Surface Reactions of Mesoporous Bioactive Glasses Monitored by

Article pubs.acs.org/JPCC

Surface Reactions of Mesoporous Bioactive Glasses Monitored by Solid-State NMR: Concentration Effects in Simulated Body Fluid Claudia Turdean-Ionescu,† Baltzar Stevensson,† Isabel Izquierdo-Barba,‡,§ Ana García,‡,§ Daniel Arcos,‡,§ María Vallet-Regí,‡,§ and Mattias Edén*,† †

Department of Materials and Environmental Chemistry, Stockholm University, SE-106 91 Stockholm, Sweden Departamento de Química Inorgánica y Bioinorgánica, Facultad de Farmacia, Instituto de Investigación Sanitaria Hospital, Universidad Complutense de Madrid, 12 de Octubre i+12, 28040 Madrid, Spain § Networking Research Center on Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN) Madrid, Spain ‡

ABSTRACT: A bone-mineral-mimicking layer of hydroxycarbonate apatite (HCA) forms at the surface of a bioactive glass on its contact with body fluids. We report a solid-state 29Si nuclear magnetic resonance (NMR) spectroscopy study of the surface reactions preceding the HCA formation at three CaO− SiO2−(P2O5) mesoporous bioactive glasses (MBGs) with distinct compositions, surface areas, and mesoporous arrangements, during their immersion in simulated body fluid (SBF) out to 30 days. The evolution of the various populations of coexisting silicate species associated with the bulk and surface portions of the pore-walls were monitored. The MBGs revealed drastically different surface alterations between the scenarios of low (0.6 g/L) and high (20 g/L) MBG concentrations in the SBF: for the low MBG dose, which is expected to be more relevant for in vivo conditions, all MBGs follow a “universal” dissolution mechanism beyond ≈24 h of SBF soaking, regardless of their precise compositions and textural properties. The only essential difference among the specimens occurs during the first hour of soaking when their variable Ca2+ reservoirs are depleted. In contrast, for high MBG concentrations, the surface reactions and their associated silicate network degradation retard for Ca-poor MBGs, whereas the reactions are completely quenched for Carich compositions. These findings rationalize previously reported discrepancies in the correlation between the HCA formation and the MBG composition for distinct concentrations during SBF testing, and simplify future MBG design by identifying which compositional and textural factors are relevant for a rapid and substantial HCA formation in vitro.

1. INTRODUCTION Bioactive glasses (BGs) are frequently employed in maxillofacial and periodontal surgery because they interface with the living tissue via a surface layer of biomimetic hydroxycarbonate apatite (HCA) that closely mimics bone mineral.1−3 SiO2-based BGs may be fabricated either by a traditional melt−quench route,1,3 or by sol−gel3−5 and evaporation-induced selfassembly (EISA)6,7 procedures. In contrast with melt-prepared BGs that are often implanted as granules with a low surface area, the sol−gel and EISA approaches yield porous BGs with substantial surface areas, notably so the EISA-derived mesoporous bioactive glasses (MBGs) that manifest an ordered arrangement of nm-sized pores.7 Several MBGs7−14 and sol− gel-derived BGs3,4 with variable compositions and textural properties are reported from the CaO−SiO2−(P2O5) systems. Hench and co-workers proposed a five-step sequence onward referred to as the “Hench mechanism” (HM)that describes the surface reactions emanating in HCA formation at melt-prepared BGs.1 The initial step involves release of network-modifier ions (e.g., Na+, K+, Ca2+) that exchange with protons from the surrounding solution to form silanol (SiOH) surface groups. The latter are also formed during stage © XXXX American Chemical Society

2 by the breaking of Si−O−Si linkages: Si−O−Si+H2O→2Si− OH. These two initial steps lead to a silanol-rich glass surface.1 The third stage is essentially a reversal of its preceding one; i.e., the silanols undergo condensation reactions (2Si−OH→Si− O−Si+H2O) that increase the network polymerization. This results in a continuously thickening hydrated silica-gel surface layer, as reported in numerous studies of BGs subjected to (simulated) body fluids.15−28 At stage 4, a new surface layer of amorphous calcium phosphate (ACP) forms, which partially incorporates additional cations (e.g., Na+, Mg2+) and anions − (e.g., CO2− 3 , F ) from the aqueous medium when the layer crystallizes into HCA (step 5).1 Many studies have explored the constitution and growth of the silica-gel/phosphate surface layers, employing X-ray diffraction (XRD),5,16,17,25,29−32 electron microscopy (often coupled with energy-dispersive X-ray spectroscopy for element mapping),7−11,19,20,24−27,32−35 particle-induced X-ray emission,28,36 as well as various spectroscopies, such as FourierReceived: December 21, 2015 Revised: February 7, 2016

A

DOI: 10.1021/acs.jpcc.5b12490 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C Table 1. MBG Compositions and Textural Propertiesa sample

mCaO

nSiO2

pP2O5

stoichiometric formulab

SBET (m2g−1)c

Vp (cm3g−1)d

dp (nm)d

mesoporous structure

S90 S85 S58

10.0 (9.6) 10.0 (10.6) 37.0 (36.6)

90.0 (90.4) 85.0 (86.5) 58.0 (59.0)

0.0 (0.0) 5.0 (2.9) 5.0 (4.4)

Ca9.6Si90.4O190.4 Ca10.3Si84.1P5.6O192.5 Ca35.1Si56.5P8.4O169.2

468 480 195

0.63 0.64 0.46

5.37 5.38 9.45

p6mm Ia3d p6mm

a Each MBG sample is denoted Sn, where n is the nominal oxide equivalent of SiO2 in mol % of the composition mCaO−nSiO2−pP2O5, where m + n + p = 100 mol %. Values in parentheses represent the respective XRF-analyzed oxide equivalents. bCharge-balanced XRF-analyzed stoichiometric composition, with cation coefficients summing to 100.0 (mol). cSpecific surface area (SBET) determined by the Brunauer−Emmett−Teller (BET) method.69 dTotal pore volume (Vp) and average pore diameter (dp). All data are reproduced from ref 31.

previous findings for nominally identical MBG samples but with a significantly higher loading in the SBF (20 g/L).41,42 The observed concentration-dependent reaction pathways are discussed in view of the drastically different HCA formation trends observed from the three MBG compositions for the two distinct MBG concentrations.31,32,41 While the dissolution and the subsequent HCA formation processes of a BG are known to depend on its concentration and particle-size distribution,57−60 we are unaware of reports that link and rationalize such results directly to the experimentally probed underlying surfacereaction dynamics. We also identify which compositional and textural parameters that (do not) influence the surface reactions preceding the HCA formation in vitro and their interplay with the choice of MBG concentration in the SBF solution.

