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Surface Transformations of Bioglass 45S5 during Scaffold Synthesis for Bone Tissue Engineering Sara Abdollahi, Alvin Chih Chien Ma, and Marta Cerruti* Biointerface Laboratory, Department of Mining and Materials Engineering, McGill University, 3610 University Street, Montreal, Quebec H3A 2B2, Canada S Supporting Information *

ABSTRACT: In physiological fluid, a layer of hydroxycarbonate apatite, similar to bone mineral, develops on the surface of Bioglass 45S5. Collagen from the surrounding tissue is adsorbed on this layer that attracts osteoblasts, and favors bone regrowth. Bioglass is therefore an osteoinductive material. Still, due to its brittleness, the glass alone cannot be used to heal large bone defects. To overcome this issue, Bioglass is used to form a composite scaffold with poly(D,L-lactide) (PDLLA), a biodegradable polymer. The goal of this work is to understand Bioglass reactivity throughout scaffold fabrication via a low-temperature route, the solvent casting and particulate leaching technique. Changes in Bioglass (especially its surface) are susceptible to occur both while in contact with the processing fluids and potentially through a reaction with the surrounding polymeric matrix. Here we analyzed the surface changes of three different Bioglass samples: (i) as-received, (ii) treated in solutions that parallel those used in scaffold fabrication, and (iii) extracted from the scaffolds. We showed that extracted, just like treated, Bioglass deviates from the as-received, but to a larger extent. X-ray photoelectron and infrared spectroscopy support the theory that Bioglass surface was modified not just through contact with the solutions in scaffold fabrication, but upon an interaction with the polymeric matrix. The polymer network slows down the Na+/ H+ exchange between Bioglass and water used to leach salt particles to create pores within the scaffold. Changes in surface properties affect the bioactivity of Bioglass and thus of the composite scaffolds, and are therefore critical to identify. which can be used as scaffolds to fill bone defects. Composite scaffolds, which are porous like bone, allow nutrients and oxygen to reach the osteoblasts seeded or migrated within them, and play a central role in bone tissue engineering.10,11 The structures are expected to temporarily sustain load at the site of injury and gradually biodegrade as the surrounding tissue regenerates. The addition of Bg in a polymeric scaffold incites host tissue regrowth,12−18 favoring cell proliferation and production of extracellular matrix components,15 and improving bone defect filling compared to scaffolds that do not contain Bg.19 Bg polymeric composites can be fabricated using several techniques.11,20−23 For example, Blaker et al.20 investigated the effect of high temperatures involved in a coextrusion and compression molding method to construct Bg-filled poly(D,Llactide) (PDLLA) scaffolds. The elevated processing temperatures triggered a reaction at the interface between Bg and the PDLLA matrix, giving rise to carboxylate salt byproducts within the scaffold. One of the simplest and most common methods to make scaffolds is solvent casting and particulate leaching. In this technique, a porogen, usually salt,24 is added to the glass-fused

1. INTRODUCTION Bioglass 45S5 (Bg) is a bioactive glass. When immersed in physiological fluid, a layer of hydroxycarbonate apatite (HCA), similar to the bone mineral component, forms on its surface.1−3 Formation of HCA on Bg surface begins by an exchange of ions. With a composition in weight percent of 45% SiO2, 24.5% Na2O, 24.5% CaO, and 6% P2O5, the first ion that leaches from Bg is sodium (Na+), which is exchanged with protons (H+) from the surrounding solution. The rapid Na+/ H+ exchange increases the solution pH, and the Bg silica network starts to dissolve. As Si−OSi bridges rupture, silanol groups form and gradually condense into a silica-rich layer. The silica-rich surface layer facilitates the migration of Ca2+ and PO43‑ from the Bg bulk to the surface and leads to the formation of a calcium phosphate (Ca−P) rich layer. Crystallization of the Ca−P layer, as well as the integration of OH− and CO32‑ from the solution, leads to the formation of HCA on Bg surface.2−7 Tissue fibrillar collagen then integrates with the HCA layer, and as osteoblasts are recruited, a biological bond is established between the glass and the newly formed bone. Bg is thus osteoinductive and is currently used in dental and orthopedic applications, for example, to promote bone regrowth in small, non-load-bearing structures, such as the inner ear bones.1,8 In spite of numerous advantages, Bg has a low fracture toughness.9 This problem can be overcome by creating composite Bg-polymeric structures, © 2013 American Chemical Society

