Evidence of Nanometric-Sized Phosphate Clusters in Bioactive

Article pubs.acs.org/JPCC

Evidence of Nanometric-Sized Phosphate Clusters in Bioactive Glasses As Revealed by Solid-State 31P NMR Franck Fayon,* Cédric Duée, Thomas Poumeyrol, Mathieu Allix, and Dominique Massiot Conditions Extrêmes et Matériaux: Haute Température et Irradiation, CNRS UPR 3079, 1D Avenue de la Recherche Scientifique, 45071 Orléans Cedex 2, France and Université d’Orléans, Faculté des Sciences, Avenue du Parc Floral, 45067 Orléans Cedex 2, France S Supporting Information *

ABSTRACT: Bioactive glasses are able to form strong bonds to bone. This property, crucial for medical applications, depends on the glass composition and structure. Dissolution of phosphates in melt-quenched silicate glasses raises the question of chemical homogeneity and possible formation of clusters. A detailed structural characterization of the bioactive glasses is thus highly desirable. In this work, the nature of the distribution of phosphate units in a melt-quenched bioactive glass is elucidated for the first time using 31P spin-counting solid-state NMR experiments. The structure of a dense bioactive calcium silicate glass with 2.6 mol % of phosphorus oxide is shown to exhibit nanometric-sized chemical and structural heterogeneities. Clear experimental evidence of the presence of phosphate clusters of five and six PO4 tetrahedral units embedded in the disordered polymeric silicate network is given.

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INTRODUCTION Melt-quenched bioactive glasses are an important class of materials used for bone repair and reconstruction in clinical applications, such as craniofacial and periodontal defects repair, ossicular prostheses, and orthopedic bone graft substitute.1,2 These dense nonporous materials, which are mostly calciumsilicate-based glasses containing a small amount of phosphorus oxide, are able to form a strong and stable bonding interface to hard tissues.1−3 The bioactivity of these glasses has been shown to depend on their compositions and structures.4,5 The structure of bioactive glasses, which reflects that of the hightemperature molten-state quenched at the glass-transition temperature, has thus been widely investigated, and it was demonstrated that it is made of a polymeric network of SiO4 tetrahedra in which phosphorus is mainly present under the form of orthophosphate (PO43−) units.6−10 Whereas molecular dynamics computer simulations suggested the presence of phosphate nanodomains in the glass structure,11,12 experimental insights about the distribution of the phosphate units (homogeneous repartition or clustering) in the disordered silicate network are still lacking. Probing the distribution of phosphate units in a silicate glass network using conventional techniques such as small-angle X-ray scattering (SAXS) or transmission electron microscopy (TEM) remains highly challenging because silicon and phosphorus atoms have very similar electron densities, requiring the use of more advanced methods like energy-filtered TEM, anomalous-SAXS, or smallangle neutron scattering (SANS). In this context, dipolar-based © 2013 American Chemical Society

multiple-quantum (MQ) spin-counting solid-state NMR experiments,13,14 which enable selective measurements of the spatial distribution of atoms in materials lacking long-range order, appear as alternative tools to study the distribution of phosphate units in disordered silicate networks. These methods rely on the observation of MQ coherences characteristic of an ensemble of nuclei correlated through their dipolar couplings. Because the magnitude of the dipolar coupling is inversely proportional to the cube of the interatomic distance, the rate at which MQ coherences develop under a pure double-quantum dipolar average Hamiltonian provides information about the nature and extent of clustering in solids. Spin-counting solidstate NMR experiments have already been successfully employed to study the distributions of organic molecules in zeolite cavities,15,16 fluoride salts in polymer films,17,18 or hydrogen atoms in hydrogenated amorphous solids.19,20

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EXPERIMENTAL SECTION The 46.1SiO2-51.3CaO-2.6P2O5 glass was synthesized from a stoichiometric mixture of CaCO3 (99%, Sigma-Aldrich), NH4H2PO4 (99%, Alfa Aesar), and SiO2 (99.9%, ChemPur) powders. The starting material was heated in a Pt/10%Rh crucible at 300 °C for 3 h to remove ammonia and water and decarbonated at 950 °C for 5 h. The mixture was then melted Received: December 13, 2012 Revised: January 11, 2013 Published: January 11, 2013 2283

dx.doi.org/10.1021/jp312263j | J. Phys. Chem. C 2013, 117, 2283−2288

The Journal of Physical Chemistry C

Article

Figure 1. (a) Solid-state 29Si MAS NMR spectrum of the 46.1SiO2-51.3CaO-2.6P2O5 melt-quenched glass recorded at 9.4 T with a spinning frequency of 20 kHz (black dots) and its corresponding simulation (red line). Individual QnSi contributions are shown as black lines. (b) Solid-state 31 P MAS NMR spectrum recorded at 9.4 T with a spinning frequency of 14 kHz (black dots) and its corresponding simulation (red line). Individual contributions of orthophosphate groups and of PO4 units bonded to the silicate network are shown as green and black lines, respectively.

