New Insight into the Physicochemistry at the Interface between Sol

Jun 3, 2008 - Joséphine Lacroix , Jonathan Lao , and Edouard Jallot. The Journal of ... E. Jallot , J. Lao , Ł. John , J. Soulié , Ph. Moretto and ...
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J. Phys. Chem. C 2008, 112, 9418–9427

New Insight into the Physicochemistry at the Interface between Sol-Gel-Derived Bioactive Glasses and Biological Medium: A PIXE-RBS Study J. Lao,† J. M. Nedelec,‡ and E. Jallot*,† Laboratoire de Physique Corpusculaire de Clermont-Ferrand CNRS/IN2P3 UMR 6533, UniVersite´ Blaise Pascal, 24 AVenue des Landais, 63177 Aubie`re Cedex, France, and Laboratoire des Mate´riaux Inorganiques CNRS UMR 6002, UniVersite´ Blaise Pascal & ENSCCF, 24 AVenue des Landais, 63177 Aubie`re Cedex, France ReceiVed: January 21, 2008; ReVised Manuscript ReceiVed: April 4, 2008

Bioactive glasses are known to quickly induce the formation of a calcium phosphate layer at their surface when in contact with biological fluids; this property is particularly interesting for clinical applications. Because the bioactivity process deeply depends on the glass composition and texture, and because little has been attempted in quantifying the reaction kinetics of these materials, we developed an unconventional methodology to highlight the influence of the phosphorus oxide content on the physicochemical properties of bioactive glasses. Sol-gel derived glasses in the SiO2-CaO and SiO2-CaO-P2O5 systems were soaked in biological fluids for varying periods. After immersion, the surface changes were characterized using particle-induced X-ray emission (PIXE) associated to Rutherford backscattering spectroscopy (RBS), which are efficient techniques for multielemental analysis. In addition, these ion beam methods permit accurate trace elements quantification. Chemical mapping of the glass/biological fluids interface at a micrometer scale reveals the bone-bonding ability of our materials. The formation of a Ca-P-Mg layer occurs after a few hours of interaction. The kinetics of the evolution of the calcium phosphate layer is estimated by modeling the Ca/P atomic ratio decrease at the glass surface, along with supersaturation studies in biological fluids. The materials composition and texture significantly affect the kinetics and amplitude of the bioactivity mechanism. Dealkalinization of the glass matrix and the first appearance of the calcium phosphate layer are delayed for SiO2-CaO-P2O5 glasses when compared to SiO2-CaO glass. Nevertheless, once the Ca-P-Mg layer formation has begun, the layer extends on greater depths and is more quickly changed into a bone-like apatitic phase for P-containing glasses. The presence of phosphorus in the primary glass matrix facilitates the transformation of the initially amorphous calcium phosphates into bone-like apatite crystals. Moreover greater quantities of magnesium are incorporated for SiO2-CaO-P2O5 glasses: this is important information for medical applications due to Mg bactericidal and anti-inflammatory properties. Introduction Biomaterials represent one of the major advances in therapeutics over the past four decades. Devoted to the replacement of deficient functions or organs of the body, biomaterials are nowadays present in many curative strategies. Millions of people are concerned; thus, biomaterials raise not only scientific issues but also economic, ethical, regulatory, and industrial demands that tie down to requirements for security, reliability, and reproducibility. The responsibility is huge because if a drug treatment can be interrupted at any time then an implanted biomaterial cannot be withdrawn without surgery. In the last few years, innovative concepts have emerged and a new generation biomaterials such as bioactive glasses are able to bond harmoniously with living tissues while actively taking part to the healing process; they would even positively stimulate the response of the body.1–4 Bioactive glasses are ceramic biomaterials that own properties of particular interest for filling osseous defects.5–8 In contact with living tissues, bioactive glasses induce a series of physi* Corresponding author. Tel: +33 (0)4 73 40 72 65. Fax: +33 (0)4 73 26 45 98. E-mail: [email protected]. † Laboratoire de Physique Corpusculaire de Clermont-Ferrand CNRS/ IN2P3 UMR 6533, Universite´ Blaise Pascal. ‡ Laboratoire des Mate ´ riaux Inorganiques CNRS UMR 6002, Universite´ Blaise Pascal & ENSCCF.

