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3D Organized Macroporous Bioactive Glasses: a Study of Pore Size Effect on Physicochemical Reactivity by Micro-PIXE-RBS Jeremy Soulié,*,†,‡,§ Adeline Hardy-Dessources,†,‡ Jean-Marie Nedelec,†,‡ and Edouard Jallot§ †

Institut de Chimie de Clermont-Ferrand, ENSCCF, Clermont Université, BP 10448, 63000 Clermont-Ferrand, France CNRS, UMR 6296, ICCF, 63177 Aubière, France § Clermont Université, Université Blaise Pascal, CNRS/IN2P3, Laboratoire de Physique Corpusculaire, BP 10448, 63000 Clermont-Ferrand, France ‡

S Supporting Information *

ABSTRACT: Macroporous biomaterials have attracted much attention during the past decade because of the large range of associated applications (from drug delivery to tissue engineering). The present study focuses on the correlations between macropore size (from 400 to 1500 nm in diameter) and the early steps of biomineralization process and the reactivity in binary (SiO2-CaO) and ternary (SiO2-CaO-P2O5) bioactive glasses. Macrostructured glasses were elaborated by combining sol−gel chemistry and an inverse opal method with polystyrene beads colloidal crystals as the template. Macroporosity of these materials has been characterized thanks to thermoporosimetry. The in vitro biomineralization process was studied using particle-induced X-ray emission (PIXE) associated with Rutherford backscattering spectrometry (RBS), which are efficient methods for a highly sensitive multielemental analysis. Thanks to elemental maps of silicon, calcium, and phosphorus obtained at a micrometer scale for various interaction times, we demonstrate that the physicochemical reactions are sensitive to macropore size, even though their kinetic is not modified. This key result is an important step to build tunable biomaterials with a highly reproducible and finely controlled response for an optimized integration in living organisms.

1. INTRODUCTION The use of porous bioceramics as bone substitutes has been widely described during the past decade. A large number of applications can be targeted, depending on the pore size, from nanometer (drug delivery systems1−3) to a few millimeters (bone tissue engineering4−6). In the present study, we focus on bioceramics with organized micrometer-scale pores. According to Ji et al.,7 in addition to their strong interface with tissues and their osteoinductive behavior, bioceramics with macropores around 1 μm in diameter induce a highly specific surface area; favor nucleation sites for phosphocalcic phases; and enhance ion exchange, protein adsorption, and cell adhesion. Moreover, beyond the direct biological interest, a calibrated macroporosity can be used to understand the dependence of the bioactive process (including mineralization reactions) upon the pore size. Among bioceramics, bioactive glasses are attractive materials because of their ability to bond to living bone through the formation of an interfacial apatite-like layer at the glass surface, the composition of which is close to the mineral phase of bone.8 This newly formed biomimetic layer improves the osteointegration properties of the implant while the degradation products from the glass can promote the bone tissue regeneration. Only a few publications,9,10 have investigated the influence of organized and micrometer-scale porosity on physicochemical reactions (dissolution, diffusion, ionic exchange and precipitation) occurring when bioactive glasses © 2013 American Chemical Society

interact with biological media. These studies demonstrated an effect of the macroporosity on the different steps from a global point of view. The present study aspires to add spatial information about the physicochemical reactions taking place, at a micrometer scale. With this goal in mind, we synthesized macrostructured glasses following the inverse opals method proposed by Stein et al.11−13 for inorganic oxides, combined with sol−gel chemistry. Polystyrene (PS) beads of three different diameters were organized into colloidal crystals and used as a hard template to synthesize binary (SiO2−CaO) and ternary (SiO2−CaO− P2O5) glasses. Advanced characterization, including microparticle-induced X-ray emission and Rutherford backscattering spectroscopy (μ-PIXE−RBS), will highlight the dependence of the mineralization upon the porous features (macropores’ diameter, macroporous volume, specific surface area, interconnection diameter) of bioactive glasses through the recording of quantitative chemical maps of the biomaterials during interaction with biological fluids. Received: January 24, 2013 Revised: March 4, 2013 Published: March 7, 2013 6702

dx.doi.org/10.1021/jp400828v | J. Phys. Chem. C 2013, 117, 6702−6711

The Journal of Physical Chemistry C

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Figure 1. Schematic outline for the synthesis of macroporous bioactive glasses.