Received: November 21, 2012 Revised: January 8, 2013 Published: January 10, 2013 1466

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% in relation to the weight of PDLLA (Table 1). Each scaffold was replicated three times for statistical analysis. The average Bg particle

polymeric solution. The mixture is then cast in a mold and, once solidified, immersed in an aqueous setting to leach out the porogen, thus creating a porous scaffold. Despite the lowtemperature procedure, Bg risks being modified through exposure to different solvents involved in the scaffold processing, in particular, acetone and water. Bg surface is well-known to change in water, and this transformation is pH dependent. Alterations of Bg surface structure have even been characterized after dissolution experiments in TRIS buffers of different pH.1,8,25 Total glass reconstruction, that is, release of cations from the glassy network and formation of both a silica rich and calcium-phosphate rich layer, is observed only for the buffer at pH 8.1 At pH values lower than 8, no calcium phosphate reprecipitation is observed; the glassy network breaks down, quickly releasing almost all of the phosphorus and calcium initially present.1 In the buffer at higher pH, an immediate precipitation of calcium phosphate after Bg immersion prevents further ion release.1 The rate and form of ion discharge from Bg has been explicitly shown to influence genes that promote the differentiation and proliferation of osteogeneic cells.26−28 Therefore, monitoring the premature reactivity of Bg during scaffold processing through a lowtemperature processing route is critical. Despite this, there are almost no studies reporting changes in Bg structure during lowtemperature scaffold processing. The only paper that mentions this issue was recently published by Cannillo et al.,29 who showed formation of calcite on the surface of Bg-rich scaffolds during the synthesis of Bg/polycaprolactone (PCL) scaffolds via the solvent casting and particulate leaching method. The prolonged Bg particle contact with the water used to remove the salt was speculated to promote calcite development. Although calcite can be used as bone substitute material,30 its bioactivity depends on its transformation into HA.31−38 Therefore, the direct transformation of Bg into HA is preferable than the deposition of calcite on Bg surface. The impact of calcite formation on the bioactivity of the composites was not further investigated by Cannillo et al., since their overall aim29 was to optimize the scaffold production parameters. In this study, we seek to determine if Bg reacts while a composite PDLLA/Bg scaffold is made by the solvent casting and particulate leaching method. We specifically focus on the glass surface modifications to establish (i) the effect of the processing solutions on Bg and (ii) if an interaction occurs between the glass and the polymer. Since Bg reacts in physiological fluids, we speculate that some changes might occur when Bg is in contact with the solutions used during processing. Therefore, we first treated Bg powders alone in solvents paralleling those used during scaffold fabrication: acetone, used to dissolve PDLLA, and water, used to leach out the salt from the scaffold. Then we compared the surface of this “treated” Bg with that of Bg extracted from the scaffold’s polymeric matrix to determine if the latter changed differently due to interactions with the polymeric matrix.

Table 1. Sample Designations of Bg Extracted, Water Treated, and the Scaffolds Based on Bg Contenta Bg (wt. %) 0 scaffold Bg extracted Bg water treated

PDLLA/ 0BG

15

30

40

50

PDLLA/ 15BG E15

PDLLA/ 30Bg E30

PDLLA/ 40Bg E40

PDLLA/ 50Bg E50

T15

T30

T40

T50

a

For Bg water treated samples, the amount of Bg in each scaffold is placed in 40 mL of water, the volume used during the salt leaching step.

