Fluoride-containing bioactive glasses: Glass design, structure, bioactivity, cellular interactions, and recent developments

Materials Science and Engineering C 58 (2016) 1279–1289

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Review

Fluoride-containing bioactive glasses: Glass design, structure, bioactivity, cellular interactions, and recent developments Furqan A. Shah ⁎ Department of Biomaterials, Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Göteborg, Sweden

a r t i c l e

i n f o

Article history: Received 29 June 2015 Received in revised form 3 August 2015 Accepted 27 August 2015 Available online 2 September 2015 Keywords: Fluoride Bioactive glass Fluorapatite Hydroxyapatite Bioactivity

a b s t r a c t Bioactive glasses (BGs) are known to bond to both hard and soft tissues. Upon exposure to an aqueous environment, BG undergoes ion exchange, hydrolysis, selective dissolution and precipitation of an apatite layer on their surface, which elicits an interfacial biological response resulting in bioactive fixation, inhibiting further dissolution of the glass, and preventing complete resorption of the material. Fluorine is considered one of the most effective in-vivo bone anabolic factors. In low concentrations, fluoride ions (F−) increase bone mass and mineral density, improve the resistance of the apatite structure to acid attack, and have well documented antibacterial properties. F− ions may be incorporated into the glass in the form of calcium fluoride (CaF2) either by partsubstitution of network modifier oxides, or by maintaining the ratios of the other constituents relatively constant. Fluoride-containing bioactive glasses (FBGs) enhance and control osteoblast proliferation, differentiation and mineralisation. And with their ability to release fluoride locally, FBGs make interesting candidates for various clinical applications, dentinal tubule occlusion in the treatment of dentin hypersensitivity. This paper reviews the chemistry of FBGs and the influence of F− incorporation on the thermal properties, bioactivity, and cytotoxicity; and novel glass compositions for improved mechanical properties, processing, and bioactive potential. © 2015 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glass design and structure . . . . . . . . . . . . . . . . . . . . . . Fluoride-containing bioactive glasses (FBGs) . . . . . . . . . . . . . . 3.1. Stability of fluorapatite . . . . . . . . . . . . . . . . . . . . . 3.2. CaF2 incorporation by part-substitution of modifier oxides . . . . . 3.3. CaF2 incorporation with constant network former-to-modifier ratios 4. Dissolution and bioactivity of fluoride-containing bioactive glasses . . . . 5. Thermal properties of fluoride-containing bioactive glasses . . . . . . . . 6. Biocompatibility and cytotoxicity of fluoride-containing bioactive glasses . 7. Recent developments . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Multi-component glasses . . . . . . . . . . . . . . . . . . . . 7.2. Oxynitride and oxyfluoronitride glasses . . . . . . . . . . . . . 7.3. Mixed-alkali effect . . . . . . . . . . . . . . . . . . . . . . . 7.4. Alkali-free glasses and glass ceramics . . . . . . . . . . . . . . 7.5. Sol–gel derived glasses and glass-ceramics . . . . . . . . . . . . 7.6. Fluorapatite-containing 3D scaffolds . . . . . . . . . . . . . . . 8. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author at: Department of Biomaterials, Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden E-mail address: [email protected].

http://dx.doi.org/10.1016/j.msec.2015.08.064 0928-4931/© 2015 Elsevier B.V. All rights reserved.