Table 1. Properties of Polystyrene Beads and Corresponding Organized Macroporous Glasses corresponding glasses beads names

nominal diameter (nm)

measured diameter (nm)

polydispersity index

binary glasses (SiO2−CaO)

ternary glasses (SiO2−CaO−P2O5)

S400 S800 S1500

430 820 1500

451 911 1561

0.027 0.037 0.081

B75−400 B75−800 B75−1500

B67.5−400 B67.5−800 B67.5−1500

2. MATERIALS AND METHODS 2.1. Synthesis. Macrostructured bioactive binary (SiO2− CaO) and ternary (SiO2−CaO−P2O5) glasses were prepared using a combination of sol−gel process and colloidal crystal templating (Figure 1). Monodisperse sulfonated polystyrene beads (Duke Scientific) with three different diameters (430, 820, and 1500 nm, respectively named S400, S800, and S1500 (Table 1)) have been used as building blocks of the crystal. To arrange the polystyrene spheres in closed-packed arrays, suspensions with weight fraction of 2% were centrifuged at 1200 rpm for 14 h. After removal of the water, the resulting solid was air-dried. Close-packed arrays of polystyrene spheres (1 g) were deposited into an Erlenmeyer flask and permeated with sols to get final macroporous glasses with two different compositions, binary (75SiO2−25CaO) and ternary (67.5SiO2−25CaO− 7.5P2O5) glasses (weight percent). To prepare corresponding sols, 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. After 2 h of infiltration, the excess of sol was removed by a slight filtration in a Buchner funnel, and the resultant solid was allowed to age at 60 °C in a capped Teflon vessel for 24 h to achieve the inorganic network formation. The polystyrene/glass composite was then treated at 125 °C for 24 h to evaporate the solvents. Finally, polystyrene template was removed from the composite by calcination using a tubular furnace under air flow from room temperature to 700 °C, with a 10 °C/min rate and a final dwell at this temperature for 12 h. Beyond polystyrene elimination, this temperature is required to remove nitrates and to achieve further densification of the glass. After the calcination and a soft grinding in a mortar, samples were obtained in powder form (grain diameters between 40 and 200 μm) and labeled following their composition and the nominal diameter of beads used for their synthesis (see Table 1). 2.2. Materials Characterization. The PS bead diameters were measured with a Malvern Zetasizer from dilute samples of PS sphere suspensions. Scanning electron microscopy (SEM) images of the samples were obtained with a JEOL 5190 microscope operating at 1 kV. Macropore size distribution and macroporous volume were measured using thermoporosimetry (TPM)14 with orthoxylene15,16 as a solvent. TPM analyses were performed by differential scanning calorimetry (DSC) measure-

ment with a Mettler-Toledo DSC 823e apparatus, using STARe software. The DSC apparatus was calibrated (both for temperature and enthalpy) with metallic standards (In, Pb) and n-heptane. About 30−40 mg of the soaked sample was introduced into an aluminum DSC pan of 160 μL with an excess of o-xylene. The measurement procedure under air atmosphere included the following steps: (i) cooling from −10 to −90 °C at a rate of 10 °C/min; (ii) heating from −90 to −28 °C at a rate of 0.7 °C/min; and (iii) a last cooling from −28 °C to −90 °C at a rate of 0.7 °C/min. A slow rate of 0.7 °C/min was chosen to allow the continuous thermal equilibrium inside the DSC cell. Recently obtained calibration covering the macropores domain was used.17,18 Nitrogen gas sorption analyses were performed to characterize the glasses’ textural properties. The samples were vacuumoutgassed at 120 °C for 12 h to remove physically adsorbed molecules from the pores. The adsorption/desorption isotherms were recorded on a Quantachrom Autosorb-1 MP 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 using 7 points in the 0.05−0.35 p/p0 range. Total mesopore volume was measured at a relative pressure P/P0 = 0.995. 2.3. In Vitro Studies and Samples Preparation. For studying the glass reactivity under biological conditions, glass powders were immersed at 37 °C for 30 min and 1, 6, 12 h in 40 mL of a standard Dulbecco’s Modified Eagle Medium (DMEM, Biochrom AG, Germany) for which concentrations of inorganic salts is close to those of human plasma.19 Mandel et al.20 demonstrated that DMEM can be regarded as a feasible alternative to using simulated body fluid (SBF) solutions for in vitro bioactivity testing of synthetic biomaterials. Unlike SBF, DMEM contains amino acids, vitamins, and glucose. Because of the amino acids, lower rates for the dissolution of materials and a subsequent delay in surface layer formation are observed in DMEM when compared with soaking in SBF.21 Indeed, amino acids from DMEM are charged species that can be attracted by the negative glass surface and coat it with a film. In terms of simulating the in vivo environment, DMEM can be a better choice because it also contains other components present in in vivo systems, in addition to inorganic salts.22 For the same reasons, DMEM is commonly used in cell culture,23−25 in contrast to SBF (because of its deficiency of nutrients26). We 6703