at 1700 °C for 30 min and quenched by partly dipping the crucible into water. A 29Si-enriched glass sample was prepared via the same protocol using 99% 29Si-enriched SiO2 (99.9%, CortecNet). All solid-state NMR experiments were performed on a Bruker Avance I spectrometer operating at 9.4 T (29Si and 31P Larmor frequencies of 79.5 and 162.0 MHz) using 3.2 mm triple-resonance and 4.0 mm double-resonance Bruker probeheads. The quantitative 29Si MAS spectra were recorded at a 20 kHz spinning frequency, with pulse duration of 1 μs (corresponding to a flip-angle of 22.5°) and a recycle delay of 60 s. 4096 and 128 transients were collected for the 29Si natural-abundance and 29Si-enriched samples, respectively, and the obtained spectra were the same. The quantitative 31P MAS spectrum was recorded at a spinning frequency of 14 kHz, with pulse duration of 0.6 μs (corresponding to a flip-angle of 22.5°) and a recycle delay of 60 s. 128 transients were collected. The 29 Si and 31P chemical shifts were referenced relative to Si(CH3)4 and a 85% H3PO4 solution, respectively. The 31P−29Si REDOR NMR experiments21 were performed for the 99% 29Si-enriched glass sample using spinning frequencies of 15 and 24 kHz. A 16-step phase cycling, allowing the selection of the spin−echo coherence pathway, was used for the 31P observe channel, and an XY-8 phase alternation of the rotor-synchronized 180° recoupling pulses was used for the 29Si channel.21,22 To account for relaxation effects, we recorded a reference experiment (S0) for each dipolar dephasing experiment (S) by acquiring the 31P spin− echo signal in the absence of the 29Si pulses. The difference signal intensity due to dipolar dephasing was normalized relative to the reference signal intensity (ΔS/S0). The 31P and 29 Si nutation frequencies were of 62.5 and 76 kHz, respectively. The recycle delay was set to 15 s, and a saturation comb was applied before the recycle delay. 128 transients were collected for each dipolar dephasing and reference experiments. The 31P static MQ NMR experiments were performed using the pulse sequence previously described by Saalwachter et al.23 The MQ excitation and reconversion blocks consist of n cycles of eight 31P 90° pulses with (X)4 (X̅ )4 phase alternation spaced by delays 2Δ and Δ.13 To refocus the 31P chemical shift anisotropy during the MQ excitation and reconversion periods, four additional 31P 180° pulses were applied during the 2Δ delays on the time scale of 3Δ.23,24 The different MQ coherence orders were separated in a phase-incremented twodimensional (2-D) experiment after Fourier cosine trans-

formation with respect to the phase. For each experiment, 32 slices with an 11.25° phase increment (excitation block) were recorded allowing the separation of MQ coherence orders from −15 up to +16. A four-step phase-cycling of the last 90° pulse was used for quadrature detection. For excitation (and reconversion) times ranging from 0.8 (4 cycles) to 8.8 ms (44 cycles), each slice was the sum of 192 transients. For longer excitation times of 10.2 (52 cycles) and 12 ms (60 cycles), 256 transients were coadded. The 31P nutation frequency was 92 kHz. The recycle delay was set to 10 s, and a saturation comb was applied before the recycle delay.

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RESULTS AND DISCUSSION

The melt-quenched bioactive glass studied here has a 46.1SiO251.3CaO-2.6P2O5 molar composition, with identical silica and phosphorus oxide contents than that of the medically approved 45S5 glass (46.1SiO2-26.9CaO-24.4Na2O-2.6P2O5).1,2 As shown in Figure 1a, the quantitative 29Si MAS spectrum of this glass exhibits an asymmetric line shape, revealing the presence of several overlapping resonances. The experimental spectrum is nicely simulated with three Gaussian lines located at −89.4, −81.8, and −75.6 ppm, which are unambiguously assigned to Q3Si, Q2Si, and Q1Si units, respectively. The 29Si isotropic chemical shifts and line widths of the QnSi units (SiO4 tetrahedral units with n bridging oxygen atoms) are in good agreement with the values previously reported for a CaSiO3 glass,25 and the relative amounts of Q3Si (25.6%), Q2Si (57.6%), and Q1Si (16.8%) units indicate that the disordered silicate network consists of cross-linked chains of SiO4 tetrahedra. The average number of nonbridging oxygen atoms per SiO4 tetrahedron determined from the proportions of QnSi units (1.91 ± 0.02) is close to that expected from the stoichiometry assuming that phosphorus mainly forms Ca2+ chargecompensated PO43‑orthophosphates groups (1.89). This result is in agreement with the experimental 31P MAS spectrum of the glass (Figure 1b). It shows a slightly asymmetric line shape nicely modeled with an intense contribution at 2.7 ppm (92%) and an additional peak of much weaker intensity at −4.9 ppm (8%). According to the QnP chemical shift ranges in calcium phosphates, the former is ascribed to Ca-orthophosphate groups (Q0P), whereas the latter cannot be directly assigned. Assignment of this weak intensity peak is achieved using more advanced solid-state NMR experiments. From 31P refocused INADEQUATE MAS experiments probing through-bond P−O−P connectivities, 2284