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1. Introduction Bioactive glass (BG) belongs to a diverse group of silico–soda–lime– phosphate materials [1] known to bond to both hard and soft tissues [2,3]. Upon exposure to an aqueous environment, these glass compositions undergo ion exchange, hydrolysis, selective dissolution and reprecipitation of an apatite layer on their surface [4]. This surface apatite elicits an interfacial biological response inducing bioactive fixation [5] and inhibits further dissolution of the glass, preventing complete resorption of the material. The interface thus established allows interaction with biological moieties such as collagen, fibronectin, plasma proteins, cells including blood cells, fibroblasts and osteoblasts; which in due course of time promotes the development of strong chemical bonding between the biomaterial and the physiological environment [6]. Such a response is verification of high levels of biocompatibility and stability of BG implants [7]. However, central to this bioactive behaviour is the controlled rates of Ca2+ and Si4+ ion release [8]. Glass dissolution behaviour is often studied using physiologically relevant test solutions such as simulated body fluid (or SBF) [9], Tris–buffer solutions [10,11], and a variety of cell culture media [12]. The dissolution kinetics and consequently the rate of formation of this surface-active apatite layer are a direct result of atomic structure [13]. Incorporation of certain therapeutic agents into the glass, such as fluoride (F−) ions in the form of calcium fluoride (CaF2) is of particular interest, as fluorine decreases their chemical reactivity by acting as a corrosion inhibitor and promotes the formation of a thin surface gel layer with a high silica concentration [14]. These may be incorporated either by part-substitution of network modifier oxides, Na2O or CaO [15], or by keeping the ratios of the other constituents relatively constant [16]. This review looks specifically at the chemistry of fluoride-containing bioactive glass (FBG) compositions; the chemical stability of fluorapatite (Ca10(PO4)6F2); how fluoride ion incorporation influences the thermal properties, bioactivity, and cytotoxicity; and novel glass compositions for improved mechanical properties, processing, and bioactive potential. 2. Glass design and structure The classic Bioglass® 45S5 contains SiO2 (46.2 mol%), Na2O (24.3 mol%), CaO (26.9 mol%) and P2O5 (2.6 mol%). Silica (SiO2) is the primary glass network former, and although high silica content is recommended, bioactivity and biodegradation decrease with N 60 mol% SiO2 content. Network modifying oxides (e.g., Na2O and CaO) disrupt the glass network, making it susceptible to dissolution in aqueous environments. And while phosphate acts as a surface nucleation site for amorphous calcium phosphate (ACP), it is not a critical component for bioactivity [17]. Indeed, phosphate-free glasses have been shown to be bioactive both in vitro [18] and in vivo [19]. The rate of glass degradation in physiological conditions has a positive compositional dependence. According to the SiO2–CaO–Na2O ternary phase diagram for glass kinetics proposed by Hench [20], compositions containing 30–60 mol% SiO2, 10–50 mol% CaO, 5–40 mol% Na2O, and a fixed P2O5 content of 6 mol%, for example Bioglass® 45S5, exhibit maximum bioactivity. Obtained via the traditional melt-quench route, glass compositions with N65 mol% SiO2 are bio-inactive (or bio-inert) owing to the high number of bridging oxygen atoms. Compositions with N 50 mol% SiO2, N 35 mol% Na2O, and b 10 mol% CaO display rapid degradation rates and are resorbed within days to weeks. Compositions with silica content below 30 mol% do not form glass and are isolated by fibrous encapsulation in vivo [21]. In contrast to the melt-quench route, glasses obtained by the sol–gel method exhibit bioactivity for up to 90 mol% SiO2 content [22], and since Na2O is not required to lower the melting temperature, they often contain fewer components than melt-quenched glasses [13]. And while, melt-quenched glasses are dense and exhibit mainly nonporous surfaces, the sol–gel texture is inherently porous and provides a specific surface area up to two orders higher in magnitude than for