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Figure 2. SEM pictures of colloidal crystals for S400 (A, D), S800 (B, E), and S1500 (C, F) beads.

Figure 3. SEM pictures of macroporous bioactive glasses after calcination: B75−400 (A, D), B75−800 (B, E), B75−1500 (C, F).

then decided to use DMEM in the present study in view of confronting with further biological results. For each sample, the powder weight-to-DMEM volume ratio was fixed at 3.3 g·L−1. This should allow only consideration of the influence of the porosity over the mineralization properties to be studied. After interaction, part of the DMEM was sampled to analyze its chemical composition by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) while the glass particles were removed from the solution and air-dried. Prior to characterization with the μ-PIXE−RBS nuclear microprobe, glass particles were embedded in resin (AGAR, Essex, England), and 400 nm thin sections of these samples were prepared by means of a Leica EM UC6 Ultramicrotome and inserted into 50 mesh copper folding grids, which were placed on a Mylar film with a hole of 3 mm in the center. Measurements were performed on the area of the section placed over the hole. 2.4. Micro-PIXE−RBS Analysis. PIXE and RBS methods are used simultaneously. The PIXE method permits the identification and the quantification of elements in sections of biomaterials grains after interaction with biological medium.27 RBS is used to determine the accurate thickness of the glass sections as well as the electric charge received by the samples during irradiation, which are to be determined for reliable PIXE spectra quantification. Analyses of our materials were carried out using nuclear microprobes at the CENBG (Centre d’Études

Nucléaires de Bordeaux-Gradignan, France). The experimental characteristics of the CENBG microbeamline have been published previously.28,29 For PIXE−RBS analyses, we chose a proton scanning microbeam of 1.5 MeV energy and 50 pA in intensity. The beam size was nearly 500 nm. Such parameters resulted in higher ionization cross sections for light elements (Z < 20) and, thus, 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 below 1 h. An 80 mm2 Si(Li) detector, orientated at 135° with respect to the incident beam axis and equipped with a 12 μm thick beryllium window, was used for X-ray detection. PIXE spectra were treated with the software package GUPIXWin. For RBS measurements, 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.

3. RESULTS 3.1. Macrostructured Glasses Synthesis and Textural Properties. S400, S800, and S1500 diameters measured by Zetasizer are slightly higher than nominal sizes. The spheres are very monodisperse, and their polydispersity indexes are below 6704



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Table 2. Textural Properties of Macroporous Bioactive Glasses thermoporosimetry oores diameter (nm)

shrinkage %

SEM macroporous volume (cm3/g)

N2 adsorption

interconnection diameter (nm)

specific area (m2/g)

total porous volume (cm3/g)

initial beads crystal

B75

B67.5

B75

B67.5

B75

B67.5

B75

B67.5

B75

B67.5

B75

B67.5

S400 S800 S1500

287 416 1079

387 577 943

36 54 30

14 36 39

1.4 2.37 3.87

4.47 7.01 8.16

55 177 242

90 170 239

108 81 76

357 230 141

0.338 0.286 0.198

0.701 0.375 0.235

Figure 4. Nitrogen adsorption−desorption isotherm plots of macrostructured glasses.