cochemical reactions at the interface between the material and host tissues, leading to the formation of an interfacial calcium phosphate layer. This layer finally develops into a hydroxycarbonate apatite similar to the mineral phase of bone.9,10 In addition, this layer provides favorable sites for the mineralization of bone cells and will therefore generate a strong interfacial bond between the bioactive glass and host tissues.11–18 The formation of this bond is characteristic of a material’s bioactivity. The bioactivity process deeply depends on the glass composition and texture; a better understanding of the bioactivity mechanism requires the collection of reliable quantitative information regarding the physicochemical reactions occurring at the bioactive glass/biological system interface. Although the bioactivity mechanism has been investigated extensively over the past few years, quantitative data on the ionic exchanges occurring at the material surface are still sparse. Aiming at this objective, we developed an unconventional methodology to highlight the influence of the phosphorus oxide on the physicochemical behavior of bioactive glasses. We elaborated sol-gel derived bioactive glasses in SiO2-CaO and SiO2-CaO-P2O5 systems. The sol-gel method allows the synthesis of nanoporous materials of excellent purity and homogeneity at low processing temperatures.19 Another advantage is the opportunity to finely tune the materials’ composition.20–22 In vitro interactions with biological fluids were conducted on

10.1021/jp800583m CCC: $40.75  2008 American Chemical Society Published on Web 06/03/2008

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TABLE 1 SiO2 P2O5 CaO

TABLE 2 B75

B72.5

B70

B67.5

72.20 ( 0.37

71.49 ( 0.40 2.48 ( 0.02 24.36 ( 0.14

68.25 ( 0.26 4.85 ( 0.06 25.76 ( 0.18

63.75 ( 0.40 6.95 ( 0.14 24.13 ( 0.11

24.50 ( 0.17

a

Composition of the sol-gel derived B75, B72.5, B70, and B67.5 glasses (weight %) as determined by ICP-AES.

the prepared materials. We obtained information on the glasses reactivity at both a local and global scale. Determining the biological fluid’s composition after interaction with bioactive glasses is very systematic when studying such types of materials: it gives access to the glass’s reactivity at a global scale. We then used these data to perform supersaturation studies in biological fluids in order to evaluate the potential for each glass to induce the formation of bone-like hydroxyapatite. The next step of our study was the collect of reliable quantitative data regarding the in vitro dissolution of the material, the ionic exchanges, and the formation and growth of the calcium phosphate layer. Achieving this goal required the following: (i) to analyze the chemical element’s distribution at the bioactive glass/biological system interface, (ii) to adopt characterization techniques providing an excellent sensitivity along with a spatial resolution high enough. On these considerations, we chose the PIXE technique (particle induced X-ray emission) associated to RBS (Rutherford backscattering spectrometry) to record elemental maps of the interface between the bioactive glass and the biological system. The PIXE-RBS nuclear microprobe has a sensitivity in the order of a few parts per million, which allows the study of trace elements (such as magnesium) involved in the bioactivity process. Furthermore, chemical mapping of the bioactive glass/biological medium interface is made possible at the micrometer scale, allowing the complete follow-up of the calcium phosphate layer formation along with accurate major and trace element quantification. It will permit important evaluation for the kinetics of the evolution of the calcium phosphate layer at the glass/biological fluids interface, and thus a useful estimation for the in vitro bioactivity of SiO2-CaO and SiO2-CaO-P2O5 glasses. Experimental Methods Preparation of the Bioactive Glass Samples. Gel-glass powders in the SiO2-CaO system, named B75, and in the SiO2-CaO-P2O5 system, named B72.5, B70, and B67.5, were prepared using the sol-gel process. We have used the following convention to label our samples: a glass named BX is of chemical composition SiO2(X wt %)-CaO(25 wt %)-P2O5(100-(X + 25) wt %)). Tetraethylorthosilicate (Si(OC2H5)4), triethylphosphate (PO(OC2H5)3), and calcium nitrate Ca(NO3)2, 4H2O were mixed in ethanol in the presence of water and HCl. The prepared sols were then transferred to an oven at 60 °C for gelification and aging. Four hours later, the obtained gels were dried at 125 °C for 24 h, then finally grinded to powder and heated at 700 °C for 24 h to achieve nitrate elimination and further densification. The dry gel powders were then compacted into discs of 13 mm diameter and 2 mm height. Materials Characterization. The chemical composition of the bioactive glass powders were determined by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The experimental glass compositions are given in Table 1. The textural characterization of the samples was performed by nitrogen gas sorption analyses. The samples were vacuum outgassed at 120 °C for 12 h to remove physically adsorbed

B75

B72.5

B70

B67.5

2

30 74 124 112 BET surface area (m /g) BJH average pore diameter (nm) 8.4 8.1 9.9 13.2 BJH modal pore diameter (nm) 4.6 4.6 6.2 8.9 total pore volume (cm3/g) 0.062 0.104 0.307 0.289 a

Textural properties of the gel-glass powders. The surface area (S) was determined from the linear portion of the BET plot, the total pore volume (V) was estimated from the amount of N2 adsorbed at P/P0 ) 0.995, the pore size distribution and the modal pore diameter were calculated by applying the BJH method to the N2 desorption branches, and the average pore diameter was calculated as r ) 2V/S.