similar compositions of melt-quenched glasses, which provides more sites for ACP nucleation [23]. Immersion in physiological solutions causes cations (Na+ and Ca2+) released from the glass to be exchanged for H+ (or H3O+) ions in the solution, thus causing a pH increase, followed by network dissolution by Si–O–Si bond breakage, or alternatively, solubilisation of entire Si–O–Si chains [24], and later condensation of SiOH as a silica-gel layer. Ca2+ and PO3− 4 ions released from the glass and those present in solution precipitate to form an ACP layer in the early reaction stages [25], which later undergoes crystallisation by incorporation of CO2− 3 , OH−, or F− anions, giving rise to a mixed hydroxy-, carbonated-, fluor-, apatite layer to which living bone can bond. Although the mechanism is widely accepted, it necessitates the presence of phosphate within the glass and therefore fails to explain the behaviour of phosphate-free compositions [18]. Alternatively, silicate glasses may be viewed as inorganic network polymers of oxygen crosslinked by silicon atoms. The surface reactivity, solubility, thermal expansion coefficient, and the probability of undergoing glass-in-glass phase separation can then be predicted from parameters such as network connectivity and crosslink density [24]. Network connectivity is the average number of bridging oxygen atoms per network forming element, while crosslink density is the average number of additional crosslinking bonds, N2, for the elements other than oxygen (i.e., Si, P, B, Al) forming the glass network backbone. Crosslink density affects the physical properties of BG in a manner similar to polymers, i.e., the lower the crosslink density, the lower the glass transition temperature (Tg), and higher the surface reactivity, dissolution and degradation. The transition from a three-dimensional network to a linear chain occurs at a crosslink density of 0 (or a network connectivity of 2). Not surprisingly, the reactivity and solubility change dramatically at the point of transition from a high molar mass crosslinked network to a low molar mass linear polymer chain. Most glass compositions that are known to bond chemically to biological tissues exhibit network connectivity values below 2, indicating an absence of crosslinking. For instance, Bioglass® 45S5 has a calculated network connectivity value of 1.9. This implies that silicate chains are able to dissolve into solution without Si–O–Si bond cleavage, without the need for hydroxyl (OH−) ion mediated Si–O–Si breakdown. Moreover, glasses with network connectivity values just above 2, and the structure being slightly more interconnected, would still be able to react without hydrolytic breakdown of the Si–O–Si bonds since the P–O bond can also be readily broken. Minor changes in composition have dramatic effects on reactivity and biological properties. While lower network connectivity makes the glass more reactive, higher network connectivity makes the glass more chemically durable and improves the mechanical properties, e.g., for high stress-bearing applications [24].

3. Fluoride-containing bioactive glasses (FBGs) Fluoride incorporation into BGs improves their bone bonding ability. A pioneering study on FBGs reported a series of glasses containing 42–60 mol% SiO2, 2.6 mol% P2O5, and a fixed Na2O-to-CaO ratio of 0.9, where 40 mol% CaO was then substituted by CaF2 [26]. Glass discs were suspended in Tris–buffer solution, at a volume-to-surface ratio of 0.1 ml/mm2. The work established that the Si–O–Si bonds and Si–O bonds with one non-bridging oxygen form early in the reaction between the glass surface and an aqueous environment. In the late stages, phosphorus ion depletion from the solution occurs as the amorphous calcium phosphate (ACP) layer crystallises over the silica gel layer. While high silica content is associated with formation of the silica-rich layer followed by the calcium phosphate layer, low silica content is associated with formation of both layers simultaneously. The rates of ion release and fluorapatite deposition diminish with increasing SiO2 content. However, the rates of ion release change dramatically at the

F.A. Shah / Materials Science and Engineering C 58 (2016) 1279–1289 Table 1 Nominal compositions of the HNaCaF2 and HCaCaF2 glasses in mol% ([38]). Glass

SiO2

P2O5

CaO

Na2O

H (45S5) HNaCaF2 5% HNaCaF2 10% HNaCaF2 15% HNaCaF2 20% HNaCaF2 24.3% HCaCaF2 5% HCaCaF2 10% HCaCaF2 15% HCaCaF2 20% HCaCaF2 26.9%

46.2 46.2 46.2 46.2 46.2 46.2 46.2 46.2 46.2 46.2 46.2

2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6 2.6

26.9 26.9 26.9 26.9 26.9 26.9 21.9 16.9 11.9 6.9

24.3 19.3 14.3 9.3 4.3 24.3 24.3 24.3 24.3 24.3

CaF2

Density (g·cm−3)