good agreement with published data (between 26% and 34%) in the case of macroporous silica.34,35 As presented in Table 2, the macropore volume increases with the diameter of the macropores for both B75 and B67.5 glasses. The average diameter of interconnection windows was measured from the SEM images for each glass. It corresponds to the contact point between PS beads, resulting in interconnection windows between macropores after calcination. It is clear that the diameter of interconnection increases when the bead size increases. The mesoporosity of all glasses as studied, as shown by to N2 adsorption/desorption measurements (Figure 4), is negligible. The shape of these isotherms is similar for all glasses, regardless of the size of the macropores. The absence of a plateau in equilibrium P/P0 = 1 confirms the presence of macroporosity. This means that only macropores will affect the biological behavior of the materials, allowing the study of the influence of macropore size and volume on the bioactivity. Specific surface area (Table 2) decreases when the macropore size increases. 3.2. μ-PIXE−RBS Quantitative Imaging of Macroporous Glass Grains. Several μ-PIXE−RBS elemental maps for each time of interaction in DMEM were recorded. In this paper, we present only a restricted selection of these maps. The observed distributions correspond to the intensity of X-rays locally emitted by the sample under proton irradiation. The Xray intensities are proportional to the concentration of their respective emitters, so the elemental concentrations can be calculated in any area of the μ-PIXE−RBS maps with a few parts per million sensitivity. This is far better than classical SEM-EDS (Scanning Electronic Microscopy-Energy Dispersive X-Ray Spectroscopy) analyses and is a direct consequence of

0.1 (Table 1). The colloidal crystals, formed after centrifugation, are shown in Figure 2. According to images at lower magnification (Figure 2A, B, and C), the crystalline organization is present throughout the whole grain and is not confined to only a few zones. At higher magnification (Figure 2D, E, and F) and despite stacking faults (1D, 2D, nonorganized areas), only two structures coexist: hexagonal (large majority) and cubic phases. In the literature, cubic phases are more often described for crystals of spheres organized by sedimentation,30,31 and Woodcock has shown through simulations that a thermodynamic cubic lattice was energetically more favorable than a hexagonal lattice.32 On the contrary, centrifugation often induces hexagonal or polycrystalline phases because the force exerted by an acceleration of several g’s results in a more compact organization.33 In this section, we will describe structures of inorganic replicas. Because these structures are similar for binary and ternary glasses, only B75−400, B75−800, and B75−1500 are presented in Figure 3. For the three glasses, their pore diameters, shapes, and interconnections are uniform. The wall thickness increases with pore diameter, from 51 nm for the lowest thickness (B67.5− 400) to 69 nm for the highest (B67.5−1500) (Supporting Information). The macroporosity is mainly organized in a compact hexagonal lattice, which is in agreement with the stacks observed in the bead crystals. Pore diameters and macroporous volumes have been measured using thermoporosimetry. The results are presented in Table 2. The pore size logically increases with the bead size for both binary and ternary glasses. Upon densification at 700 °C, a contraction of the network is observed. The corresponding shrinkage is around 30−35%, with extremes observed for B75−800 (54%) and B67.5−400 (14%) glasses. This average shrinkage ratio is in 6705



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Figure 5. Elemental maps of B75−800 glass grains before interaction and after 12 h of interaction with biological fluids.

Figure 6. Elemental maps of B67.5−800 glass grains before interaction and after 12 h of interaction with biological fluids.

Figure 7. Evolution of elemental concentrations in B75 grains. 6706



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Figure 8. Evolution of elemental concentrations in B67.5 grains.