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Figure 2. REDOR dephasing curves (ΔS/S0) of the peaks at 2.7 (green triangles) and −4.9 ppm (black circles) obtained from 31P−29Si REDOR21 experiments performed at 9.4 T with spinning frequencies of 24 (filled symbols) and 15 kHz (open symbols). In panel a, the lines correspond to fits of the initial REDOR curvatures (ΔS/S0 ≤ 0.3) to the parabolic function f(τ) = (4/3π2)τ2 M2, from which the heteronuclear dipolar second moment (M2) values are obtained.27,28 In panel b, the line corresponds to a numerical simulation performed with the SIMPSON code29 for 31P−29Si spinpairs. A normal distribution of the interatomic P−Si distance, with an average value of 3.0 Å (σ = 0.2 Å) typical of a P−O−Si bond, was considered to account for the disorder in the glass network (distribution of bond angles and bond lengths). Longer-range 31P−29Si dipolar interactions were neglected.

Figure 3. (a) Multiple-quantum coherence intensity distribution as a function of the excitation time. The distribution is symmetric about n = 0 and only positive coherence orders are shown. Longitudinal magnetization is included in zero-quantum coherences. (b) Fit of the MQ intensity distribution (excitation time of 10.4 ms) to a Gaussian function. (c) Variation of the number of correlated spins N, obtained from fits of the MQ intensity distributions, as a function of the MQ excitation time.

glass sample (Figure 2), enabling the measurements of P−Si interatomic proximities, reveal relatively large 31P−29Si dipolar couplings for this signal. Figure 2a shows the fits of the initial REDOR curvatures (ΔS/S0 ≤ 0.3) to a parabolic function,

the presence of PO4 units with P−O−P bond(s), like pyrophosphate Q1P or metaphosphate Q2P groups, can be excluded.26 31P−29Si rotational echo double-resonance21 (REDOR) experiments performed for a 99% 29Si-enriched 2285



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Figure 4. Variation of the experimental multiple-quantum intensities with the excitation time. Positive and negative coherence orders have the same intensities and only positive coherence orders are shown. The green diamonds and red circles correspond to four-quantum and double-quantum coherences, respectively. The blue squares correspond to zero-quantum coherences and longitudinal magnetization (LM). The curves correspond to numerical simulations considering model clusters of four (a), five (b), six (c) and eight (d) PO4 tetrahedral units. An example of the considered clustered atomic arrangements is shown above each curve.

distinct MQ orders were separated in a 2-D phase-incremented experiment after Fourier transformation with respect to the phase. As shown in Figure 3a, the spectral intensity is spread to higher MQ orders, up to four, with increasing the excitation time. Assuming that the dipolar couplings of the correlated spins are of the same order of magnitude and neglecting differential relaxation effects, the intensity of the nth coherence order I(n) is well-approximated by exp(−n2/N), where N is the excitation time-dependent number of correlated spins.13,14 The timedependence of N provides in turn information about the atomic arrangement. For isolated clusters of spins, N remains constant after a long enough excitation time, whereas homogeneous extended spin networks yield unbounded growth curves. Interacting clusters show a mixed behavior, where N first increases up to a saturation value, followed by an unbounded growth of slower rate at longer times.13,14 As shown in Figure 3b, the observed MQ intensity distribution is nicely fitted to a single Gaussian line for all excitation times. The corresponding number of correlated spins N increases with the excitation time to reach a plateau at N = 5.5 (Figure 3c), clearly evidencing a mixture of well-separated clustered atomic arrangements predominately composed of five and six PO4 units. Probing the longer-range intercluster distances would require much longer excitation times, which cannot be reached due to relaxation effects. This result unambiguously indicates that the glass structure is heterogeneous on a nanometric length scale and can be described as phosphate clusters embedded in a polymeric silicate network. To further confirm the above analysis and to obtain additional information about the distances between the phosphorus atoms constituting the clusters, we performed