5.0 10.0 15.0 20.0 24.3 5.0 10.0 15.0 20.0 26.9

2.719 2.737 2.761 2.774 2.788 2.800 2.703 2.679 2.641 2.599 2.536

boundary between high silica (conventional) glass and low silica (invert) glass compositions. The inclusion of CaF2 in the glass composition distorts the crystal structure, which develops when the ACP layer crystallises. Fluorapatite crystals of a rod-shaped morphology are formed instead of carbonated hydroxyapatite crystals of a flake-like morphology. Associated with the fluoride addition is an increase in Na+ and Ca2+ release from the glass [27]. Addition of CaF2 to silico–soda–lime–phosphate glasses lowers their chemical reactivity, because F− ions inhibit the formation of a thick silica gel layer on the surface [14,28]. However, the lack of a continuous silica-rich layer does not affect the ability of the FBG surface to eventually become covered by a layer of fluorapatite [15]. The formation of fluorapatite on the surface is of particular interest due to higher chemical stability than fluoride-free apatites. 3.1. Stability of fluorapatite The pH range 5.0–5.5 has been described as a “hypothetical critical decalcifying pH level” by Stephan (1944), above which Ca2+ salts do not dissolve from dental enamel in normal mouth conditions, and below which decalcification would occur readily [29]. At nearphysiological pH (6.9–7.4), hydroxyapatite in dental enamel exhibits two- and four-fold higher Ca2+ and PO3− solubility than fluorapatite 4 [30,31]. The process of dissolution and reprecipitation, in the presence of F− ions, results in incorporation of fluoride ions into enamel to yield a mineral phase that is more resistant to acid attack [32]. The ionic radius of the hydroxyl (OH−) ion in the hydroxyapatite (Ca10(PO4)6OH2) lattice is believed to be too large to fit inside the Ca(II) triangle, causing the oxygen atom to be slightly offset from the calcium plane, thus generating an electric dipole moment and raising the lattice energy. The ionic radius of the F− ion is smaller than the OH− ion, and when available, F− ions replace OH− ions in the apatite lattice to form fluorapatite. The substitution of F− ions for OH− ions allows significant reduction in the unit cell volume by positioning the F− ion at the centre of the Ca(II) triangle. The lattice energy is lowered — resulting in a crystal structure with better chemical durability [33]. The fluorapatite structure contains Ca–F, Ca–O, and P–O bonds; and based on their electrostatic bond strengths, their relative destruction

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Table 3 Nominal compositions of the high phosphate content glasses in mol%. Theoretical network connectivity is 2.08 ([42]). Glass

SiO2

P2O5

CaO

Na2O

A2 B2 C2 D2 E2 F2 H2

38.14 36.41 34.60 32.95 31.37 28.40 34.60

6.33 6.04 5.74 5.47 5.21 4.71 5.74

25.91 24.74 23.51 22.38 21.31 19.29 50.38

29.62 28.28 26.87 25.59 24.36 22.06

CaF2 4.53 9.28 13.62 17.76 25.54 9.28

rates are in the order: Ca–F N Ca(I)–O N Ca(II)–O N P–O. Therefore once all Ca–F and Ca–O bonds are broken, fluorapatite is destroyed; since P–O bond cleavage is considered unnecessary for fluorapatite − dissolution [34]. Ca2+, PO3− ion release is found to be non4 , and F stoichiometric with dissolved Ca/P and Ca/F ratios being higher than mineral stoichiometric ratios, suggesting that Ca2+ and F− ions are leached preferentially, in relation to PO3− [35]. Ca2+ and F− removal 4 is associated with PO3− hydrolysis and leads to the formation of a 4 di-calcium phosphate layer (CaHPO4), with Ca/P = 1 [36]. In B-type carbonate substituted apatites, when present in nonstoichiometric amounts, F− ions are trapped interstitially within the apatite structure, instead of at the vacancy left by the CO2− for PO3− 3 4 substitution, causing a destabilising effect on the apatite structure. The result is increased solubility and chemical reactivity of carbonated fluorapatite, particularly in acidic pH environments [37]. The rates of − Ca2+, PO3− 4 and F release decrease with increasing the pH from 2 to 6 for fluorapatite, and from 4 to 7 for carbonated fluorapatite [35]. Additionally, no chemical bonding exists between the CO2− and F− 3 ions and therefore the arrangement is chemically unstable in simulated physiological environments. Further, it has been suggested that the elimination of carbonate groups during early stages of acid-mediated demineralisation of carbonated hydroxyapatite may in fact be of benefit in forming fluorapatite [33]. 3.2. CaF2 incorporation by part-substitution of modifier oxides CaF2 may be incorporated into bioactive glasses by part-substitution of the network modifier oxides [15]. Molecular dynamics (MD) simulations of glasses designed by part-substitution of Na2O or CaO by CaF2 in Bioglass® 45S5 (Table 1) show that fluorine forms complexes with modifier cations, i.e., sodium and calcium [38], having the highest charge-to-size ratio [39]. Fluorine increases polymerisation of the silicate tetrahedra by removing modifiers from the siliceous matrix, however no appreciable amount of Si–F bonds are detected by either