molecules such as moisture from the pores. The adsorption/ desorption isotherms were recorded on a Quantachrom Autosorb-1 apparatus. The instrument determined isotherms volumetrically by a discontinuous static method at 77 K. The surface areas were obtained by applying the BET method to the N2 isotherm. The pore size distribution was determined by the BJH method on the desorption branch. Total pore volume was measured at a relative pressure P/P0 ) 0.995. In Vitro Studies. The glass discs were immersed at 37 °C for 1 h, 6 h, and 1, 2, 5, and 10 days in 45 mL of a standard Dulbecco’s Modified Eagle Medium (DMEM, Biochrom AG, Germany), whose composition is almost equal to human plasma. After interaction, part of the DMEM was analyzed by ICP-AES while the glass discs were removed from the solution, air-dried, and embedded in resin (AGAR, Essex, England). Before characterization, the glass discs were cut into thin sections of 30 µm nominal thickness using a Leica RM 2145 microtome. Then the sections were placed on a Mylar film with a hole of 3 mm in the center. Measurements are performed on the area of the section placed over the hole. PIXE-RBS Analysis. PIXE and RBS methods are used simultaneously. The PIXE method permits the identification and the quantification of major and trace elements at the biomaterial/ biological fluids interface. RBS is used to determine the electric charge received by the samples during irradiation. This parameter is absolutely necessary for PIXE spectra quantification. Analyses of the biomaterial/biological fluids interface were carried out using nuclear microprobes at the CENBG (Centre d′E´tudes Nucle´aires de Bordeaux-Gradignan, France). The experimental characteristics of the CENBG microbeam line have been published previously.23,24 For PIXE-RBS analyses, we chose a proton scanning microbeam of 1.5 MeV energy and 500 pA in intensity. The beam size was nearly 2 µm. Such parameter values resulted in higher ionization cross sections for light elements (Z e 20) and thus in a better sensitivity for PIXE analysis by using a detector without filter. Furthermore, weak intensities and the choice of protons as the ion beam allowed the target degradation to be minimized during irradiation. However, the intensities were sufficient to permit measurement duration under 1 h. An 80 mm2 Si(Li) detector was used for X-ray detection, orientated at 135° with respect to the incident beam axis, and equipped with a beryllium window 12 µm thick. PIXE spectra were treated with the software package GUPIX.25 Relating to RBS, a silicon particle detector placed at 135° from the incident beam axis provided us with the number of protons that interacted with the sample. Data were treated with the SIMNRA code.26 Results Gel-Glass Powder Characterization. Table 2 shows the results for the BET surface area, for the average and modal

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Figure 1. Elemental maps at the periphery of a SiO2-CaO B75 glass disk after 1 h of interaction with biological fluids (112 × 112 µm2).

pore diameters and for the total pore volume. The surface area and the total pore volume increase with the phosphorus content. Very wide pore size distributions were observed for all of the glasses: the pores ranged from 3 to 80 nm in diameter. However, both the modal and average pore diameters increase with the phosphorus content. These results are well interpreted by the physicochemical properties of the glass matrix elements. Calcium, as a network modifier, causes discontinuity in the glassy network, resulting in the formation of nonbridging oxygen.27 In contrast, P2O5 is a network former; but the incorporation of the phosphorus oxide into the glass matrix results in a more disordered structure compared to a glass, which would be solely composed of SiO2 and CaO. Hence, a higher distribution of “defects” and of nonbridging oxygens is obtained for ternary SiO2-CaO-P2O5 compared to SiO2-CaO (B75) binary glasses.28 Then mesopores with larger diameters are formed, causing the porosity to be more important for ternary glasses. Consequently, the surface area and the total pore volume also increase. In Vitro Activity of the Glass Discs. Chemical Mapping of the Glass/Biological Fluids Interface. Phosphorus-free SiO2-CaO B75 Binary Glass. Elemental maps for each immersion time in DMEM were recorded. In this paper, we only present a restricted selection of these maps; other ones have been provided and commented in our previous works.29,30 Figure 1 represents the elemental distribution at the periphery of a P-free B75 glass disk after 1 h of interaction with biological fluids. The observed distributions correspond to the intensity of X-rays locally emitted by the sample under proton irradiation. Figure 1 shows that the bioactivity process has already begun for the B75 glass disk after 1 h of interaction with biological fluids. One observes that phosphorus, coming from the biological medium, is already incorporated at the surface of the glass, but in a scattered way. Calcium is still distributed homogeneously in the inner regions of the glass, but this element has been