size was 75 μm. Particles of sieved sodium chloride, NaCl (Fisher Scientific, ACS grade reagent, Fair Lawn, NJ), measuring 70−355 μm were then added to the mixture; the amount of salt was 94% in relation to the total weight of solid components. The resulting paste was cast in cylindrical aluminum molds of 3/8” diameter and 6/32” height and left overnight. Once solidified, the scaffolds were removed from the molds and immersed in parafilm-sealed beakers containing 40 mL of DI water for approximately 3 days to leach out the salt. Three different repeat beakers were prepared for each PDLLA scaffold of different Bg composition. pH measurements were taken at regular intervals after 0, 30, 90, 210, and 330 min, within the first 5.5 h of immersion. The silver nitrate test was used to confirm salt removal with a 2.5% (w/v) silver AgNO3 (Sigma-Aldrich, Inc., Oakville, ON) solution. Finally, the scaffolds were dried under vacuum for 2 h. 2.2. Bioglass Extraction. Bg extraction was carried out by stirring the scaffolds in acetone for 30 min. Acetone dissolved the PDLLA matrix. The resulting solution was decanted, and the Bg washed with acetone several times to remove the polymer residues. The “extracted” Bg samples were then air-dried for approximately 15 min, and these are referred to as E15, E30, E40, and E50 depending on the scaffold composition that they were extracted from (Table 1). 2.3. Bioglass Treatment. A first batch of Bg powders was immersed in acetone overnight to analyze changes occurring in the solvent used to dissolve PDLLA. The powders were then removed from the acetone and air-dried for 15 min. Specifically, 2.5 mg/mL Bg and acetone was used. A second batch of Bg powders was treated following the same procedure as the salt leaching step used during scaffold fabrication: the same amount of Bg present in the different scaffolds (see Table 1) was weighed and placed in the same volume of water used during the salt leaching step. The beakers were then sealed with parafilm and left for 3 days to simulate the salt leaching procedure. The pH changes in the solution were monitored at the same intervals described in the scaffold preparation procedure. Samples were finally dried under vacuum for 2 h. These samples are from now on referred as “water treated” Bg samples, and specifically as T15, T30, T40, and T50 depending on the Bg/water ratio used to mimic amounts present in PDLLA/15Bg, PDLLA/30Bg, PDLLA/ 40Bg, and PDLLA/50Bg scaffolds, respectively (Table 1). Three repeats for each four different Bg concentrations were conducted for statistical analysis. 2.4. X-ray Photoelectron Spectroscopy (XPS). XPS was performed on the as-received, water treated, and extracted Bg powders. Spectra were collected using a ThermoFisher Scientific Kalpha instrument, equipped with a monochromatic Al Kα X-ray source (1486.6 eV) with an estimated 0.1% analytical sensitivity. An electron flood gun was used during the analyses to compensate for charge buildup on the samples. Elemental surveys were recorded by accumulating 2 scans, running from 1350 to 0 eV and step size of 1 eV. High resolution spectra of C1s were collected by accumulating three scans, using a step size of 0.1 eV. Measurements were taken on three spots (X-ray beam diameter of 250 μm) per sample for statistical

2. MATERIALS AND METHODS 2.1. Scaffold Preparation. PDLLA scaffolds were prepared through the solvent casting and particulate leaching technique as described in previous studies.23,39 Briefly, PDLLA (Boehringer Ingelheim Chemicals, Inc., Petersburg, VA) was weighed in a beaker and acetone (Sigma-Aldrich, Inc., Oakville, ON) was added to the polymer to create a 5% (w/v) solution. The mixture was sealed with Al foil and parafilm, and then left to dissolve overnight on a magnetic stirrer. Bg (NovaBone Products, LLC, Alachua, FL) was then combined with the solutions, the amount varying from 15 to 50 wt. 1467

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analysis. Data acquisition and processing were carried out with the Avantage XPS software. 2.5. Fourier Transform Infrared (FTIR) Spectroscopy. A Bruker Tensor 27 IR spectrometer equipped with a DTGS (deuterated triglycine sulfate pyroelectric) detector was used to record FTIR spectra of the composite Bg/PDLLA scaffolds and the Bg powders. Ground Bg powders were diluted with KBr in a 1:4 ratio before the measurements. Spectra were recorded in the 4000−400 cm−1 spectral range with 256 scans and 4 cm−1 resolution.

