GR-HA material is used in the formation of the CaP precipitates. Therefore, the calcium concentration is theoretically higher than the value given from the calcium concentration in the solution. When using SBF, there is an initial amount of calcium present in the solution. This calcium can be used for the formation of the CaP precipitates almost immediately after immersion. For this reason, the calcium concentration in the supernatant that is attributed to material dissolution obtained in these assays is higher and probably more realistic than the values given for the water assays. From all the granules tested, only those of 7.5 GRHA were analysed by FTIR-ATR because this material was the only one forming a continuous surface layer. FTIR results are shown in Fig. 5. After 3 days of immersion, CaP precipitates could be analysed and there was no significant difference compared to the untreated granules. For the longer immersion period samples, the appearance of the carbonate bands (1454, 1417 and 880 cm 1) indicates the presence of carbonate-substituted CaP precipitates because the phosphate bands are still present. Moreover, for the 60-day immersion sample, the bands characteristic of OH could also be distinguished (3330 and 2974 cm 1). As the CaP layer formed in the 7.5 GR-HA after 30 days of immersion is a carbonated one, it can be
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concluded that its origin is the SBF as NaHCO3 is used in its preparation.
4. Conclusion The continuous agitation assays indicated the presence of competing mechanisms between dissolution and precipitation that became evident because of the differences obtained when using calcium-free and SBF solutions. For these tests, it was also noted that 7.5 GR-HA is more soluble than 4.0 GR-HA, which is more soluble than HA. The ability to form a CaP layer is by far greater in the 7.5 GR-HA than in the other tested materials, although after 60 days the precipitates forming in the 4.0 GR-HA were already covering most of its surface.
Acknowledgements The authors would like to acknowledge the help provided by Instituto Geolo´gico e Mineiro and its director Prof. Machado Leite.
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