partially released from the periphery of the material, in the regions where phosphorus is detected. This corresponds to the first stage of the bioactivity process: the dealkalinization of the glass surface. Concerning the silicate network, it is still uniform at that time. Two regions can be distinguished in Figure 1: (i) the inner part of the glass, whose composition is close to that of the primary glass, resulting in uniform distributions for Si and Ca and (ii) the periphery of the material, composed of a Si-rich layer from which Ca ions were released, while P coming from the biological environment was incorporated. In Figure 1, we also observe that traces of Mg are taken from biological fluids and incorporated into the glass matrix; however, their localization is made difficult because of low count rates for this element along with a high continuum background at the characteristic Mg peak energy. After 6 h of interaction (data shown in ref 29), the two previously observed regions have evolved. Dealkalinization has advanced to the inner part of the glass matrix and therefore almost purely silica regions are observed that extend to depths up to 30 µm. However, the silicate network has retained its consistency and Si is still distributed uniformly. The peripheral layer has grown: a Sirich layer is present at the glass surface, in which Ca, P, and traces of Mg are incorporated. After 1 d of immersion in biological fluids, the silicate network appears to be broken down at the material periphery. From now on, the peripheral layer is composed of mainly Ca and P, with traces of Mg. The inner regions of the matrix still consist of the glass in its original composition. The ionic exchanges and the physicochemical reactions involved in the bioactivity process then continue to feed the growth of the Ca-P-Mg layer at the glass surface. Finally, three regions can be distinguished after 5 days of interaction (Figure 2). The primary glassy network, where high Si concentrations are detected, corresponds to the inner regions of the glass. On the periphery, a Ca-P-Mg rich layer has developed on a depth of about 10 µm. Between these two areas,

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Figure 2. Elemental maps at the periphery of a SiO2-CaO B75 glass disk after 5 days of interaction with biological fluids (256 × 256 µm2).

a narrow zone exists that is enriched in Ca; this layer results from the diffusion of the mobile Ca from the inner of the glass to its surface. P-Containing SiO2-CaO-P2O5 Ternary Glasses. Concerning ternary glasses B72.5 (containing 2.5 wt % P2O5), B70 (5 wt % P2O5), and B67.5 (7.5 wt % P2O5), the evolution of elemental distributions reveals that these materials are able to induce the formation of a Ca-P-Mg peripheral layer through a mechanism similar to that of B75 binary glass. Nonetheless, there is evidence that the Ca release is delayed with increasing P content in the glass. Indeed, after 1 h of interaction with biological fluids the Ca release was less pronounced for B72.5 and B70 glasses when compared to the B75 glass. For B67.5, the dealkalinization even seems not to have started already. However, the Ca-P layer grows rapidly and after 1 day a Ca-P rich layer is visible at the surface of the different glasses. Figure 3 illustrates the elemental distributions for the B70 glass after 5 days of immersion: a Ca-P-Mg peripheral layer has been formed, which is a few micrometers thick. The thin Ca-enriched layer, which was observed between the inner of the glass and the periphery for B75, does not exist for any of the ternary glasses. EWolution of Elemental Concentrations at the Periphery of the Glasses. Depending on the elements distributions, chemical maps were divided into various regions of interest using the SUPAVISIO analysis software. Whenever the Ca-P-rich peripheral layers were detected, thin masks of measurement were created (i.e., focusing on the X-ray spectra of some user-defined regions of interest), allowing the calculation of elemental concentrations in these areas. Depending on the region of interest, masks from 5 × 5 up to 20 × 20 µm2 were defined, as the Ca-P layer thickness increases with time of immersion in biological fluids. With this methodology, the evolution of elemental concentrations at the glass periphery can be observed and is presented in Figure 4. The results correspond to the

average of concentrations calculated in several regions of interest. These regions of interest were defined over various samples in order to be ensured of measurement reproducibility. Each point represents an average of four measurements. Errors on elemental concentrations were determined by calculating the root-mean-square of errors related to each measure. They mainly depend on four parameters: the statistical error associated with the determination of the elemental peak area, the fit error, the error due to the overlapping peak areas and the errors related to instrumental factors (mainly due to the determination of the electric charge deposited on the samples and due to the irradiation damages). During the first 6 h of interaction with biological fluids, the largest decrease in Ca concentration is observed at the periphery of the B75 binary glass. At that time, the B75 periphery is composed of 12.7% Ca (wt %). Ca is present in similar concentrations at the periphery of B72.5 and B70 ternary glasses: 14.3% and 13.4%, respectively. For the B67.5 ternary glass, which initially owns a higher P content, no decrease is observed in Ca concentration: it is kept constant at the material’s periphery until 1 h of interaction, then it increases up to 33.4% after 6 h of interaction. However, there is no doubt the dealkalinization of the glass surface has occurred (cf. Figure 6, Ca has been leached into biological fluids). In fact, the relative increase in Ca concentration is due to a sharp decrease in Si concentration during that time. This indicates that the expansion of a calcium phosphate layer, still very rich in Ca at that time, has already started. At the same time, Si concentration remains virtually the same for B75, B72.5, and B70 glasses. Regarding P concentration, Figure 4 shows that it increases at the periphery of the glasses to represent up to 3-4% after a few hours of interaction. Alternatively, it is visible that incorporation of Mg coming from the biological medium began.