transform infrared and Raman,7−11,17,24,25,29,33,37 X-ray photoelectron,37 and solid-state nuclear magnetic resonance (NMR);30−32,38−43 see Cerruti’s review for further information.44 While atomistic modeling of the BG surface (reactions) are reported,45,46 experimental investigations that directly monitor the BG-surface reactions/modifications at a subnanometer scale are very sparse.39−42 Magic-angle spinning (MAS) 29Si NMR spectra acquired directly by “single” radio frequency (rf) pulses offer quantitative profiles of the various structural groups in amorphous silicates.47,48 Its use is particularly rewarding when contrasted with results from 1H→29Si cross-polarization (CP) NMR experiments49−51 that selectively probe the silicate surface. Here NMR responses are detected solely from 29Si nuclei in close spatial proximity (in practice ≲0.4 nm) to 1H neighbors. Since the 1H content is very low in the silicate interior (e.g., inside the MBG pore-walls), application of 1H→29Si CP NMR effectively translates into a direct probing of the surface silicate speciation, provided that the through-space mediated 1H−29Si dipolar interactions responsible for the magnetization transfers are reactivated for a sufficiently short time period.41,48 We recently utilized such NMR experiments for monitoring the alterations of the bulk and surface segments of MBGs with variable compositions on their exposure to a simulated body fluid (SBF) for increasing time periods.41,42 Such 29Si NMR experimentation is also reported for probing the surface modifications of melt-prepared39,40 and sol−gel-derived30,38 BGs during SBF exposure. The Hench mechanism was originally introduced for rationalizing the evolution of melt-prepared BG surfaces in (simulated) body fluids.1 Melt-prepared glasses typically comprise substantial network-modifier contentsand thereby exhibit relatively fragmented silicate networks that readily dissolve in aqueous media; a degrading silicate network is a necessary (but not sufficient) condition for HCA formation from such glasses.20,52−56 However, while the HM was shown to be applicable/relevant also for MBGs,41,42 each element thereof may not be essential for rapid and substantial HCA generation from mesoporous structures; see Gunawidjaja et al.41,42 Here we discuss these aspects further from a more quantitative standpoint. This article reports a MAS 29Si NMR investigation that characterizes the modifications of the bulk and surface portions of the pore-walls in three MBG samples with molar compositions 37CaO−58SiO2−5P2O5,9 10CaO−85SiO2− 5P2O5,9 and 10CaO−90SiO2,11 during their immersion in SBF from 15 min to 30 days. The MBGs also exhibit distinct textural properties (see Table 1). The interconversions among the various coexisting silicate species are evaluated and compared with the predictions of the HM. Moreover, we contrast the present reaction profiles obtained for a relatively modest MBG concentration in the SBF (0.6 g/L) with our

2. MATERIALS AND METHODS 2.1. MBG Preparation and Composition Analysis. An EISA procedure6 was employed to prepare three MBG specimens of nominal molar compositions 10CaO−90SiO2 (denoted “S90”), 10CaO−85SiO 2 −5P 2O 5 (“S85”), and 37CaO−58SiO2−5P2O5 (“S58”) from tetraethyl orthosilicate (TEOS), triethyl phosphate (TEP), and Ca(NO3)2·4H2O. The synthesis was carried out at 40 °C, using the nonionic triblock copolymer P123 as structure-directing agent. Other conditions are provided in ref 9. The resulting homogeneous membranes were heated at 700 °C for 6 h to remove organic species and nitrate ions. The cation composition of each pristine S90, S85, and S58 MBG specimen was estimated by X-ray fluorescence (XRF) spectroscopy. The as-analyzed (Si, Ca, P) weight percentages were converted into the charge-balanced stoichiometric compositions listed in Table 1, which also presents the textural properties of the pristine MBG samples; see Turdean-Ionescu et al.31 for details. The batched and analyzed compositions agreed well, except for some losses of P. The currently analyzed MBGs are also close to those of nominally identical compositions discussed in refs,41,42 with which our NMR results are compared. The presented stoichiometric compositions exclude the unknown but minor hydrogen contents of each specimen, which primarily consists of physisorbed water molecules that varies among the MBG samples but do not affect their (cation) stoichiometry. The remaining hydrogen portion stems from protons of SiOH groups (see section 3) that do affect the cation/oxygen charge-balance; their relative populations are probed by the 29Si NMR experiments discussed below. 2.2. In Vitro Studies. An SBF solution was prepared according to Kokubo et al.61 by dissolving NaCl, KCl, CaCl2, MgCl2·6H2O, NaHCO3, K2HPO4·3H2O, and Na2SO4 into distilled water, with tris(hydroxymethyl)aminomethane/HCl (TRIS/HCl) for buffering the solution at pH = 7.38. The solution was filtered (0.22 μm; Millipore) to avoid bacterial B


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The Journal of Physical Chemistry C

Figure 1. Schematic depiction of the surface silicate speciations typical for the pristine (a) S58 and (b) S85 MBGs. (c) Representative MBG-τSBF surface after exposure of any S90/S85/S58 MBG to SBF for τSBF ≳ 1 day at a low concentration (mMBG/V ≈ 0.6 g/L). The OH groups in magenta color originated from Ca2+ ↔ H+ exchange of the QnCa groups in (a). The double-headed arrow highlights the very similar surface-signatures of (c) compared to those of the pristine S85 MBG in (b).

species (see section 3), by employing an in-house-prepared iterative fitting computer program. It allows for imposing restrictions of each peak position and full-width at halfmaximum (fwhm) height. Each NMR spectrum was fitted multiple times with different initial conditions, with an upper fwhm limit of 12 ppm and the chemical shift bounded within ±3−4 ppm. The resulting set of best-fit results for each parameter was then used to derive its mean value and standard deviation.

contamination. 600 mg of each pristine MBG sample in the form of a fine powder (25% of the entire Si speciation, as reflected in markedly more intense 29Si NMR peaks in the shift-range ≳−85 ppm compared with the spectra from the S85 and S90 samples (Figure 3). The Q3H silanol populations are similar for all three MBGs. Out of the total silicate speciation, they amount to ≈17% for S90/S58 and ≈23% for S85. Presumably, the Q2H population is also similar at all MBG surfaces. Note that 29Si NMR signals from Q2H and Q3Ca groups both resonate ≈−92 ppm42,47,49,63 and cannot be resolved. As discussed further below, there may be a minor contribution from Q3Ca groups to the 29Si NMR spectrum of S90, while >50% of the total NMR peak intensity centered at δSi ≈−92 ppm from the S58 counterpart may stem from Q3Ca groups in the pore-wall interior. Altogether, an increasing Q3Ca NMR signal contribution accounts for the growing sum of Q2H/Q3Ca populations (stated within parentheses) according to S85(3%)≲S90(7%)S90(68%)>S58(44%). 4.1.2. SBF-Exposed Samples. We next consider the alterations of each silicate structure on its immersion in SBF for variable time intervals up to 30 days. Figure 3 reveals a striking depletion of the 29Si NMR signal intensity in the region ≳−85 ppm from all MBG-τSBF specimens, as is obvious already after 15 min of SBF soaking. This reflects a very rapid leaching of Ca2+ from the mesoporous surface,41,42 which is devoid of