This band has been typically observed on the surface of silica glasses in the presence of other cations such as Na+.1,41 Finally, the peak at ∼500 cm−1 is also a manifestation of δSiOSi.1,41 As can be seen from Figure 1, the spectra of the acetone treated Bg and of the starting Bg powders are almost identical, which indicates that Bg does not react in acetone. FTIR spectra from the water treated samples are shown in Figure 2, together with the spectrum of the as-received Bg for reference. The spectra (T15-T50) refer to powders reacted in different Bg/water ratio, to mimic the amounts of Bg present in the composite PDLLA/Bg scaffolds during the salt-leaching step. On all water treated samples, a major change occurs in the carbonate νCO peak located at 1550−1350 cm−1: while this is an intense single peak in the as-received Bg, it decreases in intensity and develops into a double peak on the water treated Bg samples. A similar phenomenon was seen in another study, in which FTIR spectra of Bg after immersion in TRIS buffer were compared with reference doped silica samples.8 The single carbonate peak was related to carbonates bound to surface Na+ cations, while the double peak, to carbonates bound to surface Ca2+ cations. Based on the difference in position between the two components in the νCO band, we can deduce that the carbonates formed on the water treated Bg powders are a mix of ionic and monodentate surface Ca-carbonates.40 The change in surface carbonation correlates with the release of Na+ ions from the surface of the glass to the surrounding solution, and migration of Ca2+ ions from the bulk to the surface of the glass. The band at ∼1660 cm−1 indicates the presence of coordinated water on the surface of Bg water treated and could not be clearly distinguished on the starting Bg.42 Some changes are observed in the broad band in the 3800− 2400 cm−1 region. These relate to νOH of H-bonded OHbearing species, like hydroxyl groups or coordinated water, present on the surface of Bg. The maximum of the band shifts from ∼3000 cm−1 on the as-received Bg to ∼3250 cm−1 on the water treated Bg. A similar result was observed by Cerruti et al.8 who measured IR spectra, in vacuum, of Bg before and after treatment in buffered aqueous solutions. In the latter study, the band shift was associated with a decrease in H-bond strength of the water molecules interacting with surface Na+ cations on the as-received Bg, and with surface Ca2+ cations on the treated Bg.8 Although in our work we collect spectra in air and

3. RESULTS AND DISCUSSION FTIR spectra of Bg before and after acetone treatment are shown in Figure 1. The broad band encompassing the ∼3750−

Figure 1. FTIR spectra of Bg (a) before and (b) after acetone treatment, normalized with respect to the peak centered at ∼1020 cm−1.

2500 cm−1 region has been assigned to νOH that stems from free and H-bonded hydroxyl groups.8 The peaks at ∼1970 and 1701 cm−1 are overtone bands of SiO and CO vibrations, respectively.8 The sharp peak at ∼1500 cm−1 relates to νCO of carbonates, formed on the surface of Bg as a result of a reaction between atmospheric CO2 and available surface Ca2+ ions.1 The most intense and complex band spanning the ∼1300−800 cm−1 region is ascribed to the absorptions of both SiO and PO groups on the glass.1 The band at ∼730 cm−1 is due to the bending of highly distorted six-membered SiOSi rings (δSiOSi).1

Figure 2. FTIR spectra of (A) higher and (B) lower wavenumber regions of as-received and water treated Bg. (Remove in DI water for three days.) Spectra T15, T30, T40, and T50 refer to Bg powders water treated with a Bg/water ratio mimicking the ratio present in the PDLLA/15Bg, PDLLA/ 30Bg, PDLLA/40Bg, and PDLLA/50Bg scaffolds, respectively. 1468