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Figure 3. Elemental maps at the periphery of a SiO2-CaO-P2O5 B70 glass disk after 5 days of interaction with biological fluids (51 × 51 µm2).

Figure 4. Evolution of elemental concentrations at the bioactive glasses periphery with time of exposure to biological fluids, ([) B75, (X) B72.5, (•) B70, (]) B67.5.

Beyond 6 h of interaction, Ca concentration increases dramatically at the periphery of B75, B72.5, and B70 glasses. For all of the materials, a maximum value is reached after a few days. Then Ca appears to be the main constituent of the peripheral layer. Concerning P concentration, it is multiplied by 2 or even 3 between 6 h and 1 day of interaction. Then, a maximum is reached after 5 days of interaction. During that

time, the silicate network depolymerised at the periphery of the material. Indeed this can be seen in Figure 4: Si concentration decreases significantly and after 10 days of interaction, Si is only present on the order of a few percent at the glasses surface. Regarding Mg, it is incorporated into the growing Ca-P peripheral layer in a significant way (concentration greater than 0.5%).

PIXE-RBS Study

Figure 5. Evolution of elemental concentrations in the inner regions of the bioactive glasses with time of exposure to biological fluids, ([) B75, (X) B72.5, (•) B70, (]) B67.5.

Thus, after 10 days of interaction, the peripheries of the different glasses have undergone substantial changes that have importantly affected their composition. The bioactive glasses surfaces now consist of a Ca-P rich layer, containing 30 to 45 wt % Ca and 10 to 14 wt % P, in which traces of Mg are incorporated on the order of 0.5 to 1 wt %. Traces of Si from the initial glassy network still remain. EWolution of Elemental Concentrations in the Inner Regions of the Glasses. Elemental concentrations were also calculated in the inner part of the glass discs, in the regions that were not directly exposed to biological fluids. Results are presented in Figure 5. During the first 2 days of interaction with the biological environment, fluctuations are observed in Si and Ca concentrations. Si and Ca concentrations evolve in an opposite way; this is because Si and Ca oxides represent nearly 100% of the glass matrix. Hence, a variation in the concentration of one of these oxides necessarily results in an opposite change for the other one. P concentration demonstrates a slight upward trend. Traces of Mg are detected in proportions less than 0.4%. Changes in the elemental concentrations are due to the migration and diffusion of ions toward the periphery of the material; there the bioactivity process drains ions of the matrix in order to feed the growth of the calcium phosphate layer. After 10 days of interaction, for the B75 glass the elemental concentrations are close to that of the primary glass before interaction. For ternary glasses, there has been a significant decrease in Si concentration, along with an appreciable increase in P concentration: this suggests that the Ca-P-Mg layer gradually extends to greater depths, up to reach the inner regions of the glass. Composition of Biological Fluids. Changes in the biological fluids composition with time of interaction with bioactive glasses are shown in Figure 6. Ca concentration increases during the first hours of interaction. This is due to the dealkalinization of the glass matrix; first step of the bioactivity process: Ca is released from the glass surface, as observed during the microPIXE-RBS analysis of glasses. The release of Ca is faster and more abundant for the P-free B75 glass. After this rapid release,