3.3.1. Binary Glasses. Evolutions of silicon, calcium, and phosphorus concentrations for macrostructured binary glasses are presented in Figure 7. Three general trends appear, regardless of the element: (i) evolutions are clearly constituted by several steps, (ii) the pore size does not affect the kinetics of the phenomenon, (iii) the pore size does influence, however, the intensity of phenomenon. During the first step, the silicon concentration increases up to 1 h for the three glasses while at the same time, the calcium concentration is greatly reduced. As described in previous studies, these opposite evolutions can be attributed to the dealkalization of the glass.36−38 In the second step, the percentage of silicon decreases to a minimum value after 6 h of interaction while calcium and phosphorus increase and reach a maximum value after the same duration. This second step corresponds to the precipitation of calcium phosphate phases within the grain concomitantly with a partial dissolution of the silica network. Finally, during the third and last stages, the amounts of calcium and phosphorus diminish, and the silicon becomes predominant in the grain. According to other studies,38 this trend could indicate dissolution of metastable calcium phosphates. It is important to note that during the period for which a phosphocalcic phase is present, the calcium concentrations are directly correlated with the pore size. They are, indeed, more important when the pore diameter increases. The opposite effect is observed for silicon. A calcium-rich phase seems predominant in grains with bigger pores, at the expense of initial silicate network. The evolution of phosphorus concentration is the opposite of the calcium. This will be discussed later in Section 5. 3.3.2. Ternary Glasses. Elemental concentration evolutions of B67.5−400, B67.5−800, and B67.5−1500 glasses are presented in Figure 8. As for binary glasses, the kinetics seem to be independent of the pore size, and this size is clearly influencing the amplitude of variation for the concentrations. The dealkalization process also occurs between 0 and 1 h. It is, however, less important for ternary glasses because of their greater stability.37 After 1 h of interaction, the formation of calcium phosphate in the grain is demonstrated by the concomitant increase in calcium and phosphorus concentrations along with the decrease of silicon (which is also due to a partial dissolution of the silicate network, as for binary glasses). A slowdown of this phenomenon seems to occur after 6 h, which is indicated by a shift of the silicon and calcium curves and a stagnation of the

the choice of protons, rather than electrons, as a probe. The typical spatial resolution is 500 nm. Figure 5 shows how the distributions of Si, Ca, and P within a binary glass (B75−800) evolve with time of interaction with biological fluids. The spatial distributions of silicon and calcium are homogeneous within the grain before interaction with the biological medium; they remain homogeneous during the process. Phosphorus from DMEM is incorporated into the grain right after 15 min. It is still present in the material after 12 h without spatial discrimination. The three ternary glasses also behave in similar ways. The μPIXE−RBS chemical maps suggest that the elemental distributions do not evolve during interaction with biological fluids. The mapping of the B67.5−800 glass (Figure 6) illustrates this for the B67.5 glass. The key result of this part is the uniform distribution of the elements over the grains. Indeed, contrary to what could be observed for conventional sol−gel glasses,36,37 no surrounding Ca−P-rich layer is formed at the periphery of the grain, but the mineralization rather affects the whole material. 3.3. Evolution of the Concentrations Inside the Grains. Elemental concentrations were calculated inside the glass grains, and the evolutions of concentrations with time are shown in Figures 7 and 8. Each point represents an average of 10 measurements. The errors in elemental concentrations were determined classically by calculating the estimated standard deviation of the mean (type A estimation of standard uncertainties, following the NIST TN 1297 guidelines). These errors are below 5% for Si, 10% for Ca, and 7% for P. Taking into account the uncertainties, general and reliable trends can still be observed. Moreover, before interaction with DMEM, compositions of the various samples calculated thanks to PIXE−RBS measurements agree fairly well with the nominal ones (Table 3) Table 3. Nominal versus Actual Compositions (weight %) of B75 and B67.5 Glasses

B75 nominal values B75−400 B75−800 B75−1500 B67.5 nominal values B67.5−400 B67.5−800 B67.5−1500

Si

Ca

P

35.05 32.32 31.38 31.97 31.55 28.32 28.34 28.12

17.86 16.86 16.26 17.01 17.86 17.31 17.45 17.02

0 0 0 0 3.27 3.04 2.99 2.98 6707



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Figure 9. Evolution of elemental concentrations in biological fluids for B75 glasses.

Figure 10. Evolution of elemental concentrations in biological fluids for B67.5 glasses.

Figure 11. (a) SEM pictures of the B67.5 glass during interaction with DMEM and (b) schematic hypothesis of phenomena occurring on the pores wall of the macroporous glasses.