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respectively.45−47 Thus, the presence of Bg makes the PDLLA/ 50Bg scaffold more hydrophilic than the scaffold made with PDLLA alone. Since the spectra of the composite PDLLA/Bg scaffolds do not allow analysis of Bg-related bands, we extracted the Bg powders from the scaffolds by dissolving the composites in acetone. As shown earlier (Figure 1), the glass does not react with acetone, which implies that this extraction step should not interfere with changes occurred on Bg during the scaffold processing. After dissolution of the PDLLA matrix, we collected the Bg powders and washed them several times with acetone to remove any polymeric residue. The FTIR spectra of these samples are shown in Figure 4, which includes the spectrum of the as-received Bg for comparison. The changes observed in the spectra of the extracted Bg are similar to those previously described on the water treated Bg samples (Figure 2). The Nabound carbonates present in the as-received Bg are substituted by Ca-bound carbonates. However, lower amounts of Cabound carbonates are observed on the extracted Bg compared to the water treated Bg powders analyzed before. In addition, the band at ∼1660 cm−1 previously observed on the water treated Bg samples is substituted on the extracted Bg powders by a complex envelope of bands extending from ∼1650 to ∼1750 cm−1. These bands fall in the range of both νC−O and νCO absorptions. These peaks may be related to the presence of some PDLLA that remained bound to the extracted Bg powders despite the extensive rinsing. Two small peaks centered at around 2900 cm−1, superimposed on the broad νOH band, relate to νCH and confirm the presence of PDLLA. Additionally, the 1660 cm−1 band (δHOH) assigned to coordinated water is absent, and the νOH band from ∼3800 to 2000 cm−1 is less intense on the extracted Bg powder. Overall, these features indicate that the extracted Bg sample is less hydrophilic than the water treated Bg. This is another indication that the extracted Bg contains traces of the hydrophobic PDLLA scaffold matrix on its surface. As previously observed on the water treated Bg samples (Figure 2), the band located at 940 cm−1 that pertains to SiONBO decreases on all extracted Bg samples compared to the as-received Bg. The decrease, however, is different from that observed on water treated Bg powders. In particular, the relative intensity of the components in the band centered at ∼1100 cm−1, which includes both SiO and PO bonds, changes for powders extracted from the different scaffolds. The largest differences between the relative intensity of these components measured on the water treated and extracted powders are observed for spectra E15 and E30 (Figure 4) versus T15 and T30 (Figure 2), which may indicate that the silicate network in the E15 and E30 samples has changed the most compared to all the others analyzed. Finally, the band located at ∼500 cm−1 is different than in the as-received Bg. This peak correlates with νsym SiOSi. Its position shifts from ∼509 cm−1 on the as-received Bg to ∼515 and 534 cm−1 on PDLLA/50Bg and PDLLA/30Bg scaffolds, respectively. These changes were not observed on the water treated Bg samples, and may result from a rearrangement of silica rings configuration, possibly related to changes in cation concentration.25,48 Computational work by Tilocca and Cormack49,50 shows that three-membered (3M) silica rings are more abundant on the surface of both slabs and nanoparticles of Bg, and that this goes along with a larger concentration of surface Na+ compared to the bulk. Both 3M silica rings and higher Na+ concentration were suggested to be

therefore we monitor both water-water and water-surface Hbonds, the similarity of our results suggests the same explanation for this band shift. Indeed, Car-Parrinello molecular dynamics simulations show that Na+ and Ca2+ are very strong sites of interaction for water,43 and that the H-bonds formed between Na+ and/or Ca2+ and water molecules are so strong that they give rise to cation-oxygen distances close to those observed in bulk glass.44 In accordance with the interpretation of our IR band shift, Na+ generates stronger H-bonds with water molecules compared to Ca2+, as evidenced by the shorter calculated distance of Na−O compared to Ca−O.44 Finally, a decrease in the component at ∼940 cm−1 is seen on all water treated Bg powders in comparison with the as-received Bg. This band represents nonbridging SiO groups (SiONBO), SiO− in the glassy network not directly connected to another Si atom.1 A decrease in this band indicates a release of cations (Na+ especially) from the Bg bulk, which is expected to occur when Bg is immersed in aqueous solutions.1 No major differences are observed depending on the Bg/water ratio used during this treatment. Taken altogether, the changes show that immersion of Bg treated in water for 3 days, as in the salt leaching step of scaffold production, starts Bg dissolution. But how do these results compare with what happens to Bg powders inside the scaffold, throughout the actual scaffold processing? Will the presence of a polymeric matrix around Bg slow down or impede these transformations? To answer these questions, we prepared Bg-PDLLA scaffolds using the solvent casting and salt leaching technique. SEM images of scaffold sections showed a rather uniform distribution of Bg particles within the scaffolds (Figure S1 in the Supporting Information). We measured FTIR spectra of PDLLA scaffolds with and without Bg (Figure 3).

Figure 3. FTIR spectra of scaffolds (a) PDLLA/0Bg and (b) PDLLA/ 50Bg.

Spectra have been normalized to the peak at ∼1790 cm−1 and linearized to eliminate the differences in scattering. Only minor differences can be observed between the spectra of the scaffold made in PDLLA alone and the composite PDLLA/50Bg scaffold, since peaks from PDLLA overshadow those relative to Bg. The main differences are the occurrence of a peak at ∼1600 cm−1 as well as an increased absorbance in the 3600−2400 cm−1 region in the spectrum of the PDLLA/Bg scaffold. These bands refer to the δHOH and the νOH of water molecules, 1469

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Figure 4. FTIR spectra of (A) higher and (B) lower wavenumber regions of Bg as-received and extracted from scaffolds. Spectra E15, E30, E40, and E50 refer to Bg powders extracted from PDLLA/15Bg, PDLLA/30Bg, PDLLA/40Bg, and PDLLA/50Bg scaffolds, respectively.

