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Influence of dissolution medium pH on ion release and apatite formation of Bioglass® 45S5
Author's Accepted Manuscript
Influence of dissolution medium pH on ion release and apatite formation of Bioglasss 45S5 Liane Bingel, Daniel Groh, Natalia Karpukhina, Delia S. Brauer
www.elsevier.com/locate/matlet
PII: DOI: Reference:
S0167-577X(14)02301-5 http://dx.doi.org/10.1016/j.matlet.2014.12.124 MLBLUE18276
To appear in:
Materials Letters
Received date: 8 November 2014 Accepted date: 25 December 2014 Cite this article as: Liane Bingel, Daniel Groh, Natalia Karpukhina, Delia S. Brauer, Influence of dissolution medium pH on ion release and apatite formation of Bioglasss 45S5, Materials Letters, http://dx.doi.org/10.1016/j.matlet.2014.12.124 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Influence of dissolution medium pH on ion release and apatite formation of Bioglass® 45S5 Liane Bingel1, Daniel Groh1, Natalia Karpukhina2, Delia S. Brauer1,* 1Otto
Schott Institute of Materials Research, Friedrich Schiller University Jena, Fraunhoferstr. 6, 07743
Jena, Germany 2Dental
Physical Sciences, Oral Growth and Development, Institute of Dentistry, Barts and The London
School of Medicine and Dentistry, Queen Mary University of London, Mile End Road, London E1 4NS, UK
*Corresponding author: phone: +49-3641-948510; fax: +49-3641-948502; e-mail: [email protected]
Abstract Bioactive glasses, particularly Bioglass® 45S5, have been used to clinically regenerate human bone since the mid-1980's, owing to their ability to degrade in physiological solutions, release ions and form an apatite surface layer, which cells adhere to and proliferate on. Although low pH conditions do occur in the human body, e.g. during bacterial infections, in vitro dissolution experiments are usually performed at a physiological pH or 7.3 exclusively. Here, we investigated the dissolution behaviour of 45S5 at low pH (5) and high pH (9) in addition to pH 7.3. The results show that ion release occurs significantly faster at low pH, resulting in significantly faster apatite formation (3 hours vs. 6 hours at pH 7.3). By contrast, at pH 9 low ion exchange rates were observed, resulting in no significant apatite formation during the time period studied. Results suggest that low pH caused by bacterial infection is unlikely to inhibit apatite formation and, thus, bioactive glass clinical performance.
Keywords apatite; dissolution; glass structure; bioactive glass; bioactivity
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1. Introduction The reason for the clinical success of Bioglass® 45S5 [1], which has been in clinical use since the mid1980s [2], is its ability to degrade in aqueous solution, release ions, form a hydroxycarbonate apatite (HCA) surface layer and allow for the formation of an intimate bond to bone [1, 3]. We have recently shown that fluoride-containing bioactive glasses (BG) formed apatite fast in low-pH cell culture medium [4] and explained this by the release of fluoride ions into solution, which is known to lead to formation of low-solubility fluorapatite [5, 6] rather than HCA. In a later study, however, it was shown that Bioglass® 45S5 also formed apatite in low pH culture medium, albeit more slowly than fluoridecontaining BG [7]. As in vitro dissolution and cell culture experiments are usually performed at a normal physiological pH (i.e. around pH 7.3) [8], but low pH conditions are relevant during a range of clinical conditions (e.g. during bacterial infections [9]), a better understanding of BG dissolution in various pH environments can help us to improve bioactive glass performance.
2. Materials and methods Bioglass® 45S5 (46.1 SiO2, 2.6 P2O5, 26.9 CaO, 24.4 Na2O; in mol%) was prepared using a melt-quench route and obtained amorphous; nominal and analysed compositions agreed well, as described previously [10]. 0.062 mol L-1 tris(hydroxymethyl)amino methane (Tris) solution was prepared as described previously [10]. pH was adjusted using 1 mol L-1 HCl solution to either pH 9 ± 0.15 or pH 7.35 ± 0.15. 0.1 mol L-1 acetic acid/sodium acetate (HAc/NaAc) buffer was prepared as described previously [11] and the pH adjusted to pH 5 ± 0.05 using 1 mol L-1 sodium hydroxide solution. 75 mg 45S5 powder (<32µm) was immersed in 50 mL solution for 15 min (HAc/NaAc only), 3 hours, 6 hours, 1 day and 3 days in a shaking incubator at 37°C. Before and after each time period, pH was measured (pH meter HI 8314 with pH electrode HI 1217 D, HANNA Instruments), samples were filtered through medium porosity filter paper (5 µm particle retention, VWR International) and acidified using nitric acid (69%). Elemental concentrations were analysed using inductively coupled plasma optical emission spectroscopy (ICP-OES, Varian Liberty 150, Agilent Technologies). Experiments were performed in triplicates, and results are presented as a percentage of the ions originally present in the glass (mean ± standard deviation, SD). The retained powders were rinsed with acetone, dried and characterised by Fourier transform infrared spectroscopy (FTIR; Alpha, Bruker Daltonic GmbH) and powder X-ray diffraction (XRD; AXS-Bruker D8-Discover, CuKα, data collected at room temperature).