Figure 2. Schematic representation of the distribution of disordered calcium phosphate (CaP) clusters across the pore-walls of (a) S85 and (b) S58. The white and blue color represents the pores (voids) and the silicate portion of the pore-walls, respectively. Note that S58 exhibits smaller CaP clusters than S85. The pore-wall scenario of (b) likely also applies to Ca-rich sol−gel-derived BGs.

sizes estimated as ≈20−40 PO3− 4 groups in S85 (i.e., a CaP cluster diameter of ≈1.5 nm) and ≈6 groups in S58.65,66 The Ca accumulation at the surface of the silicate pore-wall segmentcoupled with the presence of CaP clusters facilitates HCA formation,41,63 thanks to the high MBG surface area and mesoporous channel system that provides a high accessibility of both Ca2+ and PO3− 4 species to the surrounding fluids.8−10,31,32,41 However, the large CaP clusters in S85 [Figure 2(a)] are overall in much closer contact with the surrounding solution than their small counterparts in the S58 structure, where a non-negligible (perhaps major) part of the total CaP reservoir is embedded inside the silicate network. Although the precise structural role of P has not yet been been probed experimentally in sol−gel BGs, it is likely that Ca-rich specimens thereof (as opposed to melt-prepared BGs; see refs 65 and 66) conform to the model for S58 depicted in Figure 2(b). As discussed in sections 5.1 and 5.2, the different extents of solution-exposed clusters in S85 versus S58 have consequences for their HCA-generation in vitro, which becomes strongly dependent on the particular MBG concentration in the SBF. A direct consequence of the presence of orthophosphate anions in the CaP clusters is a consumption of (3/2)Ca2+ 2+ cations per PO3− 4 group: this restricts the availability of Ca in 41,42,63 the silicate pore-wall portion, meaning that the Ca-poor S85 exhibits pore-walls of essentially neat mesoporous SiO2, whereas the Ca-rich S58 structure involves a lower (average) polymerization of the CaO−SiO2 pore-wall component, accompanied by a surplus of surface-associated Ca 2+ cations.42,63 The structural consequences of an increased silicate network polymerization from the entrapment of Ca2+ by the PO3− 4 groups is well-known in the context of melt-prepared BGs.35,54,55,67 D


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Figure 3. Experimental 29Si MAS NMR spectra (black lines) recorded by single pulses from the pristine S85, S90, and S58 specimens (top row). All other spectra were recorded after SBF immersion from the as-indicated MBG-τSBF samples when using mMBG/V = 0.6 g/L. Deconvoluted peak components are plotted with gray lines; their assignments are displayed at the top of each column. Note that the NMR peak around −92 ppm has contributions from both Q2H groups (sole component for S85 and major component for S90) and Q3Ca moieties (minor and major components for S90 and S58, respectively). The curve beneath each spectrum represents the difference between the experiment and best fit.

QnCa groups after 15 min and 60 min for the cases of S90 and S58, respectively. The evolution of the silicate speciations discussed below should be viewed in light of the following dissolution trends reported in ref 31 for the same sets of MBG-τSBF specimens: (i) During the first 15 min of immersion, the pH of the solution increases rapidly from 7.38 and then remains essentially constant out to ≈8 h at the values 7.45, 7.50, and 7.60 for S85, S90, and S58, respectively. (ii) A similar trend is observed for the Ca2+ concentration: after an initially steep increase at τSBF = 15 minwhich is much larger for S58 than for the Capoor S85/S90 specimensthe [Ca2+] remains nearly constant for τSBF ≲ 8 h.31 (iii) For the S90/S85 MBGs, the Ca reservoir is fully depleted during the first hour of MBG exposure to the solution, whereas that for S58 is reduced drastically, but with ≈50% of the initial Ca content remaining thereafter. However, a significant (but unknown) fraction of the Ca ensemble resides in a separate ACP layer that forms very rapidly due to the

substantial Ca leaching from S58. Note that significant HCA deposits were evidenced both by powder XRD and by 31P MAS NMR within each MBG series after 24 h. Owing to the HCA formation that proceeds from τSBF ≈ 24 h, the net amount of Ca in the solid phase (comprising the Ca-leached MBG and the ACP/HCA layer) increases steadily over the first week of SBF immersion.31 The 29Si NMR results discussed below allows for a separate probing of the Si-associated Ca reservoir. (iv) Consistent with a continuos silicate network degradation, the Si concentration of the solution elevates monotonically out to ≈3 days throughout, after which [Si] saturates at ≈2.2 mmol/L. The increase of [Si] is largest for the S58 soaking, whereas the S85/S90 samples manifest solutions with nearly equal Si concentrations.31 The evolution of the Ca contents in each solid phase and its accompanying SBF solution31 matches well the herein deduced alterations of the fractional populations of Q1Ca and Q2Ca groups in each MBG-τSBF sample. Figure 4 plots the various x(QnE) data E


Article

The Journal of Physical Chemistry C Table 2. Best-Fit 29Si MAS NMR parameters (single-pulse excitation)a Q4

Q2H + Q3Ca b

Q3H

Q2Ca

sample

−δ (ppm)

fwhm (ppm)

fraction (%)

−δ (ppm)

fwhm (ppm)

fraction (%)

−δ (ppm)

fwhm (ppm)

fraction (%)

S85 S85-0.25h S85-1h S85-4h S85-8h S85-7d S85-30d

110.4 110.5 110.6 110.7 110.6 111.0 111.3

10.0 9.5 9.3 9.2 9.0 8.7 8.7

74.5 75.6 76.9 75.2 73.6 75.8 78.6

101.5 101.0 101.2 101.2 101.2 101.3 101.6

9.2 7.6 7.3 7.6 7.6 6.9 6.6

22.5 21.6 20.9 22.8 23.6 21.7 19.5

91.8 91.9 92.1 91.9 91.5 91.6 92.0

9.8 7.2 5.6 5.5 5.8 5.8 5.7

3.0 2.8 2.2 2.0 2.8 2.5 1.9

S90 S90-0.25h S90-1h S90-1d S90-7d S90-30d

109.9 110.6 110.7 110.8 111.0 111.0

10.8 9.4 9.7 9.0 8.7 8.6

68.0 74.2 77.3 75.6 75.9 77.0

100.2 100.9 100.8 101.3 101.4 101.4

8.5 7.8 7.6 7.1 7.1 6.8

17.1 22.6 20.6 22.4 22.3 20.8

91.7 91.0 92.1 92.1 91.8 92.0

9.4 7.2 4.6 5.5 5.5 5.8

S58 S58-0.25h S58-1h S58-4h S58-1d S58-7d S58-30d

108.8 110.7 110.7 110.7 111.0 111.2 111.4

11.0 9.1 8.7 8.5 8.3 8.3 8.4

44.4 64.6 70.5 69.8 71.7 74.8 78.1

99.5 100.7 101.0 101.1 101.2 101.5 101.7

7.9 7.1 6.8 6.8 6.6 6.8 6.9

17.5 25.4 25.2 26.3 25.6 23.0 20.8

92.0 92.0 91.9 91.8 92.1 91.4 92.4

9.5 8.5 6.5 6.5 6.0 4.5 4.3

Q1Ca

−δ (ppm)

fwhm (ppm)

fraction (%)