The calcium concentration continuously increases up to 6 h, and then the curves reach a plateau (or even decrease for B75− 1500 glass) up to 12 h. Contrary to what might be expected, considering the evolution of silicon, the amount of calcium is more important for the B75−400 glass. This point will be discussed in Section 5. Curves of phosphorus are, however, consistent with those of silicon, since the ionic exchange between the material and the medium is also favored for glass whose pore diameter is the largest (B75−1500). 3.4.2. Ternary Glasses. Silicon and phosphorus changes in DMEM during the immersion of the ternary glasses (Figure 10) are very similar to that of the binary glasses: an increase in the silicon concentration and a decrease for phosphorus. These trends are amplified when the pore diameter increases.

phosphorus one. In the case of ternary glasses, elemental concentrations are also correlated with the pore diameter because calcium and phosphorus percentages are higher for the B67.5−1500 glass and lower for the B67.5−400 glass. 3.4. Elemental Evolution of the Biological Medium. 3.4.1. Binary Glasses. As for the concentrations measured within the grains, the elemental evolutions of the biological medium demonstrate an undeniable relationship between the diameter of the pores and the changes in the DMEM composition. Time evolutions of Si, Ca, and P for binary glasses are shown in Figure 9. Because of the dissolution of the glass network, the amount of silicon in the medium gradually increases over time. This phenomenon seems more important for the B75−1500 sample. 6708



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Table 4. Ca/Si and P/Si Weight Ratios in B75 and B67.5 Grains after 12 h of Immersion in DMEM Ca/Si P/Si

B75−400

B75−800

B75−1500

B67.5−400

B67.5−800

B67.5−1500

0.31 0.030

0.66 0.024

2.36 0.048

0.81 0.209

1.77 0.391

4.00 0.906

Figure 12. Evolution of Ca/P atomic ratio for B75 and B67.5 glasses.

whose pore diameters are larger. The (Ca−P phase volume)/ (silicate matrix volume) ratio (VCa−P/VSi) is then higher for 1500-glasses than for 800-glasses and 400-glasses, for which the lowest value is observed. Assuming that the density of each of these two phases (Ca−P phase and silica matrix) is constant for a given category of glass (binary or ternary), the mass ratios are also higher, which is consistent with our results (Table 4). The second question raised by our results is related to the increased release of silicon and calcium for the ternary 1500glass compared to the 800- and 400-glasses (Figure 10). These results may seem paradoxical: since dealkalization and dissolution of the network are favored by a large surface area, it should be higher for the B67.5−400 glass than for the B67.5− 1500 glass. A possible explanation would be to consider the interconnection diameter: it increases when the pores size increases (Table 2). For a given thickness of precipitates onto the pores’ surface, the interconnection windows for small-pore samples is more easily blocked, thus limiting diffusion and slowing down the dissolution. Dissolution of systems with larger macropores would then be enhanced, although the latter have a lower specific surface. The third and final point to clarify is the opposite trends of calcium release between binary and ternary glasses. In the previous paragraph, we underlined that release is favored for glasses with larger pores. This assumption is verified for ternary glasses, but not in the case of binary glasses. This could be linked to the nature of phases formed in the pores. Indeed, a massive release of calcium associated with a limited source of phosphorus preferentially leads to the formation of insoluble calcium-rich phases,36 which can be amorphous or crystallized as calcium oxide (CaO), Portlandite (Ca(OH)2) or calcium carbonate (CaCO3), at the expense of metastable calcium phosphates, in the case of conventional or mesoporous sol−gel glasses immersed in biological medium, formed as octacalcium phosphate, dicalcium phosphate, or hydroxyapatite.36,39 Considering that the phosphorus amount is the same for each glass (B75−400, B75−800, B75−1500) and that the macropore volume is higher for glasses with bigger macropores (Table 2),

Contrary to what happens for binary glasses, the amount of calcium released into the biological medium follows the same trend as silicon and phosphorus: it is higher for B67.5 glass. For the three glasses, the calcium concentration quickly rises up to 1 h, and then a plateau is reached between 1 and 12 h.