3. Results and discussion In all three dissolution media, 45S5 caused the typical dramatic pH rise [12] during the first minutes to hours after immersion (Fig. 1), owing to an ion exchange between modifier ions (Na+ and Ca2+) in the glass and protons (H+) in the dissolution medium [12, 13]. Accordingly, ionic concentrations in solution increased dramatically (Fig. 1).
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Results at pH 5 (Fig. 1a) show that this ion release occurred at very early time points, with about 90% of calcium ions being present in solution at 15 min. (HAc/NaAc buffer contained large amounts of Na+ ions, which masked Na+ released from the glass, but sodium release can be expected to be no less than calcium release.) Similarly, phosphate concentrations were very high at early time points (90% at 15 min) but decreased afterwards. At pH 7.3 (Fig. 1b) and pH 9 (Fig. 1c), pH rise and ion release were not quite as fast as for pH 5. pH and Na+ concentrations increased steadily over the entire time of the experiment (3 days). Na+ concentrations in solution were significantly higher in pH 7.3 than pH 9 solution (77 vs. 36%), suggesting that the ion exchange happened much slower at higher pH. From experimental pH values the H+ concentrations in solution can be calculated, allowing for comparison of initial H+ concentration (c0(H+)) before 45S5 immersion and at later time points. In Fig. 1d this difference, c0(H+)-c(H+), is presented, and the results show that the overall ion exchange was indeed much more pronounced with lower pH of the dissolution medium. This should not come as a surprise, as a larger concentration of protons in solution can be expected to result in more ion exchange occurring. FTIR spectra showed significant changes after 45S5 immersion in pH 5 and pH 7.3 solution compared to the unreacted glass (Fig. 2a,b). The non-bridging oxygen (NBO, Si-O- alkali+) band at 915 cm-1 disappeared (owing to ion exchange [5]) and a new band appeared at about 790 cm-1, which is usually assigned to Si— O—Si vibrations between silicate tetrahedra [14] after treatment of bioactive glasses in Tris buffer [15] or simulated body fluid (SBF) [12], indicating formation of a silica-gel surface layer after leaching of Ca2+ and Na+ ions and formation of Si—OH groups. At later time points (3 and 6 hours for pH 5 and pH 7.3 solutions, respectively), a sharp, high intensity band appeared at 1025 cm-1 as well as a split band at 560 and 600 cm-1. These bands correspond to the formation of a crystalline phosphate surface layer, and particularly the split band is the most characteristic region for apatites. With time, these bands sharpened and increased in intensity. Formation of apatite was also confirmed by XRD (patterns shown for 3 days in Fig. 2d). The fact that 45S5 seems to form apatite well in low pH conditions is of great interest for clinical applications, as it shows that even under low pH conditions 45S5 can still be expected to perform its apatite forming action, e.g. during inflammation [9] or in the oral cavity after consumption of acidic beverages [16]. In addition, the pH rise caused during ion release may help to counteract the pH drop during bacterial infections. Indeed, this pH rise has also been suggested to cause antibacterial properties of BG [17]. FTIR spectra of 45S5 treated in pH 9 solution (Fig. 2c) did not show pronounced changes. The intensity of the NBO band at 915 cm-1 decreased, suggesting that some ion exchange had occurred, in agreement with ICP-OES and pH results (Fig. 1). The typical apatitic features discussed above (at 560, 600 and 1025 cm-1), however, did not appear, in agreement with absence of apatite-related reflections in XRD patterns at 3 days (Fig. 2d). Instead, FTIR spectra showed a broad, low-intensity band at 590 cm-1, which is commonly taken as an indication of presence of either amorphous calcium phosphates or calcium apatites of poor crystallinity [18], suggesting that the process of apatite formation had started, but that apatite was forming at much slower rates than at lower pH. This agrees with ICP-OES results, which showed that in all 3