−δ (ppm)

fwhm (ppm)

fraction (%)

7.3 3.2 2.1 2.0 1.8 2.2

83.0

9.5

5.0

75.0

9.7

2.6

16.1 7.2 4.3 3.9 2.7 2.2 1.1

83.8 81.6

9.3 8.5

16.0 2.8

77.0

9.4

6.0

a

Deconvolution results of the 29Si MAS NMR spectra obtained by direct single-pulse excitation. The fractional populations are representative for the total silicate speciation of the MBG. The data uncertainties (±1σ) are as follows: chemical shifts (δ; ± 0.25 ppm); full width at half-maximum height (fwhm; ± 0.3 ppm); fractional populations (“fraction”; ± 2 percentage units). bBoth Q2H and Q3Ca species contribute to this resonance: in the pristine S90 and S58 MBGs, a significant part of the total fractional population stems from Q3Ca groups, whereas this contribution is negligible in the pristine S85 sample and all S85-τSBF/S90-τSBF specimens, as well as in the S58-τSBF samples with τSBF ≳ 24 h. 1 + 6H+ → 2Q 3 + 2H O + 3Ca 2 + [2Q 1 →2Q 3 ] 2Q Ca H H Ca 2

for increasing SBF immersion intervals up to 1 week. In this section, we only consider the results represented by the solid symbols and relevant for the MBG concentration of 0.6 g/L. All silicate groups at the S90 surface have released their Ca2+ ions within the first 15 min. In the S58 structure, the Q1Ca and Q2Ca species are completely removed during 15 and 60 min, respectively. The Q2H population at the S85 surface remains constant (≈3%) during the first 15 min of SBF exposure (see Figure 4). In contrast, for the S90 and S58 specimensfor which the underlying NMR signal (δSi ≈ −92 ppm) also carries contributions from Q3Ca speciesthe net Q2H/Q3Ca population is reduced to half that of the corresponding pristine S90 (7%) and S58 (16%) samples. Yet, the absolute change is only drastic at the S58 surface. Note that each Ca2+ cation released must be balanced by an exchange with two protons to form either one Q2H group or two Q3H groups [see Figure 1(a, c)]: 3 + 2H+ → 2Q 3 + Ca 2 + 2Q Ca H

(1)

2 + 2H+ → Q 2 + Ca 2 + Q Ca H

(2)

(4)

Equations 3 and 4 reflect stage 3 of the HM. By the coupling of eqs 1−4, the Ca2+ release leads to an increased Q3H population. In contrast with the S85 surface that is essentially devoid of Ca-associated groups (as witnessed by insignificant changes in its Q3H and Q2H populations after 15 min of SBF soaking), an increase of the Q3H reservoir is indeed observed in the S90-0.25h and S58-0.25h specimens relative to their pristine counterparts (Figure 4). Although the extents of completion of the condensation reactions 3 and 4 are unknown, the increase in the Q3H fraction matches reasonably well the sum of Q1Ca and Q2Ca populations initially present in S90. However, the corresponding elevation Δx(Q3H) ≈ 0.08 in the S58-0.25h sample is significantly smaller than the total {QnCa} content in S58. Regarding the two potential Q3Ca/Q2H contributions to the NMR peak around −92 ppm, we interpret the difference in the total signal fraction between the pristine S90/S58 samples and their S90-0.25h/S58-0.25h counterparts in Table 2 as reflecting the fraction of Q3Ca groups present at the surface out of the total amount of Q3Ca and Q2H species in the corresponding pristine MBG. The remaining of the S58-0.25h structure comprises Q2H groups, as well as Q3Ca/Q2Ca species within the pore-wall, whose n⩽3 Ca2+ ions served as network modifiers to drive Q4→QCa 2+ conversions. These Ca species are released much slower because they only become exposed to the solution gradually as the silicate network degrades/“dissolves”: the slow dissolution process followed by the reaction eq 3 account for the “tail” of the x(Q2H) curve in Figure 4e for τSBF > 1 h, which has no

The consumption of H+ ions accounts for the concurrent elevation of the pH-value of the solution.31 Note, however, that the Q H2 groups thereby formed may undergo further condensation reactions into Q3H groups, according to 2SiO2 (OH)2 → 2SiO3(OH) + H 2O [2Q H2→2Q H3]

(3)

whereas Q1Ca species may condense in several ways when their Ca2+ ions are removed, e.g. F


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Figure 4. Plots of the as-indicated QnE populations against the SBF exposure interval τSBF. The solid symbols correspond to the data listed in Table 2 from the specimens deriving from S85 (left panel), S90 (mid panel), and S58 (right panel), each soaked at a concentration of mMBG/V = 0.6 g/L. The open symbols represent data for mMBG/V = 20 g/L, reproduced from Gunawidjaja et al.41,42 Note the use of a horizontal logarithmic time scale and that the vertical scale of all plots employs the same span of values (except for (k) that uses a twice as large vertical range). Note that the data in (d) and (e) represent populations from both Q2H and Q3Ca species; see Table 2. No data are available for the S58-τSBF series with mMBG/V = 20 g/L.