Figure 5. Atomic % concentration measured by XPS for P, Si, and Na (A) and Ca and C (B) on the surface of Bg as-received (black solid), T30 (white solid), and E30 (gray striped). O was removed from the calculations.

responsible for the high bioactivity of Bg.49 However, since on the extracted Bg the surface Na+ concentration is lower than on the as-received Bg, it is hard to know which rearrangement of silica configuration the νsym SiOSi peak frequency change relates to, and if the newly formed species induce a change in the overall Bg bioactivity. FTIR spectra show that extracted Bg is unlike the as-received. Some PDLLA is strongly bound to the former and cannot be removed thorough rinsing. Spectra for the water treated (Figure 2) and the extracted Bg (Figure 4) demonstrate that the two samples reacted to a different extent. Therefore, the acetone/ water treatment does not fully mimic the transformations occurring on Bg during scaffold processing. To better understand the differences, we analyzed both water treated and extracted Bg by XPS. Figure 5 illustrates the XPS atomic concentrations of Na, Si, Ca, P, and C on the surface of the as-received, water treated, and extracted Bg powders. Since the results obtained on the water treated and extracted Bg samples at different concentrations were not statistically different, we report only the values obtained for samples E30 and T30. Both water treated and extracted Bg have less Na on their surface than the asreceived powders (Figure 5A). This conforms to the IR results (Figures 2 and 4). Specifically, the immersion of Bg in water,

both by itself or as a component of PDLLA/Bg scaffolds, starts the Bg dissolution process, which leads to the release of Na+ ions from the glass. Both water treated and extracted Bg show a higher amount of surface Ca and P than the as-received powders (Figure 5). This is an indication of the formation of a Ca/P rich layer on the water treated and extracted Bg surface. The developing Ca/P rich layer was not observed by IR spectroscopy, which indicates that this layer is thin and only a highly surface-sensitive technique such as XPS can detect its presence. More Ca and P are found on the surface of Bg water treated (∼17% for Ca and ∼11−12% for P) than the extracted (∼11% for Ca and 7% for P), which shows that the Ca/P layer must be thicker on the water treated than the extracted Bg powders. An opposite trend is observed for Si, found in higher amount on the as-received powders than on the extracted Bg, and lowest on the water treated Bg samples. The layer of calcium phosphate that precipitates on the surface of Bg probably covers the silica rich matrix of the Bg, or the silica-rich layer formed during Bg dissolution.1 Hence, while the thickest Ca/P layer is observed on water treated Bg, the sample shows the smallest amount of Si. The amount of C on the surface of the as-received and water treated Bg is approximately the same (∼17 at. %), whereas a higher atomic percent of C is present on Bg extracted (22 at. 1470

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interaction between Bg and PDLLA is not dependent on the amount of Bg in the scaffold. Both the FTIR and XPS analysis indicate that Bg changes during scaffold processing. Furthermore, the alterations cannot be completely simulated by simply immersing Bg powders in the same solvents and for the same time periods used during scaffold processing. With regards to the mechanism of Bg reactivity proposed in earlier studies and summarized in the introduction, the pH of the solution is expected to increase when Bg is present.1,3,7 To determine why extracted and water treated Bg react differently despite similar treatments in the fluids of scaffold processing, we monitored the pH changes during the water immersion step used for the Bg powder treatment (Figure 7A) and the leaching step of PDLLA/Bg scaffolds (Figure 7B). The differences in pH changes for these two sets of samples are remarkable. Much higher pH values are reached from the very first few minutes of immersion in water for all the water treated Bg powders (Figure 7A) than when Bg powders are present inside the scaffolds (Figure 7B). Overall, the increase in pH is much smaller and slower when lower amounts of Bg are present in the scaffolds (Figure 7B). No changes in pH are observed in the absence of Bg in the PDLLA scaffolds (Figure 7B, spectrum PDLLA/0Bg), as expected because PDLLA does not degrade within a couple of hours, but instead requires weeks to release lactic acid byproducts that would decrease the pH.52,53 The overall increase in pH on all samples including Bg is to be related to the exchange between Na+ ions from Bg bulk and H+ from the solution, which explains why larger amounts of Bg lead to higher pH. Both HCA and CaCO3 are less soluble at higher pH. Consequently, the higher surface Ca-bound carbonates (compare FTIR spectra in Figures 2 and 4) and thicker Ca/P layer (see Figure 5) on the water treated than on the extracted samples result from exposure to a higher pH during the water treatment. This is in agreement with a previous study that analyzed the reactivity of Bg at different pH.1 The larger changes observed in the IR bands in the 940− 1100 cm−1 region for samples E15 and E30 (Figure 4) indicate that the structure of these samples changed more than all others analyzed and can also be related to the observed changes in pH for these samples. Indeed, these samples were exposed to pH values in the range of 6.4−7.4 during contact with water in the leaching step of the scaffold processing. Such values are close to the optimal pH values found earlier to trigger Bg dissolution and reprecipitation.1 At higher pH, the earlier formation of HCA and calcium carbonates on Bg surface acts as a protective layer and prevents further glass dissolution. These differences in pH are therefore crucial in explaining the different extent of Bg reactivity observed by FTIR and XPS for water treated and extracted samples. We propose that the slower pH increase observed in the solutions containing PDLLA/Bg composite scaffolds is due to the hydrophobic