three solutions phosphate concentrations decreased after an initial maximum at early time points, which usually coincides with apatite precipitation [6]. While a higher pH is thought to be favourable for apatite formation [19], here apatite formation was very low at pH 9, owing to slow ion release and thus low concentrations of calcium and phosphate ions in solutions. This probably only delayed apatite formation, as with time an increase in concentrations can be expected, allowing for more apatite precipitation. Presence of carbonate substitution in the apatite [20] was indicated by a band at about 870 cm-1 in FTIR spectra, present for 45S5 treated at pH 7.3 for 1 or 3 days (Fig. 2b). For all samples, broad CO32- bands were present from 1400 cm-1, indicating B-type substitution (i.e. carbonate replacing a phosphate group). These bands were most pronounced for 45S5 treated in pH 9 solution (despite low apatite formation) and lowest for samples treated at pH 5, suggesting that carbonate was incorporated into apatite more readily at higher pH values. This agrees with findings that in alkali-containing solutions both alkali and carbonate incorporation into apatite increased with increasing pH of the solution [19]. According to Hench's original mechanism of BG degradation and apatite formation [21] the exchange of alkali cations from the glass for protons from the surrounding medium is the first step in BG dissolution. During the dissolution studies here, however, the concentrations of calcium ions in solution, while being lower than those of sodium ions (Fig. 1b,c), appeared also at very early time points, suggesting that while some differences in release rates may exist (owing to differences in field strength), calcium ions are also released fast. However, as calcium is consumed (together with phosphate ions) during apatite precipitation, which occurred at very early time points (Fig. 2), it is not possible to make any statements on Ca2+ release, except that it is likely to be higher than indicated by the concentrations detected in solution. It is therefore possible that no differences exist between the release rates of alkali and calcium ions, which is also confirmed by alkali-free glasses forming apatite no slower than alkali-containing ones [6]. A later step of BG degradation and apatite formation according to Hench [21] is alkaline hydrolysis of Si— O—Si bonds. This hydrolysis is known to occur at high pH values (usually above pH 10), resulting in congruent dissolution of silicate glasses [22]. The pH range detected during in vitro dissolution or cell culture studies is usually well below that (Fig. 1), and ICP-OES results suggest that no congruent dissolution occurred up to pH 9. It has been noted that low network connectivity BG, e.g. 45S5, may release silicate chains without the need for Si—O—Si hydrolysis [23]. However, the fact that bioactive phospho-silicate glasses, even more polymerised compositions such as S53P4 [24], degrade completely in the body [25-28] suggests that Si—O—Si hydrolysis does occur to some extent. One also needs to keep in mind that even if the overall pH detected is well below pH 10, it may be significantly higher locally, e.g. very near the BG/water interface, allowing for Si—O—Si hydrolysis to occur. Solubility of amorphous silica has been shown to increase rapidly for pH values above 8, while possibly going through a minimum between pH 7 and 8 [29]. Our data here seem to agree with this, with concentrations of silicon species at 3 days being higher at pH 9 (3.2±0.1 mmol L-1) than at pH 7.3 (1.8±0.1 mmol L-1) and pH 5 (2.1±0.1 mmol L-1). Si concentrations increased with time, possibly as pH also increased with time (Fig. 1c). 4
4. Conclusion Bioglass® 45S5 formed apatite significantly faster at low pH than at physiological, caused by faster ion exchange. Ion release at alkaline pH was slow, by contrast, resulting in significantly delayed apatite precipitation. These results are of interest for clinical applications of 45S5, as they suggest that low pH conditions (e.g. during inflammation) are unlikely to inhibit apatite formation and, thus, bioactive glass clinical performance.
Acknowledgements The authors acknowledge Ms Brunhilde Dreßler, Institute of Geography, University Jena, for support with ICP-OES measurements and funding by the Carl Zeiss Foundation, Germany.
References [1] Hench LL, Splinter RJ, Allen WC, Greenlee TK. Bonding mechanisms at the interface of ceramic prosthetic materials. J Biomed Mater Res. 1971;5:117-41. [2] Hench LL, Hench JW, Greenspan DC. Bioglass®: A short history and bibliography. J Aust Ceram Soc. 2004;40:1-42. [3] Hench LL, Polak JM, Xynos ID, Buttery LDK. Bioactive materials to control cell cycle. Materials Research Innovations. 2000;3:313-23. [4] Shah FA, Brauer DS, Wilson RM, Hill RG, Hing KA. Influence of cell culture medium composition on in vitro dissolution behavior of a fluoride-containing bioactive glass. J Biomed Mater Res A. 2014;102:64754. [5] Brauer DS, Karpukhina N, O'Donnell MD, Law RV, Hill RG. Fluoride-containing bioactive glasses: Effect of glass design and structure on degradation, pH and apatite formation in simulated body fluid. Acta Biomater. 