Table 2 Nominal compositions of the low phosphate content glasses in mol%. Theoretical network connectivity is 2.13 ([41]). Glass

SiO2

P2O5

CaO

Na2O

A B C D E F G H

49.47 47.12 44.88 42.73 40.68 36.83 33.29 44.88

1.07 1.02 0.97 0.92 0.88 0.80 0.72 0.97

23.08 21.98 20.94 19.94 18.98 17.18 15.53 44.87

26.38 25.13 23.93 22.79 21.69 19.64 17.75

CaF2 4.75 9.28 13.62 17.76 25.54 32.71 9.28

Fig. 1. 19F MAS NMR spectra of glasses B, C, D, E, F, G, H (Table 1). Spinning side bands are marked by an asterisk [16].

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domains that remain separated from the phosphosilicate network [28]. Importantly, in contrast to MD simulations, 19F MAS NMR and 19F{23Na} REDOR experiments suggest the presence of FNax clusters in the glass structure, characterised by a well resolved band at −211 ppm [40]. 3.3. CaF2 incorporation with constant network former-to-modifier ratios

Fig. 2. 29Si MAS NMR spectra of glasses A, C, E, F, G (Table 1). Spinning side bands are marked by an asterisk [16].

29

Si magic angle spinning nuclear magnetic resonance (MAS NMR) [38] or 29Si{19F} REDOR experiments [40]. More specifically, ab initio MD simulations of the composition ‘HCaCaF2 10%’ indicate that fluorine atoms are associated with both Na and Ca atoms, and a preference for fluorine to coordinate to either sodium or calcium is not observed, since each F− is located within a mixed shell of modifier Na+ and Ca2+ cations, the composition of which directly corresponds to the relative Na/Ca prevalence. Moreover, a high affinity of Ca2+ for PO3− 4 ions leads to calcium-rich phosphate aggregates separated from calciumpoor silicate regions in non-fluoridated glasses, while Na+ and Ca2+ cations in FBGs are attracted towards F− to give rise to Na–Ca–F-rich

CaF2 may also be added without altering the network former-tomodifier ratio, rather than substituting for the modifier oxides, and therefore without affecting the glass network connectivity. Since the number of bridging vs. non-bridging oxygen atoms per silicate structural unit remains relatively constant [24], the dissolution behaviour too remains predictable. Two such series of low phosphate (Table 2) [41] and high phosphate (Table 3) [42] compositions are discussed. 19F (Fig. 1) and 29Si (Fig. 2) MAS NMR experiments have shown that CaF2 addition also does not give rise to Si–F bonds (the proportion of Si–F bonds and non-bridging fluorine atoms is small, if not zero) [16]. It is therefore proposed that F− ions exist as charged CaF+ species, consistent with previous molecular dynamics simulation studies which suggested F− ions to be exclusively bonded to modifier cations [38]. Further, an absence of Si–O–P bonds indicates that phosphate does not contribute to the glass network and exists as phase separated ortho+ phosphate (PO3− 4 ) or phosphate clusters charged balanced by Na and 2+ Ca ions [16]. 4. Dissolution and bioactivity of fluoride-containing bioactive glasses Na2O substituted glasses (HNaCaF2) are more compact in structure than CaO substituted glasses (HCaCaF2) since mobile Na+ ions are replaced by relatively immobile Ca2+ ions; and on immersion in Dulbecco's

Fig. 3. pH of Tris–buffer solution after immersion of (a) low phosphate and (b) high phosphate glasses vs. time and (c) low and (d) high phosphate content glass powder vs. CaF2 content in the glass [42].

F.A. Shah / Materials Science and Engineering C 58 (2016) 1279–1289

Fig. 4. XRD patterns of F4, F9, F17 (glasses B, C, and E, respectively, in Table 1), and 45S5 (a, c, e, and g, respectively) after 3 days and F4, F9, F17 and 45S5 (b, d, f, and h, respectively) after 7 days immersion in A-MEM [45].