which provide reliable information about dipolar couplings in multispin systems of unknown geometry.27,28 From this analysis of short dephasing times, a heteronuclear dipolar second moment value of 5.0 ± 0.1 × 105 rad·s−2 is obtained for the high intensity peak (at 2.7 ppm) assigned to orthophosphate groups. For the weak intensity peak at −4.9 ppm, a much larger value of 10 ± 1 × 105 rad·s−2, very close to that calculated for a P−Si interatomic distance of 3.0 Å characteristic of a P−O−Si bond (10.1 × 105 rad·s−2), is obtained. Additional numerical simulations29 of the entire REDOR dephasing curve considering a distribution of 31P−29Si spin-pairs are also consistent with an average P−Si distance of 3.0 Å (Figure 2b). These results allow us to unambiguously assign this signal to PO4 units having one P−O−Si bond and being covalently bonded to the polymeric silicate network under the form of end-chain groups. As shown above, phosphorus atoms are mainly present in the glass structure under the form of Ca2+ charge-compensated orthophosphate PO43− groups that are not bonded to the disordered polymeric network of SiO4 units. Whereas the formation of crystalline Si-doped phosphate nanodomains has been shown in bioactive porous sol−gel glasses from highresolution TEM and electron diffraction experiments,30 the nature of the distribution of orthophosphate units in a dense melt-quenched glass remains unclear. To highlight the spatial distribution of phosphate units in the glass structure, we employed 31P static MQ NMR experiments, which enable measurements of the number of P atoms correlated by throughspace dipolar interactions. Creation and reconversion of even MQ coherences was achieved using a specific pulse sequence, which refocuses isotropic and anisotropic chemical shifts and creates a double-quantum dipolar average Hamiltonian.23 The 2286


The Journal of Physical Chemistry C numerical simulations29 of the intensity evolution of the individual MQ orders with excitation time. For these simulations, P-clusters of different sizes (from four up to ten P atoms) were constructed from the 3-D packing arrangements encountered in the crystalline calcium orthophosphate compound α-Ca3(PO4)2, the structure of which contains 12 inequivalent P sites.31 To partially account for the local structural disorder in the glass (reflected by distribution of 31P isotropic chemical shift observed in the broad 31P MAS spectrum), we considered 12 clusters with the same number of P atoms but slightly different geometries (each of them being centered on a distinct P site of the α-Ca3(PO4)2 structure), and the resulting simulations were summed up. The results of these simulations are compared with the experimental built-up curves of the different MQ orders in Figure 4. A mismatch between the experimental curves and the simulations performed for the 4- and 8-P cluster models is clearly observed (Figure 4a,d), where the intensities of the fourquantum coherences order are significantly underestimated and overestimated in the simulations, respectively. In contrast, a nice agreement between the experimental data and numerical simulations for the 5-P and 6-P clusters is observed (Figure 4b,c). This result not only confirms the validity of the previous data analysis but also shows that the geometric arrangement of the PO4 units and the P−P distances within the P-clusters involved in the glass structure are both relatively similar to the model clusters considered here. As mentioned above, the 5- and 6-P cluster-models correspond to a three-dimensional packing arrangement of neighboring PO4 units, similar to the one found in crystalline calcium orthophosphates and having a size of ∼1 nm. It can thus be concluded that the sizes of the orthophosphate clusters found in the glass structure are on the order of 1 nm.

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REFERENCES

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CONCLUSIONS We have demonstrated that 31P MQ NMR experiments enable measurements of the spatial distribution of the phosphate units in melt-quenched bioactive glasses. The structure of the 46.1SiO2-51.3CaO-2.6P2O5 bioactive glass is shown to exhibit chemical and structural heterogeneities and is composed of phosphate clusters having a size of ∼1 nm embedded in a polymeric silicate network. Because the structure of the glass reflects the high-temperature molten-state structure quenched at the glass-transition temperature, the observed heterogeneities likely originates from a liquid−liquid phase separation on the nanometer length scale. ASSOCIATED CONTENT

S Supporting Information *

Sketches of the pulse sequences used for the 31P−29Si REDOR and 31P static multiple-quantum NMR experiments. This material is available free of charge via the Internet at http:// pubs.acs.org

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ACKNOWLEDGMENTS

The authors acknowledge CNRS, Université d’Orléans and ANR NT09_514015 “Nanoshap” for financial support.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (+33) 238 25 55 25. E-mail: franck.fayon@cnrs-orleans. fr. Notes

The authors declare no competing financial interest. This work was presented at the 54th Rock Mountain Conference on Analytical Chemistry (July 15th-19th, 2012). 2287



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Evidence of Nanometric-Sized Phosphate Clusters in Bioactive

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