2010;6:3275-82. [6] Mneimne M, Hill RG, Bushby AJ, Brauer DS. High phosphate content significantly increases apatite formation of fluoride-containing bioactive glasses. Acta Biomater. 2011;7:1827-34. [7] Shah FA, Brauer DS, Desai N, Hill RG, Hing KA. Fluoride-containing bioactive glasses and Bioglass® 45S5 form apatite in low pH cell culture medium. Mater Lett. 2014;119:96-9. [8] Cannillo V, Pierli F, Ronchetti I, Siligardi C, Zaffe D. Chemical durability and microstructural analysis of glasses soaked in water and in biological fluids. Ceram Int. 2009;35:2853-69. [9] Punnia-Moorthy A. Evaluation of pH changes in inflammation of the subcutaneous air pouch lining in the rat, induced by carrageenan, dextran and staphylococcus aureus. J Oral Pathol Med. 1987;16:36-44. [10] Groh D, Döhler F, Brauer DS. Bioactive glasses with improved processing, Part 1: Thermal properties, ion release and apatite formation. Acta Biomater. 2014;10:4465–73. [11] Chen X, Brauer DS, Karpukhina N, Waite RD, Barry M, McKay IJ, et al. ‘Smart’ acid-degradable zincreleasing silicate glasses. Mater Lett. 2014;126:278-80. [12] Jones JR, Sepulveda P, Hench LL. Dose-dependent behavior of bioactive glass dissolution. J Biomed Mater Res. 2001;58:720-6. [13] Fagerlund S, Hupa L, Hupa M. Dissolution patterns of biocompatible glasses in 2-amino-2hydroxymethyl-propane-1,3-diol (Tris) buffer. Acta Biomater. 2013;9:5400-10. [14] Filgueiras MRT, La Torre G, Hench LL. Solution effects on the surface reactions of three bioactive glass compositions. J Biomed Mater Res. 1993;27:1485-93. [15] Brauer DS, Mneimne M, Hill RG. Fluoride-containing bioactive glasses: Fluoride loss during melting and ion release in tris buffer solution. J Non-Cryst Solids. 2011;357:3328-33. [16] Burwell AK, Litkowski LJ, Greenspan DC. Calcium sodium phosphosilicate (NovaMin): remineralization potential. Adv Dent Res. 2009;21:35-9. 5
[17] Gubler M, Brunner TJ, Zehnder M, Waltimo T, Sener B, Stark WJ. Do bioactive glasses convey a disinfecting mechanism beyond a mere increase in pH? International Endodontic Journal. 2008;41:670-8. [18] Sepulveda P, Jones JR, Hench LL. In vitro dissolution of melt-derived 45S5 and sol-gel derived 58S bioactive glasses. J Biomed Mater Res. 2002;61:301-11. [19] Elliott JC. Structure and chemistry of the apatites and other calcium orthophosphates. 1st ed. Amsterdam, New York, London, Tokyo: Elsevier; 1994. [20] Lu X, Leng Y. Theoretical analysis of calcium phosphate precipitation in simulated body fluid. Biomaterials. 2005;26:1097-108. [21] Hench LL, Andersson Ö. Bioactive glasses. In: Hench LL, Wilson J, editors. An introduction to bioceramics. Singapore: World Scientific Publishing; 1993. p. 41-62. [22] Hench LL, Clark DE. Physical chemistry of glass surfaces. J Non-Cryst Solids. 1978;28:83-105. [23] Hill RG, Brauer DS. Predicting the bioactivity of glasses using the network connectivity or split network models. J Non-Cryst Solids. 2011;357:3884-7. [24] Lindfors NC, Koski I, Heikkila JT, Mattila K, Aho AJ. A prospective randomized 14-year follow-up study of bioactive glass and autogenous bone as bone graft substitutes in benign bone tumors. J Biomed Mater Res B. 2010;94B:157-64. [25] Wheeler DL, Stokes KE, Hoellrich RG, Chamberland DL, McLoughlin SW. Effect of bioactive glass particle size on osseous regeneration of cancellous defects. J Biomed Mater Res. 1998;41:527-33. [26] Froum S, Cho SC, Rosenberg E, Rohrer M, Tarnow D. Histological comparison of healing extraction sockets implanted with bioactive glass or demineralized freeze-dried bone allograft: A pilot study. Journal of Periodontology. 2002;73:94-102. [27] Ducheyne P. Effect of bioactive glass particle size on osseous regeneration. J Biomed Mater Res. 1999;46:301-3. [28] Hill R, Rawlinson S, Davis G, Nehete S, Shahdad S. A clinical case study: Using a strontium substituted bioactive glass - StronBone® - to fill alveolar sockets. Eur Cells Mater. 2014;28:51. [29] Iler RK. The chemistry of silica: solubility, polymerization, colloid and surface properties, and biochemistry. New York: Wiley; 1979.
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Figures a
b
c
d
Figure 1: Ionic concentrations in solution (black, left axis) and pH (blue, right axis) over time for initial pH of (a) 5, (b) 7.3 and (c) 9. (c) Difference between initial H+ concentration in solution and H+ concentration at later time points.
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a
b
c
d
Figure 2: FTIR spectra of 45S5 after treatment in solution for various time periods. Initial pH: (a) 5, (b) 7.3 and (c) 9. (d) XRD patterns of untreated glass and powders after immersion at pH 5, 7.3 and 9 for 3 days. (Hydroxycarbonate apatite reflections are marked by asterisks; JCPDS 00-019-0272.)