J. Phys. Chem. C, Vol. 112, No. 25, 2008 9423 there is a gradual decrease in the Ca concentration. Ca is taken from the biological fluids and incorporated at the glass’s periphery, in order to supply the growth of the calcium phosphate layer. Nevertheless, after 10 days of interaction B75 binary glass has released great quantities of Ca so that we note an overall increase in the Ca concentration of biological fluids. This could indicate the final dissolution of the previously formed Ca-P layer. In contrast, for ternary glasses, the amount of Ca taken from biological fluids is important and Ca concentration is steadily declining with time. Regarding P concentration, it decreases rapidly with time of interaction. Binary and ternary glasses incorporate a significant P amount from the biological medium. After 10 days of interaction, the P concentration in biological fluids is divided by 2 or even 3. The higher the initial P content in the glass, the higher the amount of P taken from the solution. Concerning Si concentration, binary and ternary glasses exhibit a common trend. Initially, biological fluids are silicon free. During the first hours of interaction, low quantities of Si are detected in the biological medium. Then, as the silicate network is depolymerized as a result of the Ca-P layer extension to greater depths, larger concentrations of Si are released in biological fluids. An extremum is reached after 10 days of interaction. At that time Si concentration is equal to 56 to 59 ppm. From Figure 6, the slow decrease in Mg concentration with increasing time of interaction demonstrates that low quantities of Mg are taken from the biological medium. After 10 days of immersion, 1 to 2 ppm Mg are incorporated into the glasses matrices. Greater quantities of Mg are taken for ternary glasses. For the B75 binary glass, a final increase in Mg concentration is observed, which would confirm that for this material parts of the Ca-P-Mg layer are dissolved after 10 days of interaction. Calculation of the Ca/P and (Ca+Mg)/P Atomic Ratios at the Glass/Biological Fluids Interface. To better understand the glass’s behaviors, we calculated the Ca/P and (Ca+Mg)/P atomic ratios at the surface of the glass discs by creating thin masks of measurement about 1 µm thick at the glass/biological fluids interface. Results are presented in Figure 7. They shall be compared to the 1.67 Ca/P value of Ca10(PO4)6(OH)2 stoichiometric hydroxyapatite (HA) and to the 1.09 (Ca + Mg)/P value of adult-human calcified bone.31–33 For all glasses, the Ca/P atomic ratio decreases with increasing immersion time. A limit value is reached after 5 days. The Ca/P decrease is faster for P-containing ternary glasses. Moreover, the final Ca/P value is lower for SiO2-CaO-P2O5 glasses. Indeed, after 10 days of interaction, the Ca/P ratio is between 1.8 up to 1.9 for ternary glasses; for the B75 binary glass, the Ca/P ratio is equal to 2.1. Another indication concerning the influence of the initial P content is deduced from the evolution of the (Ca + Mg)/P atomic ratio. This ratio is lower for glasses with higher P content. After 10 days of interaction, greater quantities of Mg are present at the surface of P-containing glasses: the (Ca+Mg)/P final values are close to that of adult-human calcified bone and range from 1.1 to 1.2 for B72.5, B70, and B67.5 glasses, up to 1.3 for the P-free B75 glass. Discussion We have obtained information both on the surface reactivity of glasses, by using PIXE-RBS microprobes and on their global reactivity by determining the biological fluids composition. Now it is proposed to compare the materials reactivity at both these local and global scales. First, from a thermodynamic concern, we will establish for each glass the potential of forming a hydroxyapatite layer. This is made possible through supersatu-

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Figure 6. Evolution of elemental concentrations in biological fluids with time of interaction, ([) B75, (X) B72.5, (•) B70, (]) B67.5.

Figure 8. evolution of the supersaturation degree for hydroxyapatite (HA) formation in biological fluids with time of interaction ([) B75, (X) B72.5, (•) B70, (]) B67.5. Figure 7. Evolution of Ca/P and (Ca+Mg)/P atomic ratios at the glass/ biological fluids interface, ([) B75, (X) B72.5, (•) B70, (]) B67.5. Data are compared to the characteristic ratios of stoichiometric hydroxyapatite (HA) and of inorganic phases of adult-human calcified bone.31–33

ration studies of biological fluids. Second, studying the Ca/P atomic ratios decrease will permit an evaluation for the kinetics of evolution of the calcium phosphate layer at the glass/ biological fluids interface. Changes in the Degree of Supersaturation in DMEM. The bioactivity process induces the formation of apatitic precipitates. Physicochemical reactions results in intense ionic exchanges that cause major changes in the supersaturation degree for the mineral formation in biological fluids. Hence, calculating the supersaturation degree provides valuable information about the ability of a glass to form an apatitic layer.34 We have calculated the supersaturation degree by considering the formation of stoichiometric HA. Then a criterion for comparing the in vitro bioactivity of glasses will consist of observing the evolution trends for the supersaturation degree.

The formation of HA is given by: 10Ca2++ 6PO34 + 2OH T Ca10(PO4)6(OH)2. The ionic activity product of HA is defined 6 · [PO3-]6 · γ -2 · [OH-]2, where as: Q ) γCa2+10 · [Ca2+]10 · γPO34 OH 4 γi is the activity coefficient. The activity coefficients have been determined in previous works:35 for a solution at physiological ionic strength, γi is equal to 0.36 for Ca2+, 0.06 for PO43-, and 0.72 for OH-. Therefore, the ionic activity product Q can be calculated because Ca and P concentrations were measured, while the amount of hydroxyl ions was deduced from pH measurements. Then we can define SD, supersaturation degree for HA formation in the solution: SD ) Q/Ksp, where Ksp is the solubility product of HA in aqueous solution. Ksp is reported to be 10-117.2 at 37 °C.36,37 Knowing the SD helps to predict the evolution of the system insofar as it tends to thermodynamic equilibrium. The solution and the HA solid phase reach equilibrium when SD ) 1. For SD < 1, dissolution of the HA mineral is favored. For SD > 1, the solution is supersaturated with respect to the HA mineral and precipitation of HA is favored. The evolution of SD in biological fluids is presented in Figure 8. As is well-known, body fluids are initially supersaturated with