%) (Figure 5B). In order to understand the reason behind this difference, we analyzed the high-resolution C spectra for these samples (Figure 6).

Figure 6. High resolution C1s XPS spectra on Bg powders (a) asreceived, (b) T30, and (c) E30.

The spectra measured can be fit with three components, centered at 288.8, 286.5, and 284.6 eV, which correspond to carbon in a CO, C−O, and C−C/C−H environment,51 respectively. The carbonates (CO32‑) present on the glass surface, as confirmed by the FTIR analysis (Figures 2 and 4), give rise to the CO peak. C−C/C−H bonds are due to hydrocarbonaceous impurities that typically contaminate the sample surface.8 The C−O component can be partially due to O-containing impurities and to surface carbonates on the asreceived and water treated Bg. However, the relative intensity of the C−O component compared to the C−C/C−H peak notably increases on the extracted Bg (Table 2). Therefore, the C−O peak of extracted Bg must be related to different species, not present on either the as-received or water treated Bg. We suggest that the increase of the C−O component confirms the observations made with IR spectroscopy; that is, despite the acetone rinse, some polymeric matrix (PDLLA) remained adsorbed on the extracted Bg powders. C−O bonds are in fact present in the PDLLA backbone. The increase of the C−O component is paralleled by a higher CO/C−C peak ratio on the extracted Bg (Table 2), which is further indication of the presence of PDLLA on the extracted powders. These results suggest a strong interaction takes place between the Bg surface and the polymeric matrix of the scaffold. The interaction likely involves the formation of covalent bonds between the carboxyl end groups from the PDLLA and the hydroxyl groups on the Bg surface. Similar results are obtained for powders extracted from all the composite scaffolds (Table 2). Thus, the

Table 2. Relative Percent of the Three Components Used to Fit the C1s High Resolution XPS Spectra for Bg powders AsReceived, Water Treated, and Extracted C−C/C−H (relative %) Bg wt %

as-received

15 30 40 50

50.6 ± 0.1

water treated 64.2 62.4 60.4 59.9

± ± ± ±

3 3.4 0.4 2.1

CO (relative %) extracted

as- received

± ± ± ±

44.7 ± 0.6

50.3 51.5 45.6 49.9

2 3.7 2.2 4.8

water treated 26.8 27.7 30.6 30.8 1471

± ± ± ±

2 1.5 0.6 1.9

C−O (relative %) extracted

as- received

± ± ± ±

4.7 ± 0.5

32.9 31.1 33.4 32.7

2.4 2.3 2.1 1

water treated 9.1 9.9 9 9.3

± ± ± ±

1.1 1.9 0.9 0.5

extracted 16.8 17.4 21 17.3

± ± ± ±

1.5 3 3 3.9

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Figure 7. pH measured in DI water during immersion of Bg powders alone (A) or in the presence of PDLLA/Bg composite scaffolds (B).