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Modified Eagle's Medium, attributable to the relative ease of Na+ for H+ exchange in solution, Na2O substitution by CaF2 results in lowered pH rise than CaO substituted glasses — an effect that may be beneficial for in vivo applications where there is a risk of pH associated tissue damage. However, Na2O substitution also results in high glass Ca2+ content, which in combination with high HCO− 3 concentration of the medium induces calcite (CaCO3) formation at the expense of apatite [15]. Upon immersion of the same glasses in artificial saliva, the formation of crystalline apatite is much slower than in Tris–buffer solution owing to the presence of a protein called mucin, which though does not inhibit the crystallisation of calcium phosphate but significantly delays the process. Reportedly, mucin uptake is relatively quicker for Na2O-substituted glasses as a result of the higher Ca2+ content. Conversely, CaO-substituted glass exhibits higher ion release profiles and quicker fluorapatite formation in artificial saliva. Further, the lower pH increment associated with FBGs has in fact been assigned to deprotonation reactions involved in the formation of calcite [43]. Upon dissolution, lower pH increments are reported for FBGs compared to non-fluoridated compositions. One possible explanation of this effect is an exchange of fluoride ions in BG for hydroxyl (OH−) ions in the solution, thus diminishing the pH increase observed upon dissolution (Fig. 3) [41]. However, it has been demonstrated that the lowered pH increment is not due to the increased fluoride content, but in fact by lower mole fraction of silica in the glass and subsequently lower OH− ion concentration. Dissolution experiments conducted in Tris–buffer solution have shown that the pH is controlled by the silicate matrix (via ion exchange processes), and that fluoride ion release contributes less to the overall pH [44]. The amounts of fluoride and phosphate ions in the glass influence the relative amounts of the various crystalline phases that form

Fig. 5. (a–b) FTIR spectra and (c–d) XRD patterns of low (top) and high (bottom) phosphate content glass powders at 1 week immersion in Tris–buffer [42].

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Fig. 6. Concentrations of (a) Ca, (b) P, (c) Si, and (d) F in culture media A-, H-, HC-, and HS-MEM [46].

following immersion in physiological test solutions. While F− ions in low concentrations facilitate fluorapatite formation even at low pH conditions, e.g., acetate-buffered and nominally carbonate-free Eagle's Minimal Essential Medium at pH 4.6) [45], higher fluoride content results in the formation of fluorite (Fig. 4). For low phosphate glasses, higher concentrations of fluorine in the glass support the formation of fluorite instead of apatite, due to an unfavourable Ca/P ratio [41]. Not surprisingly, increasing the glass phosphate content tends to offset the formation of fluorite, supporting the formation of fluorapatite, and enabling apatite nucleation at lower pH levels [42]. However, increasing glass fluoride content results in the formation of fluorite and a progressive reduction in fluorapatite, presumably due to a calcium phosphate imbalance. Although, both the low- and high phosphate series show similar trends with the progressive increase in the glass fluoride content, the apatitic phosphate bands observed on Fourier transform infrared spectroscopy (FTIR) are better defined for high phosphate glasses than for low phosphate glasses with similar fluoride content (Fig. 5). The composition of the test solution affects the dissolution behaviour of glasses. In Eagle's Minimal Essential Medium (MEM) of varying complexity, e.g., the absence of carbonate ions (A- and H-MEM), the presence of carbonate ions (HC-MEM), and the presence of carbonate ions and serum proteins (HS-MEM) [46], it has been demonstrated that the composition of the test solution not only affects glass dissolution kinetics (Fig. 6), but also the rates at which the different crystalline phases form (Fig. 7) as well as the crystal habit (Fig. 8). This effect is particularly pronounced in the presence of serum proteins where in vitro apatite formation is considerably delayed [47]. Although low phosphate glasses form apatite in Tris–buffer solution; phosphate concentration acts as the limiting factor, resulting in the formation of fluorite for high-fluoride glasses and the formation of calcite

for fluoride-free glass [44]. However, the formation of calcite (instead of fluorite) is reported for high-fluoride glasses following immersion in Dulbecco's Modified Eagle's Medium. High HCO− 3 ion concentration in cell culture medium, as well as a discrepancy in the Ca/P ratio (relative Ca2+ excess) causes the formation of crystalline CaCO3 [15]. SiO2–P2O5–CaO–Na2O–CaF2 glasses (0 b CaF2 ≥ 17.76 mol%) prepared via the conventional melt-quench route lose F− ions in the form of hydrofluoric acid (HF) after reaction with atmospheric water during melting. However, the nominal and analysed CaF2 contents in the

Fig. 7. XRD patterns of untreated glass, UT; and after immersion for 7 days in media A-, H-, HC-, and HS-MEM (○ apatite, ▼ calcite) [46].