4. DISCUSSION There is a direct relationship between the diameter of macropores and the physicochemical processes involved in the in vitro bioactivity mechanism leading to mineralization. The correlation of the results to build a hypothesis is not trivial, however. Moreover, even if structural measurements could be useful to characterize the nature of Ca- and CaP-rich phases within the macroporous glasses, the use of X-ray diffraction is not adapted to the study of poorly crystallized phases embedded in amorphous silica, and they did not provide further informations. Considering our results, the composition is the same (Table 3) for a category of glass (binary and ternary), but (i) the specific surface area increases inversely to the macropores diameter, (ii) the macroporous volume increases with the macropore diameter (Table 2), and (iii) the maximal variation (18 nm) of the wall thickness (comparing the two extreme materials B67.5−400 and B67.5−1500 can be considered as negligible, considering the pore diameter). The first interesting phenomenon is the increase in the percentage of the phosphocalcic phase formed in glasses when the diameter of the macropores increases (Figures 7 and 8). Even if the precipitation of the phosphocalcic phase is a surface phenomenon at the very beginning of the interaction (Figure 11, 1 h), this hypothesis does not seem globally valid. Indeed, the specific surface is higher for glasses with smaller macropores (Table 2), and the Ca and P concentrations should be higher for the latter if the hypothesis is correct. SEM pictures suggest that the precipitation is a volume phenomenon over time (Figure 11, 12 h). As detailed in Table 2, the macropore volume increases when the pore diameter increases. Consequently, a higher macropore volume means a higher volume of Ca−P precipitates for glasses 6709



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(6) Rezwan, K.; Chen, Q. Z.; Blaker, J. J.; Boccaccini, A. R. Biodegradable and Bioactive Porous Polymer/Inorganic Composite Scaffolds for Bone Tissue Engineering. Biomaterials 2006, 27, 3413− 3431. (7) Ji, L.; Jell, G.; Dong, Y.; Jones, J. R.; Stevens, M. M. Template Synthesis of Ordered Macroporous Hydroxyapatite Bioceramics. Chem. Commun. 2011, 47, 9048−9050. (8) Hench, L. L. Bioceramics. J. Am. Ceram. Soc. 1998, 81, 1705− 1728. (9) Zhang, K.; Francis, L. F.; Yan, H.; Stein, A. Apatite Converted from 3-D Ordered Macroporous Sol−Gel Bioactive Glass (3DOMBG) Particles. J. Am. Ceram. Soc. 2005, 88, 587−592. (10) Zhang, K.; Yan, H.; Bell, D. C.; Stein, A.; Francis, L. F. Effects of Materials Parameters on Mineralization and Degradation of Sol−gel Bioactive Glasses with 3D-ordered Macroporous (3DOM) Structures. J. Biomed. Mater. Res., Part A 2003, 66A, 860−869. (11) Holland, B. T.; Blanford, C. F.; Stein, A. Synthesis of Highly Ordered Three-Dimensional Mineral Honeycombs with Macropores. Science 1998, 281, 538−540. (12) Holland, B. T.; Blanford, C. F.; Do, T.; Stein, A. Synthesis of Highly Ordered Three-Dimensional Macroporous Structures of Amorphous or Crystalline Inorganic Oxides, Phosphates, and Hybrid Composites. Chem. Mater. 1999, 11, 795−805. (13) Stein, A. Sphere Templating Methods for Periodic Porous Solids. Microporous Mesoporous Mater. 2001, 44−45, 227−239. (14) Nedelec, J.-M.; Grolier, J.-P. E.; Baba, M. Thermoporosimetry: A Powerful Tool to Study the Cross-linking in Gels Networks. J. SolGel Sci.Technol. 2006, 40, 191−200. (15) Baba, M.; Nedelec, J.-M.; Lacoste. Porous Volume of Inorganic Materials and Degree of Swelling of Elastomers Monitored by DSC Measurements. J. Phys. Chem. B 2003, 107, 12884−12890. (16) Billamboz, N.; Baba, M.; Grivet, M.; Nedelec, J.-M. A General Law for Predictive Use of Thermoporosimetry as a Tool for the Determination of Textural Properties of Divided Media. J. Phys. Chem. B 2004, 108, 12032−12037. (17) Elimelech, H.; Nedelec, J. M.; Hardy-Dessources, A.; Babonneau, F.; Avnir, D. Cross-linked polyethylene@silica: The First Full Interpenetrating Network Hybrid Particles. J. Mater. Chem. 2010, 20, 9515. (18) Hardy-Dessources, A.; Hartmann, S.; Baba, M.; Huesing, N.; Nedelec, J.-M. Multiscale Characterization of Hierarchically Organized Porous Hybrid Materials. J. Mater. Chem. 2012, 22, 2713−2720. (19) Lee, J. T. Y.; Leng, Y.; Chow, K. L.; Ren, F.; Ge, X.; Wang, K.; Lu, X. Cell Culture Medium as an Alternative to Conventional Simulated Body Fluid. Acta Biomater. 2011, 7, 2615−2622. (20) Mandel, S.; Tas, A. C. Brushite (CaHPO4·2H2O) to Octacalcium Phosphate (Ca8(HPO4)2(PO4)4·5H2O) Transformation in DMEM Solutions at 36.5 °C. Mater. Sci. Eng., C 2005, 30, 245−254. (21) Clupper, D. C.; Gough, J. E.; Hall, M. M.; Clare, A. G.; LaCourse, W. C.; Hench, L. L. In vitro Bioactivity of S520 Glass Fibers and Initial Assessment of Osteoblast Attachment. J. Biomed. Mater. Res., Part A 2003, 67A, 285−294. (22) Lee, J. T. Y.; Leng, Y.; Chow, K. L.; Ren, F.; Ge, X.; Wang, K.; Lu, X. Cell Culture Medium as an Alternative to Conventional Simulated Body Fluid. Acta Biomater. 2011, 7, 2615−2622. (23) Webster, T. J.; Ejiofor, J. U. Increased Osteoblast Adhesion on Nanophase Metals: Ti, Ti6Al4V, and CoCrMo. Biomaterials 2004, 25, 4731−4739. (24) Gough, J. E.; Notingher, I.; Hench, L. L. Osteoblast Attachment and Mineralized Nodule Formation on Rough and Smooth 45S5 Bioactive Glass Monoliths. J. Biomed. Mater. Res., Part A 2004, 68A, 640−650. (25) Ong, J. L.; Villarreal, D. R.; Cavin, R.; Ma, K. Osteoblast Responses to As-deposited and Heat-treated Sputtered CaP Surfaces. J. Mater. Sci. 2001, 12, 491−495. (26) Kundu, P.; Waghode, T.; Bahadur, D.; Datta, D. Cell Culture Approach to Biocompatibility Evaluation for Unconventionally Prepared Hydroxyapatite. Med. Biol. Eng. Comput. 1998, 36, 654−658.