Our results also showed that some PDLLA remained adsorbed on Bg powders extracted from the scaffolds despite thorough rinsing with acetone. This indicates the formation of a strong bond between Bg and the polymeric matrix of the scaffold, possibly related to reactivity between surface hydroxyl groups from the Bg and terminal carboxylate groups from PDLLA. NMR studies could perhaps complement our findings to characterize the exact nature of the glass-polymer interactions. The observed surface transformations occurring in Bg during PDLLA/Bg scaffold synthesis are important, since they may alter the course of Bg reactivity in later stages, when the scaffold is implanted. For example, we have observed changes in the νsymSiOSi peak position on the extracted Bg, which suggests changes in silica ring configuration. Although more experimental work (for example, including NMR or small-angle Xray scattering, SAXS) and computational analysis are necessary to understand the exact nature of this change; it is possible that such differences alter Bg bioactivity. Also, we showed that the extracted Bg contained a thin layer of Ca/P on its surface, and that the process of network remodeling had already started on some PDLLA/Bg scaffold compositions. The preformed Ca/P layer may act as a protective barrier and prevent or slow down Bg dissolution when the scaffold is immersed in physiological solutions. While prevention of Bg dissolution would be detrimental to its bioactivity, slowing the dissolution down might be beneficial. In fact, this might eliminate the sudden increase in pH observed when Bg is implanted, which causes inflammation in the surrounding tissues. This study thus suggests that Bg transformations during scaffold processing are important to evaluate before scaffold implantation in vivo.

PDLLA polymer network that surrounds the Bg, which is partially bound to its surface, as shown by both FTIR (Figure 4) and XPS (Figure 5). The hydrophobic polymer network slows down water diffusion from the solution toward the Bg particles present inside the scaffold and decreases the rate of Bg dissolution, thus leading to a slower increase in pH. This slower increase in pH ends up leading to a larger overall network reconstruction of Bg during scaffold processing than if it is treated with acetone and water in the absence of a surrounding polymeric matrix.

4. CONCLUSION Composite Bg/PDLLA scaffolds are often prepared using the solvent casting and salt leaching technique. Despite the relatively large amount literature on this subject, no studies have so far analyzed the changes occurring to Bg during the processing of this type of scaffolds. This paper fills this gap, focusing on surface transformation of Bg throughout scaffold processing and its interaction with the PDLLA matrix. Specifically, we analyzed the transformations of Bg powders immersed in acetone and water (the two solvents used during scaffold preparation), and we compared the results obtained on these samples with those obtained on Bg powders extracted from composite PDLLA/Bg scaffolds containing up to 50% weight Bg. Our results showed that Bg did not react in acetone, while (as expected) it transformed when immersed in water. The analysis of the powders extracted from the scaffolds, though, showed a different extent of reactivity compared with those treated in acetone and water: a thinner Ca/P-rich surface layer was formed on the extracted samples, as well as fewer surface carbonates. Larger changes in network connectivity were detected on powders extracted from scaffolds containing up to 30% weight Bg compared to all other samples analyzed. We explained these results based on the different pH values to which the Bg powders immersed in water by themselves and those immersed in water but surrounded by the polymeric scaffold were exposed. The presence of a polymeric matrix around the Bg powders slowed down the Na+/H+ exchange known to occur when Bg is exposed to water, and allowed Bg present in the scaffolds to react at pH values closer to 6−8 (the optimal pH range for Bg reactivity) for a longer period compared to the Bg immersed in water by itself.

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ASSOCIATED CONTENT

* Supporting Information S

SEM image of a scaffold section. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +1 514 398 5496. Fax: +1 514 398 4492. E-mail: marta. [email protected]. Notes

The authors declare no competing financial interest. 1472

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ACKNOWLEDGMENTS We thank the Fonds de recherche du Québec − Nature et technologies (FQRNT) (“Nouveaux Chercheurs” program) for funding this work, as well as Boehringer Ingelheim for kindly providing the poly(D,L-lactide) (PDLLA), and NovaBone Products for supplying Bioglass 45S5 powders. We also thank Prof. Sylvain Coulombe for allowing us to use the FEI Phenom Desktop SEM instrument.

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ABBREVIATIONS Bg, Bioglass 45S5; PDLLA, poly(D,L-lactide); FTIR, Fourier transform infrared spectroscopy; XPS, X-ray photoelectron spectroscopy; SEM, scanning electron microscopy

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