mesoporous silica. Moreover, all three MBGs manifest very similar induction periods (≈24 h) for HCA formation: the pristine MBG composition only influences the amount of HCA formed, which increases proportionally with the initial Caand particularly Pcontents of the MBG.31 We stress that the surface areas and the mesoporous arrangements differ among all S90/S85/S58 specimens (see Table 1), meaning that the HCA formation rate is (somewhat counterintuitively) independent on these textural properties.31 The nearly identical set of {QnE} populations at all MBG-τSBF surfaces partially rationalizes the invariance of the qualitative HCA formation characteristics. Hence, whereas a suf ficiently large surface area and a continuous mesoporous network ensure that the initial HM reactions are accelerated relative to those of “bulk” meltprepared BGs of low surface area,41 further expansions of the MBG surface area do not shorten the induction period for HCA formation significantly. The present findings also firmly establish and rationalize the inferences of Garcı ́a et al.11 that the HCA formation is independent of the precise mesoporous arrangement (note that those evaluations employed the high mMBG/V = 20 g/L loading, where HCA formation was largely restricted by the insufficient P amount in the medium; see section 5.2). Also pertaining to the nearly identical long-term networkdegradation characteristics among the S90/S85/S58 structures is the very similar {δ(QnE), fwhm} NMR parameters of their respective QnE groups; see Table 2. Indeed, Figure 3 reveals almost indistinguishable 29Si NMR spectra from the three

counterpart during the soaking of S90 and S85 due to the absence of Q2Ca (and Q3Ca) moieties within their pore-walls. Noteworthy, during prolonged SBF immersion, the QH2 groups are gradually depleted at all MBG-τSBF surfaces (see Figure 4). However, a strong net reduction of the Q2H/Q3Ca populations is only observed for the S58-deriving specimens. After one month of SBF exposure, the Q2H population levels out at ≈2% out of the entire silicate speciation for all three MBG series; see Table 2. The equalization tendency of the silicate speciation among the MBGs at longer immersion intervals (≳7 days)regardless of their initial cation compositionsalso applies to the Q4 and Q3H populations, whose “asymptotic” values are ≈78% and ≈21%, respectively (Table 2). This implies net increases (decreases) of the Q3H (Q4) populations of the S90-τSBF and S58-τSBF structures with τSBF ⩾ 7 days (which are very significant for the S58-deriving MBGs), whereas the “asymptotic” {x(QnH)} values are close to those of the pristine S85 composition, as depicted by Figure 1. This feature is also mirrored by relatively modest variations in the fractional populations of the silicate species of the S85-τSBF samples throughout the entire SBF soaking period (Figure 4). 4.1.3. Network Degradation is Independent of MBG Composition and Texture. The fast leaching of Ca2+ cations from the silicate surfacesupplemented by the release of Ca2+/ PO3− 4 species from the CaP clustersboth of which occurs within the first hour of MBG immersion, implies that already from the first day of soaking, the silicate-network degradation closely mimics a congruent dissolution of essentially pure G


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Figure 5. 1H→29Si CPMAS NMR spectra (black traces) from pristine (top row) and SBF-exposed MBG-τSBF specimens of S85 (left panel), S90 (mid panel), and S58 (right panel). Other labels are as in Figure 3.

confirming that all MBG surfaces are dominated by interconnected Q3H groups both before and after SBF treatment. Figure 6 plots the alteration of each QnE fractional population (solid symbols) for progressively increased τSBF periods up to 1 week. All qualitative trends are identical to those inferred from the single-pulse-derived NMR data shown in Figure 4. However, they now emerge more clearly once the NMR signals from the dominating Q4 species that are located inside the pore-walls are essentially removed. The 29Si NMR responses appearing at δ≈−111 ppm (Figure 5) now stem from 29Si nuclei in Q4 species at/near the surface, where they interconnect with the various silanol groups. Throughout the SBF exposure period, the Q4 populations remains relatively constant and typically amount to 21−26% out of the surface {QnE} speciation. The observed net trends for τSBF ≳ 7 days are as follows: (i) a complete Ca2+ leaching during the first 15 min (60 min for S58); (ii) a decrease of the QH2 populations and an accompanying increase of the Q3H counterparts for periods up to ≈1 h, with the more evident decrease of the Q2H species also at the S85 surface being the primary distinction between the data of Figure 6 and that of Figure 4 discussed previously; (iii) all three S90/S85/S58 sets of SBF-exposed samples revealing very similar NMR parameters for each given QnE group; see Table 3.

MBG-30d specimens. Across the 30 days of MBG soaking, only a minor decrease by around 1−1.5 ppm are observed for the (average) chemical shift of the Q4 groups within each MBG series. Since the 29Si chemical shift decreases concomitantly with the Si−O−Si bond angle,47,48 the trend likely reflects a slight reduction in the bond angles during the silicate-network degradation. For the S90 and S58 samples, a similar trend is discernible for the chemical shift of the Q3H groups (see Table 2), possibly originating from minor bond-angle reductions accompanying the release of Ca2+ from the surface; indeed, the shift alterations are largest for the Ca-richest S58 sample. 4.2. MBG Surface Signatures. Here we discuss the evolution of the surface silicate speciation of each S90/S85/S58 deriving specimen for increasing τSBF intervals. Owing to the Ca gradient across the pore-wall (section 3), the dominance of the Q4 groups coupled with a low 29Si NMR spectral resolution mask the signals from the low-abundance {QnH} and {QnCa} moieties. Hence, corroboration of the previous findings from the directly excited 29Si NMR spectra by the surface-specific probing of 1H→29Si CP NMR experimentation is valuable. A selection of 29Si NMR spectra recorded by 1H→29Si CP are collected in Figure 5, which also displays the peaks of the various QnE components. Table 3 compiles the best-fit NMR parameters. All NMR spectra emphasize signals from the Q3H (major population) and Q2H (minor population) surface groups, H


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The Journal of Physical Chemistry C Table 3. Best-Fit 29Si MAS NMR parameters (1H→29Si CP)a Q4

Q2H + Q3Ca b

Q3H

Q2Ca

Q1Ca

sample

−δ (ppm)

fwhm (ppm)

fraction (%)

−δ (ppm)

fwhm (ppm)

fraction (%)

−δ (ppm)

fwhm (ppm)

fraction (%)

−δ (ppm)

fwhm (ppm)

fraction (%)

−δ (ppm)

fwhm (ppm)

fraction (%)