Highlights • • •
Enhanced ion release of Bioglass® 45S5 at low pH Enhanced apatite formation of Bioglass® 45S5 at low pH Negligible apatite formation of Bioglass® 45S5 at pH 9
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Influence of dissolution medium pH on ion release and apatite formation of Bioglasss 45S5 Liane Bingel, Daniel Groh, Natalia Karpukhina, Delia S. Brauer
www.elsevier.com/locate/matlet
PII: DOI: Reference:
S0167-577X(14)02301-5 http://dx.doi.org/10.1016/j.matlet.2014.12.124 MLBLUE18276
To appear in:
Materials Letters
Received date: 8 November 2014 Accepted date: 25 December 2014 Cite this article as: Liane Bingel, Daniel Groh, Natalia Karpukhina, Delia S. Brauer, Influence of dissolution medium pH on ion release and apatite formation of Bioglasss 45S5, Materials Letters, http://dx.doi.org/10.1016/j.matlet.2014.12.124 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Influence of dissolution medium pH on ion release and apatite formation of Bioglass® 45S5 Liane Bingel1, Daniel Groh1, Natalia Karpukhina2, Delia S. Brauer1,* 1Otto
Schott Institute of Materials Research, Friedrich Schiller University Jena, Fraunhoferstr. 6, 07743
Jena, Germany 2Dental
Physical Sciences, Oral Growth and Development, Institute of Dentistry, Barts and The London
School of Medicine and Dentistry, Queen Mary University of London, Mile End Road, London E1 4NS, UK
*Corresponding author: phone: +49-3641-948510; fax: +49-3641-948502; e-mail: [email protected]
Abstract Bioactive glasses, particularly Bioglass® 45S5, have been used to clinically regenerate human bone since the mid-1980's, owing to their ability to degrade in physiological solutions, release ions and form an apatite surface layer, which cells adhere to and proliferate on. Although low pH conditions do occur in the human body, e.g. during bacterial infections, in vitro dissolution experiments are usually performed at a physiological pH or 7.3 exclusively. Here, we investigated the dissolution behaviour of 45S5 at low pH (5) and high pH (9) in addition to pH 7.3. The results show that ion release occurs significantly faster at low pH, resulting in significantly faster apatite formation (3 hours vs. 6 hours at pH 7.3). By contrast, at pH 9 low ion exchange rates were observed, resulting in no significant apatite formation during the time period studied. Results suggest that low pH caused by bacterial infection is unlikely to inhibit apatite formation and, thus, bioactive glass clinical performance.
Keywords apatite; dissolution; glass structure; bioactive glass; bioactivity
1
1. Introduction The reason for the clinical success of Bioglass® 45S5 [1], which has been in clinical use since the mid1980s [2], is its ability to degrade in aqueous solution, release ions, form a hydroxycarbonate apatite (HCA) surface layer and allow for the formation of an intimate bond to bone [1, 3]. We have recently shown that fluoride-containing bioactive glasses (BG) formed apatite fast in low-pH cell culture medium [4] and explained this by the release of fluoride ions into solution, which is known to lead to formation of low-solubility fluorapatite [5, 6] rather than HCA. In a later study, however, it was shown that Bioglass® 45S5 also formed apatite in low pH culture medium, albeit more slowly than fluoridecontaining BG [7]. As in vitro dissolution and cell culture experiments are usually performed at a normal physiological pH (i.e. around pH 7.3) [8], but low pH conditions are relevant during a range of clinical conditions (e.g. during bacterial infections [9]), a better understanding of BG dissolution in various pH environments can help us to improve bioactive glass performance.
2. Materials and methods Bioglass® 45S5 (46.1 SiO2, 2.6 P2O5, 26.9 CaO, 24.4 Na2O; in mol%) was prepared using a melt-quench route and obtained amorphous; nominal and analysed compositions agreed well, as described previously [10]. 0.062 mol L-1 tris(hydroxymethyl)amino methane (Tris) solution was prepared as described previously [10]. pH was adjusted using 1 mol L-1 HCl solution to either pH 9 ± 0.15 or pH 7.35 ± 0.15. 0.1 mol L-1 acetic acid/sodium acetate (HAc/NaAc) buffer was prepared as described previously [11] and the pH adjusted to pH 5 ± 0.05 using 1 mol L-1 sodium hydroxide solution. 75 mg 45S5 powder (<32µm) was immersed in 50 mL solution for 15 min (HAc/NaAc only), 3 hours, 6 hours, 1 day and 3 days in a shaking incubator at 37°C. Before and after each time period, pH was measured (pH meter HI 8314 with pH electrode HI 1217 D, HANNA Instruments), samples were filtered through medium porosity filter paper (5 µm particle retention, VWR International) and acidified using nitric acid (69%). Elemental concentrations were analysed using inductively coupled plasma optical emission spectroscopy (ICP-OES, Varian Liberty 150, Agilent Technologies). Experiments were performed in triplicates, and results are presented as a percentage of the ions originally present in the glass (mean ± standard deviation, SD). The retained powders were rinsed with acetone, dried and characterised by Fourier transform infrared spectroscopy (FTIR; Alpha, Bruker Daltonic GmbH) and powder X-ray diffraction (XRD; AXS-Bruker D8-Discover, CuKα, data collected at room temperature).