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Figure 9. Modelling (dashed line) of the Ca/P decrease for the B75 glass.

respect to HA under normal conditions,38 and Figure 8 shows that the situation is the same in DMEM. Thus, HA precipitation is encouraged and will preferentially occur on the bioactive glasses surfaces, which provide favorable sites for HA nucleation, especially because low interfacial energies are granted by the development of a silica gel on the surface of the glasses (3rd stage of the bioactivity process).35 During the first periods of interaction, there is an increase in SD due to the release of Ca ions from the glass matrix. After the dealkalinization of the glass surface, SD decreases with increasing time of interaction: it indicates that Ca2+, PO43-, and OH- are incorporated at the glass surface, supplying the HA mineral growth. Different behaviors are observed, depending on the glass type. SD decreases more rapidly for P-containing glasses. The higher the P content in the glass, the larger the decrease slope. P-containing glasses are therefore more likely to reach quickly thermodynamic equilibrium, which means that HA formation is accelerated when compared to B75 binary glass. Nevertheless, other parameters are to be considered in assessing the in vitro bioactivity of the materials; hence, we will have to compare these results with those obtained with the micro-PIXE-RBS analyses. Kinetics of Evolution of the Ca-P Layer at the Glass/ Biological Fluids Interface. The crystallization of insoluble HA is believed to involve the formation of meta-stable phosphocalcic phases. In fact, amorphous calcium phosphates are often encountered as a transient phase during the formation of HA: usually, they are the first phase that is precipitated from a supersaturated solution containing calcium and phosphate ions. However, at physiological pH, HA is thermodynamically the most stable and the least soluble of all calcium phosphates. Hence, the formation of HA experiences a number of kinetics processes, which take place at different rates and involve more than one phosphocalcic phase during the bioactivity mechanism. In this sense, studying the Ca/P atomic ratios at the glass’s surface gives essential indication on the evolution of the phosphocalcic layer.

For all of the materials, we can assume that the Ca/P atomic ratios calculated at the glass surface decrease exponentially with time of immersion in biological fluids. A limit value, close to the 1.67 value of HA, is reached after a few days. In order to establish a comparison between the in vitro bioactivity of the different glasses, the kinetics of evolution of the Ca/P ratio were modeled with an exponential law: RCa/P ) A exp(-t/τ) + Rlim, where RCa/P is the Ca/P value at the glass/biological fluids interface after a period of interaction t, τ is a constant parameter, Rlim is the Ca/P asymptotic value, and A is the Ca/P amplitude. Figure 9 presents one example of Ca/P measurements fitted with the exponential function using least-squares regression. For each sample, Table 3 shows the initial value and the limit of the fitting function at infinity. Modeling the Ca/P decrease allows us to determine the τ parameter: τ represents the speed at which the calcium phosphate layer is transformed into a phosphocalcic phase. Table 3 exhibits the τ values with respect to the P2O5 content in the initial glass matrix. For SiO2-CaO-P2O5 glasses, τ is half that of the SiO2-CaO B75 glass. At the surface of P-containing glasses, the calcium phosphate layer evolves more quickly into a phosphocalcic phase. Local and Global Reactivity of the Bioactive Glasses. The bioactivity process consists of a well-identified group of physicochemical reactions occurring at the surface of the material. Briefly, the alkaline and alkaline-earth ions present in the glass matrix are first exchanged with H+ of the solution; then, polycondensation reactions of surface silanols create a high-surface-area silica gel. This porous hydrated silica layer provides a large number of sites for the formation and growth of calcium phosphates that will progressively crystallize into a biologically reactive hydroxycarbonate apatite equivalent to the mineral phase of bone.39,40 These physicochemical reactions are common to all of the glasses we studied but the materials’ composition and texture significantly affect the kinetics and amplitude of the phenomena. Dealkalinization of the glass matrix and the first appearance of the CaP layer are undeniably delayed for SiO2-CaO-P2O5 glasses when compared to SiO2-CaO B75 glass. These conclusions are based on several findings. First, higher Ca concentrations are detected at the periphery of ternary glasses during the early stages of interaction; therefore, lower quantities of Ca are released from the surface of SiO2CaO-P2O5 glasses. Analyses of the biological fluids composition confirm this: lower Ca concentrations are detected for ternary glasses during the first hours of interaction. Nevertheless, once the Ca-P layer formation has begun, the layer extends on greater depths and is more quickly changed into an apatitic phase for P-containing glasses. This is stated by (i) larger amounts of P and Ca taken from the biological fluids for SiO2-CaO-P2O5 glasses, (ii) faster changes in the supersaturation degree for HA formation in the solution, and (iii) faster kinetics of evolution of the CaP layer. Moreover, the Ca/P ratio finally reached for P-containing glasses is closer to that of stoichiometric hydroxyapatite. These observations can be attributed to the lower Si content in ternary glasses compared to the B75 binary glass. The higher