S85 S85-0.25h S85-1h S85-1d S85-7d

109.8 110.3 110.4 110.7 111.0

10.4 9.1 8.7 8.4 8.0

24.0 21.8 21.0 21.2 21.9

100.4 100.7 100.7 100.9 101.1

7.4 6.8 6.8 6.4 6.3

58.0 64.8 66.5 67.7 67.6

91.4 91.8 91.8 91.9 91.9

7.3 6.7 6.7 6.1 5.9

15.4 13.4 12.5 11.1 10.5

82.0

7.0

2.6

S90 S90-0.25h S90-1h S90-1d S90-7d

109.7 110.3 110.5 110.7 111.0

10.0 9.2 8.9 8.2 8.2

21.1 24.3 22.8 21.4 22.8

100.8 100.6 100.7 101.0 101.1

7.7 7.0 7.0 6.6 6.4

52.4 63.2 66.0 67.6 67.3

91.4 91.8 91.9 91.9 91.9

7.4 7.1 6.8 6.3 5.7

16.3 12.5 11.2 11.0 9.9

83.5

8.5

7.7

76.4

7.3

2.5

S58 S58-0.25h S58-1h S58-4h S58-8h S58-1d S58-7d S58-30d

108.2 110.1 110.6 110.6 110.6 110.8 111.1 111.2

11.5 8.6 8.6 8.5 8.5 8.4 8.0 8.3

18.1 21.3 24.2 24.4 25.0 24.6 24.0 27.1

100.0 100.8 101.0 101.1 101.1 101.2 101.3 101.3

7.5 6.6 6.5 6.3 6.3 6.3 6.3 6.3

33.4 56.3 64.1 64.4 64.4 65.8 66.6 64.8

91.1 92.0 92.0 92.1 92.1 92.0 92.1 92.2

10.1 8.2 7.2 7.1 7.0 6.4 6.2 6.0

28.0 16.8 11.7 11.2 10.6 9.6 9.4 8.1

82.4 83.0

9.0 8.4

15.4 4.1

76.0 76.0

8.1 8.3

5.1 1.5

a

Deconvolution results of the MAS 29Si NMR spectra obtained by 1H→29Si CP. The fractional populations are representative for the MBG surface. The notation and experimental uncertainties are as in Table 2. bBoth Q2H and Q3Ca species contribute to this resonance: for the pristine S90 and S58 MBGs, a significant part of the total fractional population stems from Q3Ca groups, whereas this contribution is negligible at the surfaces of the pristine S85 sample and all S85-τSBF, S90-τSBF, and S58-τSBF specimens with τSBF≳1 h.

Altogether, the silicate surface-reactions active during τSBF ⩽ 1 h lead to net conversions of QnCa and Q2H groups into Q3H moieties [see eqs 3 and 4], reflecting the “repolymerization processes” constituting the third HM stage.1 These reactions are essentially completed during the first 30−60 min of immersion and only minor extents of (net) interconversions among the various QnE species occur for longer time periods. While they underlie a “silica-gel” formation at melt-prepared BG surfaces, a modifier-leached MBG surface is already “gellike” and the first three HM stages do not constitute ratelimiting reactions for MBGs41,42 (provided that the MBG dose is not too high; see below); this hypothesis of Gunawidjaja et al.41 was subsequently also reached by a DFT modeling study of gel-prepared glasses.46 Noteworthy, as for the quantitative {QEn} populations representative for the entire MBG-τSBF structure (see Table 2 and Figure 4), the mesoporous surface after SBF exposure periods beyond 24 h manifests very similar silicate speciations, regardless of the precise cation composition of the pristine MBG. After 7 days of immersion, the data in Table 3 reveals a common {x(QnE)} profile for the three MBG-30d specimens, which comprise approximately 10%, 67%, and 22% of Q2H, Q3H, and Q4 moieties, respectively. For extended immersion intervals τSBF ⩾ 7 days and disregarding the Q4 species, the surface involves exclusively Q3H and Q2H groups, with around 6−7 Q3H species per Q2H moiety, as depicted by Figure 1c.

NMR-derived fractional populations when employing a significantly higher MBG loading of mMBG/V = 20 g/L but otherwise identical experimental procedures/conditions; the data are reproduced from Gunawidjaja et al.41,42 Noteworthy, for the MBG loading condition of mMBG/V = 20 g/L, the HCA formation was observed to depend strongly on each initial MBG cation composition: solely S85 formed HCA, which was detected both by 31P NMR and powder XRD after 24 h of SBF soaking.41 However, the ACP→HCA turnover was significantly lower41 relative to that observed for the corresponding mMBG/V = 0.6 g/L soaking.31 For the latter condition, 31P MAS NMR estimated roughly equal relative (molar) fractions of ACP and HCA after 24 h of soaking,31 whereas the corresponding numbers at the high concentration were 22% HCA and 78% ACP.41 Similar HCA fractions were only observed at τSBF = 7 days for the two distinct mMBG/V cases.31,41 However, while the S85 specimen exhibited delayed HCA formation when immersed at a high concentration, neither of the S90 nor S58 MBGs revealed any trace of HCA during one week of SBF exposure (as concluded independently by 31P NMR and powder XRD32), notwithstanding that ACP deposits were observed at both S58 and S90 surfaces. Yet, for the more dilute soaking, a short induction period of ≈24 h for significant HCA formation resulted for all MBG samples, while the Ca/Prichest S58 composition (Table 1) revealed the largest amount of HCA after 7 days of immersion compared with the S85/S90 specimens.31 In the following, we examine the trends of the various 29Si NMR-derived {x(QnE)} populations observed for the mMBG/V = 20 g/L MBG-loading condition relative to those obtained for 0.6 g/L. We focus our comparisons on the 1H→29Si CPMAS data of Figure 6 because they provide more transparent trends for the minor QnH and QnCa species.

5. DISCUSSION 5.1. MBG Concentration Effects of the Silicate Network Degradation. All silicate speciations discussed thus far concerned MBG immersions with a mass over volume ratio of mMBG/V = 0.6 g/L. The open symbols in Figures 4 and 6 represent the corresponding single-pulse and 1H→29Si CP I


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Figure 6. QnE populations reflecting the silicate surface and obtained by deconvoluting the 1H→29Si CPMAS NMR spectra of Figure 5; see Table 3. Other labels are as in Figure 4. Solid and open symbols represent results for concentrations of mMBG/V = 0.6 g/L and mMBG/V = 20 g/L, respectively. Note that the span of all vertical scales are identical throughout, except for the data of S58 in the right panel, whose range is twice that of the left/mid panels.

For the Ca-poor S85 and S90 samples, qualitatively similar trends are observed for the SBF soaking at both low and high mMBG/V ratios. The main distinction constitutes an overall retardation of the surface reactions for the 20 g/L scenario: while all MBG-τSBF surfaces are devoid of Ca2+-associated groups after 1 h for the low MBG-loading case, Q1Ca/Q2Ca surface species persist up to ≈24 h of soaking at mMBG/V = 20 g/L (even at the S85 surface); see Figure 6. Incidentally, the much slower Ca2+ release allows for monitoring the expected Q2Ca → Q2H conversion (eq 2) at the S90-τSBF surfaces; it manifests as a minor increase of the Q2H population between 30 min and 4 h (Figure 6e; open symbols). For the more dilute soaking, this process proceeds much faster and remains undetected as it becomes obscured by the concurrent reduction of the Q2H population when these groups condense into Q3H moieties via eq 3. The Q2H→Q3H repolymerization reactions that are very pronounced already beyond 15 min for the dilute mMBG/V = 0.6 g/L case are not obvious until τSBF ≈ 4 h for the more concentrated solutions involving S85 or S90. Moreover, the net increase of the Q3H population over 7 days of immersion is lower for the 20 g/L scenario, particularly for the S90-τSBF series; see Figure 6(g, h). The corresponding single-pulse derived {QnE} populations of Figure 4 overall corroborate the inferences from the CP NMR data, notably that the silicate speciations associated with S85 (whose surface displays negligible amounts of QnCa groups because nearly all Ca2+ is located in the CaP clusters) are less affected by the precise