3. Results and discussion In all three dissolution media, 45S5 caused the typical dramatic pH rise [12] during the first minutes to hours after immersion (Fig. 1), owing to an ion exchange between modifier ions (Na+ and Ca2+) in the glass and protons (H+) in the dissolution medium [12, 13]. Accordingly, ionic concentrations in solution increased dramatically (Fig. 1).
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Results at pH 5 (Fig. 1a) show that this ion release occurred at very early time points, with about 90% of calcium ions being present in solution at 15 min. (HAc/NaAc buffer contained large amounts of Na+ ions, which masked Na+ released from the glass, but sodium release can be expected to be no less than calcium release.) Similarly, phosphate concentrations were very high at early time points (90% at 15 min) but decreased afterwards. At pH 7.3 (Fig. 1b) and pH 9 (Fig. 1c), pH rise and ion release were not quite as fast as for pH 5. pH and Na+ concentrations increased steadily over the entire time of the experiment (3 days). Na+ concentrations in solution were significantly higher in pH 7.3 than pH 9 solution (77 vs. 36%), suggesting that the ion exchange happened much slower at higher pH. From experimental pH values the H+ concentrations in solution can be calculated, allowing for comparison of initial H+ concentration (c0(H+)) before 45S5 immersion and at later time points. In Fig. 1d this difference, c0(H+)-c(H+), is presented, and the results show that the overall ion exchange was indeed much more pronounced with lower pH of the dissolution medium. This should not come as a surprise, as a larger concentration of protons in solution can be expected to result in more ion exchange occurring. FTIR spectra showed significant changes after 45S5 immersion in pH 5 and pH 7.3 solution compared to the unreacted glass (Fig. 2a,b). The non-bridging oxygen (NBO, Si-O- alkali+) band at 915 cm-1 disappeared (owing to ion exchange [5]) and a new band appeared at about 790 cm-1, which is usually assigned to Si— O—Si vibrations between silicate tetrahedra [14] after treatment of bioactive glasses in Tris buffer [15] or simulated body fluid (SBF) [12], indicating formation of a silica-gel surface layer after leaching of Ca2+ and Na+ ions and formation of Si—OH groups. At later time points (3 and 6 hours for pH 5 and pH 7.3 solutions, respectively), a sharp, high intensity band appeared at 1025 cm-1 as well as a split band at 560 and 600 cm-1. These bands correspond to the formation of a crystalline phosphate surface layer, and particularly the split band is the most characteristic region for apatites. With time, these bands sharpened and increased in intensity. Formation of apatite was also confirmed by XRD (patterns shown for 3 days in Fig. 2d). The fact that 45S5 seems to form apatite well in low pH conditions is of great interest for clinical applications, as it shows that even under low pH conditions 45S5 can still be expected to perform its apatite forming action, e.g. during inflammation [9] or in the oral cavity after consumption of acidic beverages [16]. In addition, the pH rise caused during ion release may help to counteract the pH drop during bacterial infections. Indeed, this pH rise has also been suggested to cause antibacterial properties of BG [17]. FTIR spectra of 45S5 treated in pH 9 solution (Fig. 2c) did not show pronounced changes. The intensity of the NBO band at 915 cm-1 decreased, suggesting that some ion exchange had occurred, in agreement with ICP-OES and pH results (Fig. 1). The typical apatitic features discussed above (at 560, 600 and 1025 cm-1), however, did not appear, in agreement with absence of apatite-related reflections in XRD patterns at 3 days (Fig. 2d). Instead, FTIR spectra showed a broad, low-intensity band at 590 cm-1, which is commonly taken as an indication of presence of either amorphous calcium phosphates or calcium apatites of poor crystallinity [18], suggesting that the process of apatite formation had started, but that apatite was forming at much slower rates than at lower pH. This agrees with ICP-OES results, which showed that in all 3