the phosphorus concentration in the newly formed phases will be lower for high-diameter glasses (phosphorus curves, Figure 7). The [(Ca phases)/(metastable Ca−P phase)] proportion logically seems more important in the high porosity glasses. The dissolution of calcium phosphates will then result in an increased release for glasses with smaller pore size. This is confirmed by changes in the Ca/P atomic ratio for the binary glass (Figure 12). After 1 h of interaction, this ratio is much higher for glass B75−1500 than for the B75−800 and B75−400 glasses. Regarding ternary glasses, the evolution of these ratios is very similar, suggesting that the precipitated phases are similar.

5. CONCLUSION Interactions of macrostructured bioactive glasses with a biological medium have been characterized by using μ-PIXE− RBS quantitative mapping. The first key result is that spatial distributions of chemical elements stay homogeneous within the grain during in vitro interaction: the mineralization occurs here as a homogeneous process due to the well-organized porous structure and the highly interconnected network. Three chronological steps take place: release of calcium, dissolution of the silicate network, and precipitation of calcium phosphate phases. The demonstration that the precipitation of phosphocalcic phases takes place inside the whole material, while it is limited to only the periphery in conventional porous glasses, is a great improvement of the mineralization properties of bioactive glasses. The kinetics of these phenomena are not affected by the macropores’ size, but their intensity is: it is a key point showing how the amplitude of reactions could be finely tuned simply by controlling the macropore diameters. It is an important step forward in building advanced biomaterials with a highly reproducible and finely controlled response for an optimized integration in living organisms.

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ASSOCIATED CONTENT

S Supporting Information *

TEM images of B67.5-400 and B67.5-1500 glasses thin cut. This information is available free of charge via the Internet at http://pubs.acs.org

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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REFERENCES

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