MBG concentration in the solution relative to the corresponding {QnE} profiles for S90 [Figure 4(i, j)]. However, while the mMBG/V-dependent surface-reaction profiles are qualitatively similar for each Ca-poor S90/S85 MBG, a completely different SBF-soaking behavior results for the Ca-rich S58 specimen at each concentration (Figure 6). For the 20 g/L scenario, an insignificant Ca leaching is observed over the entire week of soaking: while the Q1Ca population decreases slightly for τSBF ≤ 3 days, that of Q2Ca remains essentially constant (or even manifests a minor increase). The negligible Ca2+ release is reflected in a minute increase in the Q2H silanol reservoir, whereas that of Q3H is merely decreased. Moreover, the silanol repolymerization processes are quenched completely: notwithstanding a minor reduction of the networkpolymerization degree over 1 week, as opposed to the 0.6 g/L scenario, the low-connectivity QnCa (n ⩽ 2) groups do not enter the supersaturated solution but merely remains at the solid surface, where they hamper further network degradation. The herein observed inhibiting of the silicate reactions is analogous to the report by He et al.68 on the dissolution mechanism of mesoporous silica in SBF, where precipitation of Ca/Mg silicates were shown to significantly retard further SiO2 degradation after a few hours. Consequently, the schematic S58 surface of Figure 1a remains much more representative for all S58-τSBF counterparts with mMBG/V = 20 g/L than the “typical” MBG surface observed at long SBF immersion periods and conveyed by Figure 1c. As such, the surface at any S58-τSBF J


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The Journal of Physical Chemistry C phase may be viewed as a “slab” of Q1Ca, Q2Ca, Q2H, and Q3H species, where the Q3H silanols no longer dominate the surface speciation for τSBF ≳ 7 days, but Q2H and Q3H groups merely coexist in nearly equal amounts; see Figure 6(f, i). 5.2. Consequences for the HCA Formation. Here we comment on the consequences of the mMBG/V-dependent surface reactions for the subsequent ACP formation (step 4 of the HM) and its crystallization into HCA (stage 5). Obviously, an improper choice of a too concentrated in vitro medium may drastically alter the silicate surface reaction from a “universal” long-term behavior (∼days) that is independent of the precise MBG composition (whose Ca2+ content is reflected solely by the extent of Ca2+ release during the first hour of SBF soaking) to either markedly delayed (for S90/S85) or even absent (for S58) silicate network transformations. To understand the distinctly different HCA formation characteristics among the three MBG specimens on their contact with solutions at low/high concentrations (mMBG/V), one must consider two main aspects: (i) their potentially distinct mechanisms for release of Ca2+/PO3− ions, which 4 depend both on the amount of surface-associated Ca species and the different location of the CaP clusters in Ca-rich/poor MBGs, and (ii) the respective relative {Ca, P} reservoirs initially present in the MBG versus the SBF. For the high loading condition mMBG/V = 20 g/L, nearly all of each total Ca2+ and PO3− 4 reservoir resides in the MBG itself, regardless of its precise cation composition: considering the experimentally analyzed cation composition of Table 1, the Ca contents of S85/S90 and S58 accounts for ≈93% and 98%, respectively, whereas >95% of P stems from the P-bearing MBGs. Consequently, the formation of the ACP/HCA surface layer critically relies on an efficient ion release from the MBG. In contrast, for the lower concentration of mMBG/V = 0.6 g/L, the aqueous medium mainly contributes with the building blocks of the ACP/HCA phases: now ≈29% and ≈59% of the total Ca reservoir stems from the S90/S85 and S58 MBGs, respectively, whereas the corresponding numbers for PO3− 4 are 36% for S85 and 46% for S58. The significant, yet not detrimental, reduction in the HCA amount formed at the S85 surface for the high MBG-loading condition is readily explained by the rapid dissolution of the relatively large (≈1 nm) surface-associated CaP clusters (that account for all P, and nearly all Ca2+, within S85). They dissolve and reform as a surface layer of ACP, which subsequently converts into HCA, albeit overall slower and in a significantly ions may be lower amount, as very few additional PO3− 4 incorporated from the solution. The absence of HCA at the S90 surface for the high 20 g/L concentration is readily understood by its absence of P: the release of Ca2+ from the surface over the first few hours leads to a complete consumption of the (insignificant) amount of PO3− 4 ions present in the initially supersaturated SBF. As confirmed by weak and broad 31P MAS NMR signals (not shown), a thin layer of ACP forms across the large S90 surface. However, the total depletion of PO3− 4 ions in the SBF impedes the ACP→HCA crystallization; see refs 31, 32, 57 for further discussions. Yet, while the main reason for the hampered crystallization was identified in our earlier work,32 some of the underlying reasons for the distinct S85/S58 responses and their dependence on MBG concentration in the SBF only became transparent with the availability of the distinct evolution of the {x(QnE)} populations against τSBF at both low and high mMBG/V values.