three solutions phosphate concentrations decreased after an initial maximum at early time points, which usually coincides with apatite precipitation [6]. While a higher pH is thought to be favourable for apatite formation [19], here apatite formation was very low at pH 9, owing to slow ion release and thus low concentrations of calcium and phosphate ions in solutions. This probably only delayed apatite formation, as with time an increase in concentrations can be expected, allowing for more apatite precipitation. Presence of carbonate substitution in the apatite [20] was indicated by a band at about 870 cm-1 in FTIR spectra, present for 45S5 treated at pH 7.3 for 1 or 3 days (Fig. 2b). For all samples, broad CO32- bands were present from 1400 cm-1, indicating B-type substitution (i.e. carbonate replacing a phosphate group). These bands were most pronounced for 45S5 treated in pH 9 solution (despite low apatite formation) and lowest for samples treated at pH 5, suggesting that carbonate was incorporated into apatite more readily at higher pH values. This agrees with findings that in alkali-containing solutions both alkali and carbonate incorporation into apatite increased with increasing pH of the solution [19]. According to Hench's original mechanism of BG degradation and apatite formation [21] the exchange of alkali cations from the glass for protons from the surrounding medium is the first step in BG dissolution. During the dissolution studies here, however, the concentrations of calcium ions in solution, while being lower than those of sodium ions (Fig. 1b,c), appeared also at very early time points, suggesting that while some differences in release rates may exist (owing to differences in field strength), calcium ions are also released fast. However, as calcium is consumed (together with phosphate ions) during apatite precipitation, which occurred at very early time points (Fig. 2), it is not possible to make any statements on Ca2+ release, except that it is likely to be higher than indicated by the concentrations detected in solution. It is therefore possible that no differences exist between the release rates of alkali and calcium ions, which is also confirmed by alkali-free glasses forming apatite no slower than alkali-containing ones [6]. A later step of BG degradation and apatite formation according to Hench [21] is alkaline hydrolysis of Si— O—Si bonds. This hydrolysis is known to occur at high pH values (usually above pH 10), resulting in congruent dissolution of silicate glasses [22]. The pH range detected during in vitro dissolution or cell culture studies is usually well below that (Fig. 1), and ICP-OES results suggest that no congruent dissolution occurred up to pH 9. It has been noted that low network connectivity BG, e.g. 45S5, may release silicate chains without the need for Si—O—Si hydrolysis [23]. However, the fact that bioactive phospho-silicate glasses, even more polymerised compositions such as S53P4 [24], degrade completely in the body [25-28] suggests that Si—O—Si hydrolysis does occur to some extent. One also needs to keep in mind that even if the overall pH detected is well below pH 10, it may be significantly higher locally, e.g. very near the BG/water interface, allowing for Si—O—Si hydrolysis to occur. Solubility of amorphous silica has been shown to increase rapidly for pH values above 8, while possibly going through a minimum between pH 7 and 8 [29]. Our data here seem to agree with this, with concentrations of silicon species at 3 days being higher at pH 9 (3.2±0.1 mmol L-1) than at pH 7.3 (1.8±0.1 mmol L-1) and pH 5 (2.1±0.1 mmol L-1). Si concentrations increased with time, possibly as pH also increased with time (Fig. 1c). 4
4. Conclusion Bioglass® 45S5 formed apatite significantly faster at low pH than at physiological, caused by faster ion exchange. Ion release at alkaline pH was slow, by contrast, resulting in significantly delayed apatite precipitation. These results are of interest for clinical applications of 45S5, as they suggest that low pH conditions (e.g. during inflammation) are unlikely to inhibit apatite formation and, thus, bioactive glass clinical performance.
Acknowledgements The authors acknowledge Ms Brunhilde Dreßler, Institute of Geography, University Jena, for support with ICP-OES measurements and funding by the Carl Zeiss Foundation, Germany.
References [1] Hench LL, Splinter RJ, Allen WC, Greenlee TK. Bonding mechanisms at the interface of ceramic prosthetic materials. J Biomed Mater Res. 1971;5:117-41. [2] Hench LL, Hench JW, Greenspan DC. Bioglass®: A short history and bibliography. J Aust Ceram Soc. 2004;40:1-42. [3] Hench LL, Polak JM, Xynos ID, Buttery LDK. Bioactive materials to control cell cycle. Materials Research Innovations. 2000;3:313-23. [4] Shah FA, Brauer DS, Wilson RM, Hill RG, Hing KA. Influence of cell culture medium composition on in vitro dissolution behavior of a fluoride-containing bioactive glass. J Biomed Mater Res A. 2014;102:64754. [5] Brauer DS, Karpukhina N, O'Donnell MD, Law RV, Hill RG. Fluoride-containing bioactive glasses: Effect of glass design and structure on degradation, pH and apatite formation in simulated body fluid. Acta Biomater. 2010;6:3275-82. [6] Mneimne M, Hill RG, Bushby AJ, Brauer DS. High phosphate content significantly increases apatite formation of fluoride-containing bioactive glasses. Acta Biomater. 