TABLE 3: Ca/P Initial Values, Final Experimental Values, Limits of the Fitting Function at Infinity, and τ Parameter Values for All of the Materials modeling: RCa/P ) A exp (-t/τ) + Rlim B75 B72,5 B70 B67,5

Ca/P experimental

initial value (t ) 1 h)

limit (t ) ∞)

initial value (t ) 1 h)

final value (t ) 10 days)

τ parameter (hours)

2,95 ( 0,14 2,84 ( 0,37 2,82 ( 0,50 2,70 ( 0,35

1,99 ( 0,07 1,85 ( 0,16 1,70 ( 0,21 1,96 ( 0,15

2,89 ( 0,45 2,62 ( 0,31 2,55 ( 0,18 2,65 ( 1,04

2,05 ( 0,27 1,85 ( 0,13 1,75 ( 0,17 1,89 ( 0,10

59 33 31 37

9426 J. Phys. Chem. C, Vol. 112, No. 25, 2008 the Si content in the glass, the thicker the hydrated, porous silicagel layer formed after surface silanols polycondensation. This generates a large active surface area, which speeds up both the dissolution process and the ionic exchanges for B75.41 However, once the Ca-P layer is formed, its growth and evolution are faster for ternary glasses because the presence of phosphorus in the primary glass matrix facilitates the transformation of the initially amorphous calcium phosphates into apatite crystals. In fact Vallet-Regı´ et al. recently showed that addition of P2O5 in a SiO2-CaO glass leads to the formation of a silicon-doped calcium phosphate matrix: nanocrystallized phosphates smaller than 10 nm, containing the Ca2+ ions, are embedded into the silicate network.42 The nanocrystals’ own lattice parameters are close to β-TCP and they lie on the glassy matrix for further growing. Hence, for SiO2-CaO-P2O5 glasses the apatitic layer may initially be limited to some scattered sites but it then quickly extends to great depths because the crystallized calcium phosphates can act as nucleation agents, increasing the kinetics of new layer formation.43 An additional explanation for the possible role of the primary phosphate nanocrystals as nucleation centers is their solubility under in vitro conditions, which may locally increase the ionic activity product and thus the supersaturation of the solution. Finally, the (Ca + Mg)/P atomic ratio at the glass/biological fluids interface is lower for P-containing glasses. Low Ca/P atomic ratios are related to low (Ca + Mg)/P values. Because the calcium phosphate layer is wider for ternary glasses, more Mg substitutions for Ca can occur, resulting in lower (Ca + Mg)/P ratios for SiO2-CaO-P2O5 glasses. The presence of appreciable amounts of Mg at the glass/biological fluids interface is important because of its well-recognized bactericidal and antiinflammatory properties.44–47 Conclusions An unconventional methodology has been successfully developed that has highlighted the influence of the phosphorus oxide content on the physicochemical properties of bioactive glasses, at both a local and a global scale. Accurate quantitative analyses of the glass/biological fluids interface have been performed, that are of paramount importance to develop a deep understanding of the bioactivity mechanism; the final view is to control the surface reactions through the optimization of determinant parameters such as the composition and the texture of the glass. Chemical mapping of the glass/biological fluids interface at a micrometer scale reveals the formation of an interfacial Ca-P-Mg layer after a few hours of interaction. Kinetics of evolution of the phosphocalcic layer have been estimated by modeling the Ca/P atomic ratios decrease at the glasses surface, along with supersaturation studies in biological fluids. As expected, the materials composition and texture significantly affect the kinetics and amplitude of the bioactivity mechanism. Dealkalinization of the glass matrix and the first appearance of the calcium phosphate layer are delayed for SiO2-CaO-P2O5 glasses when compared to SiO2-CaO glass. Nevertheless, once the Ca-P-Mg layer formation has begun, the layer extends to greater depths and is more quickly changed into a bone-like apatitic phase for P-containing glasses. The presence of phosphorus in the primary glass matrix facilitates the transformation of the initially amorphous calcium phosphates into bonelike apatite crystals, which contain greater quantities of magnesium.

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