A scenario analogous to that of S90 applies to S58 at high concentrations: despite that its P content is larger than that of S85, a non-negligible fraction of much smaller CaP clusters are embedded within the silicate network (Figure 2). Notwithstanding that some PO3− and Ca2+ species are released 4 immediately on the exposure of S58 to the SBF, the substantial Ca2+ release yields a very rapid ACP formation that (analogously to the case of S90) consumes all PO3− 4 available phosphate ions from the solution. The insignificant surface reactions preclude further dissolution, which becomes a crucial prerequisite for exposure of the (small) CaP clusters to the SBF and thereby further release of phosphate species. In contrast, at the lower mMBG/V = 0.6 g/L concentration, the ACP→HCA conversion is not impeded due to the continuous silicate network dissolution that occurs readily due to the fragmented S58 silicate network,31,42 thereby ensuring that a gradually expanded number of CaP clusters enter the solution for prolonged time intervals. The results discussed herein underscore the importance of our recent suggestion31,32 that the m/V ratio employed for SBF testing of a biomaterial must be selected such that the total Ca and P contents of the solution (far) exceeds their counterparts in the biomaterial itself. Only then an unrestricted HCA growth may occur that is independent (or only weakly dependent) on the cation composition of the tested biomaterial, thereby avoiding potentially very misleading outcomes of the in vitro bioactivity assessment. See Turdean-Ionescu et al. for further discussions.31 5.3. Bearings for Future MBG Design. Future MBG design may benefit from further exploration of the two “S58” and “S85” base compositions: both provide efficient HCA formation and they only respond differently during the first few hours of contact with surrounding solutions, stemming from their distinct Ca contents and their differing CaP cluster locations relative to the pore-wall surface (see Figure 2). Nonetheless, the S58/S85 long-term responses are identical, provided that the MBG concentration in the solution is not too high. The incorporation of P likely also assists the HCA formation by increasing the supersaturation of the plasma under in vivo conditions, although the effects are presumably less pronounced in a circulating and replenished medium than those occurring for our testing condition. Indeed, the silicate reactions and subsequent HCA formation observed for the low mMBG/V = 0.6 g/L ratio are expected to be more representative for in vivo applications than the 20 g/L counterpart. “S58”9 is a direct MBG analogue of a previously introduced sol−gel BG composition,5 with the ordered porous network of the MBG constituting its primary distinction to its predecessor. Regardless of the precise preparation route, this cation formulation is well-documented to be advantageous both in vitro and in vivo applications3−5,7−9 and further bioactivity improvements are unlikely to be achieved by minor composition alterations. However, a promising MBG series that is hitherto essentially unexplored concerns “S85-like” MBGs that matches the relative Ca2+ and PO3− 4 contents to the Ca3(PO4)2 stoichiometry. The formulation in oxide equivalents (mol %) is 3xCaO−(100 − 4x)SiO2−xP2O5, or equivalently, (1 − 4x) SiO2·x[Ca3(PO4)2]. Yan et al.7,8 reported one such MBG composition: 15CaO−80SiO2−5P2O5. A slight excess of Ca might assist the ACP formation further. The limiting amount x of incorporable CaP clusters is unknown and its determination is one promising line for future investigations. We note that the original “S85” composition9 is clearly nonideal, because its Ca2+ K


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a similar silicate surface-reaction scenario as S85, no HCA forms for the same reasons as for S58. We stress that the observed mMBG/V-dependence of the surface-reaction mechanism and its subsequent HCA formation are artifacts of the SBF testing itself: it reminds that care should be exercised when selecting the (M)BG load in the solution during assessments. At high MBG concentrations, the favorable property of a large surface area merely becomes a drawback, because the outcome of the SBF testing is strongly perturbed when too few PO43− ions are available in the solution. Nevertheless, the m/V-dependent Ca release and QnE-group interconversions are expected to be less pronounced in a circulating medium than what was observed herein in the absence of a replenished solution. The silicate reactions and the subsequent HCA creation resulting for the low mMBG/V = 0.6 g/L concentration presumably better mimics those encountered in vivo. It is also likely that the dependence of the HCA formation on the MBG composition31 becomes less pronounced under in vivo conditions. Moreover, provided that the specific surface area is suf f iciently large (presumably ≳200 m2/ g) and the ordered and continuous mesoporous channel system allows the solution to reach most/all of the surface rapidly, then the details of the surface area and textural properties become immaterial.

content is too low to charge-balance all P as orthophosphate groups, meaning that the entire P reservoir may not be exploited as (desirable) CaP clusters. The excess phosphate content partially form bonds to Si (i.e., P−O−Si),63 while the remaining is lost during synthesis, which is probably the reason why higher P losses are consistently observed in the synthesis of S85 relative to that of S58; see Table 1 and refs 41, 42, and 63.

6. CONCLUDING REMARKS The present results highlight the complex interplay between (i) the Ca content of the MBG and the associated size/location of the CaP clusters of amorphous calcium phosphate in the porewalls and (ii) the mass of MBG powder per unit SBF volume (mMBG/V) used for in vitro testing: these factors together dictate the relative contributions from the MBG and the SBF to the total Ca and P reservoirs available for ACP/HCA growth. Two concentration regimes may be identified: (A) For a low mMBG/V ratio, the Ca/P species mainly stem from the SBF, while the low MBG concentration in the solution allows for both an unlimited silicate network degradation and ACP formation, as well as an unrestricted ACP→HCA crystallization. The precise release mechanisms of Ca2+/PO3− 4 ions differ between the Ca-poor S85 and the Carich S58 MBGs evaluated herein. In the case of S85, the Ca leaching from the silicate surface-portion is essentially absent, but both Ca and phosphate species are released from the surface-located and rapidly dissolving CaP clusters. For S58, on the other hand, Ca2+ ions are primarily released from the QnCa surface groups, but the much smaller CaP clusters within the silicate pore-walls provide both Ca and P as they gradually becomes exposed to the solution when the pore-walls degrade. However, once the majority of the various Ca contents from the distinct sources have entered the solution during the first 60 min, all S90/S85/S58 MBGs then manifest identical surfacereaction mechanisms and a “universal” network degradation behavior, representative for the dissolution of essentially neat mesoporous silica. This scenario apparently applies regardless of the precise initial composition of the MBG and textural properties, although more systematic studies are required to firmly prove this hypothesis. The total amount of HCA generated over the first few days increases concurrently with the precise Ca and P contents of the MBG because it contributes differently to the total {Ca, P} reservoirs available for ACP/HCA formation.31 (B) In contrast, for a high mMBG/V ratio, the Ca and P species predominantly reside in the MBG and their efficient release becomes a critical prerequisite for ACP/HCA generation. For the S85 structure, this is not detrimental as the Ca2+/PO3− 4 components of the large and surface-associated CaP clusters are readily released; nevertheless, all silicate surface reactions retard markedly. However, a high loading of the Ca-rich S58 specimen in the SBF precludes any significant release of both Ca and silicate species from the surface into the already highly supersaturated aqueous medium: subsequent to a very rapid (yet partial) Ca2+ leaching during the first few minutes, the substantial number of nucleation sites at the MBG surface leads to an immediate ACP formation, accompanied by a total consumption of the Ca2+/PO3− 4 species available in the solution, whereupon the ACP→HCA conversion inhibits due to an insufficient number of phosphate ions.31,32,57 Moreover, also the surface reactions at S58 quench and no further silicatenetwork degradation occurs. While the P-free S90 MBG reveals

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AUTHOR INFORMATION

Corresponding Author

*(M.E.) E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Swedish Research Council (Projects VR-NT 2010-4943 and 2014-4667), the Faculty of Natural Sciences at Stockholm University, Ministerio de Ciencia e Innovación (Project MAT2012-35556), Ministerio de Economı ́a y Competitividad (Project MAT2013-43299-R) and the Agening Network of Excellence (CSO2010-11384-E). C.T.-I. was supported by a postdoctoral grant from the Carl Trygger Foundation. We gratefully acknowledge NMR equipment grants from the Swedish Research Council and the Knut and Alice Wallenberg Foundation. We thank Renny Mathew for experimental help.

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REFERENCES

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