2011;7:1827-34. [7] Shah FA, Brauer DS, Desai N, Hill RG, Hing KA. Fluoride-containing bioactive glasses and Bioglass® 45S5 form apatite in low pH cell culture medium. Mater Lett. 2014;119:96-9. [8] Cannillo V, Pierli F, Ronchetti I, Siligardi C, Zaffe D. Chemical durability and microstructural analysis of glasses soaked in water and in biological fluids. Ceram Int. 2009;35:2853-69. [9] Punnia-Moorthy A. Evaluation of pH changes in inflammation of the subcutaneous air pouch lining in the rat, induced by carrageenan, dextran and staphylococcus aureus. J Oral Pathol Med. 1987;16:36-44. [10] Groh D, Döhler F, Brauer DS. Bioactive glasses with improved processing, Part 1: Thermal properties, ion release and apatite formation. Acta Biomater. 2014;10:4465–73. [11] Chen X, Brauer DS, Karpukhina N, Waite RD, Barry M, McKay IJ, et al. ‘Smart’ acid-degradable zincreleasing silicate glasses. Mater Lett. 2014;126:278-80. [12] Jones JR, Sepulveda P, Hench LL. Dose-dependent behavior of bioactive glass dissolution. J Biomed Mater Res. 2001;58:720-6. [13] Fagerlund S, Hupa L, Hupa M. Dissolution patterns of biocompatible glasses in 2-amino-2hydroxymethyl-propane-1,3-diol (Tris) buffer. Acta Biomater. 2013;9:5400-10. [14] Filgueiras MRT, La Torre G, Hench LL. Solution effects on the surface reactions of three bioactive glass compositions. J Biomed Mater Res. 1993;27:1485-93. [15] Brauer DS, Mneimne M, Hill RG. Fluoride-containing bioactive glasses: Fluoride loss during melting and ion release in tris buffer solution. J Non-Cryst Solids. 2011;357:3328-33. [16] Burwell AK, Litkowski LJ, Greenspan DC. Calcium sodium phosphosilicate (NovaMin): remineralization potential. Adv Dent Res. 2009;21:35-9. 5
[17] Gubler M, Brunner TJ, Zehnder M, Waltimo T, Sener B, Stark WJ. Do bioactive glasses convey a disinfecting mechanism beyond a mere increase in pH? International Endodontic Journal. 2008;41:670-8. [18] Sepulveda P, Jones JR, Hench LL. In vitro dissolution of melt-derived 45S5 and sol-gel derived 58S bioactive glasses. J Biomed Mater Res. 2002;61:301-11. [19] Elliott JC. Structure and chemistry of the apatites and other calcium orthophosphates. 1st ed. Amsterdam, New York, London, Tokyo: Elsevier; 1994. [20] Lu X, Leng Y. Theoretical analysis of calcium phosphate precipitation in simulated body fluid. Biomaterials. 2005;26:1097-108. [21] Hench LL, Andersson Ö. Bioactive glasses. In: Hench LL, Wilson J, editors. An introduction to bioceramics. Singapore: World Scientific Publishing; 1993. p. 41-62. [22] Hench LL, Clark DE. Physical chemistry of glass surfaces. J Non-Cryst Solids. 1978;28:83-105. [23] Hill RG, Brauer DS. Predicting the bioactivity of glasses using the network connectivity or split network models. J Non-Cryst Solids. 2011;357:3884-7. [24] Lindfors NC, Koski I, Heikkila JT, Mattila K, Aho AJ. A prospective randomized 14-year follow-up study of bioactive glass and autogenous bone as bone graft substitutes in benign bone tumors. J Biomed Mater Res B. 2010;94B:157-64. [25] Wheeler DL, Stokes KE, Hoellrich RG, Chamberland DL, McLoughlin SW. Effect of bioactive glass particle size on osseous regeneration of cancellous defects. J Biomed Mater Res. 1998;41:527-33. [26] Froum S, Cho SC, Rosenberg E, Rohrer M, Tarnow D. Histological comparison of healing extraction sockets implanted with bioactive glass or demineralized freeze-dried bone allograft: A pilot study. Journal of Periodontology. 2002;73:94-102. [27] Ducheyne P. Effect of bioactive glass particle size on osseous regeneration. J Biomed Mater Res. 1999;46:301-3. [28] Hill R, Rawlinson S, Davis G, Nehete S, Shahdad S. A clinical case study: Using a strontium substituted bioactive glass - StronBone® - to fill alveolar sockets. Eur Cells Mater. 2014;28:51. [29] Iler RK. The chemistry of silica: solubility, polymerization, colloid and surface properties, and biochemistry. New York: Wiley; 1979.
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Figures a
b
c
d
Figure 1: Ionic concentrations in solution (black, left axis) and pH (blue, right axis) over time for initial pH of (a) 5, (b) 7.3 and (c) 9. (c) Difference between initial H+ concentration in solution and H+ concentration at later time points.
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a
b
c
d
Figure 2: FTIR spectra of 45S5 after treatment in solution for various time periods. Initial pH: (a) 5, (b) 7.3 and (c) 9. (d) XRD patterns of untreated glass and powders after immersion at pH 5, 7.3 and 9 for 3 days. (Hydroxycarbonate apatite reflections are marked by asterisks; JCPDS 00-019-0272.)
Highlights • • •
Enhanced ion release of Bioglass® 45S5 at low pH Enhanced apatite formation of Bioglass® 45S5 at low pH Negligible apatite formation of Bioglass® 45S5 at pH 9
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