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with a considerable reduction in fluorapatite crystalline phases when the CaF2 content exceeds 13–14 mol% [41]. 5. Thermal properties of fluoride-containing bioactive glasses

Fig. 8. Scanning electron microscopy images after immersion for 7 days in (a, b) A-MEM; (c, d) H-MEM; (e, f) HC-MEM; and (g, h) HS-MEM. Scale bar: 1 μm [46].

glass tend to correlate linearly, while the analysed CaO contents increase [44]. But whether CaF2 is incorporated by substitution of modifier oxides [15], or by keeping the ratios of all of the other constituents constant [41,42], an increase in CaF2 causes an increase in the amount of Ca2+ ions that may be leached from the glass, subsequently supersaturating the solution and causing a Ca/P imbalance. This Ca/P ratio discrepancy gives rise to the formation of fluorite when the available phosphate levels are low [41], or alternatively to calcite when the available carbonate levels are high. The impact is significant with CaF2 ≥ 10 mol% [15], and when CaF2 is added at the expense of Na2O (HNaCaF2 10%; [38]), the combined CaO and CaF2 content may be as high as 36.9 mol%. A 50/50 ratio of fluorite to fluorapatite crystalline phases is observed when the CaF2 content is 13–14 mol%,

Increasing the fluoride content progressively lowers the glass transition (Tg) and crystallisation (Tc) temperatures, and the viscosity, indicating a less strongly bound network. This weaker bonding is related to weaker ionic crosslinking between modifier cations and nonbridging oxygen atoms, since F− ions form hypothetical CaF+ structural units that provide less ionic crosslinking than Ca2+ ions [48]. A similar effect has been reported for glass compositions in the SiO2–Al2O3– P2O5–CaO–CaF2 system [49]. These glasses undergo bulk nucleation and crystallise to fluorapatite and mullite. The introduction of nonbridging fluorine atoms into the glass network to replace bridging oxygen atoms, the Tg is lowered and crystallisation takes place at lower temperatures. Similarly, certain compositions in the SiO2–P2O5–CaO– MgO–CaF2 system crystallise to apatite (by bulk nucleation) and wollastonite (by surface nucleation). Fluorine reduces the temperature range over which viscous flow sintering may occur [50] and increases the thermal expansion coefficient (TEC) [51] causing the glass to crystallise relatively easily. The glass phosphate content also influences the crystallisation behaviour of fluoride-containing glasses. In the SiO2–Na2O–CaO–P2O5– CaF2 system, low-phosphate (b 1.1 mol% P2O5) glasses undergo spontaneous crystallisation of fluorite with increasing fluoride content [16,52]. In contrast, an opposite effect has been described for high-phosphate content (≥6 mol% P2O5) glasses [48], where increasing fluoride content favours the amorphous state and prevents spontaneous crystallisation during quenching and casting to obtain monoliths. It is believed that phosphate content does not change the glass structure significantly and remains under a certain critical size for nucleation, presumably as nanometre-sized droplets of orthophosphate dispersed in a silicate matrix. The crystallisation temperature decreases with decreasing particle size in low-phosphate glasses [16,52]. Here, the main crystalline phase is combeite (Na2Ca2Si3O9), which crystallises through a surfacenucleated process; and no crystalline orthophosphate is detected upon heat treatment. Conversely, peak crystallisation temperatures (Tp) are independent of particle size in high-phosphate glasses [48] owing to differences in the formed crystalline phases, which crystallise to sodium–calcium–fluoride–orthophosphate phases with increasing CaF2 content (N9 mol%). In a similar glass system containing 2.6 mol% P2O5, but where CaF2 was incorporated by part substitution of Na2O and CaO, the formation of fluorapatite, as well as fluorite, cuspidine (Ca4Si2O7F2), and nacaphite (Na2CaPO4F) phases was observed upon heat treatment [38]. This suggests that a minimum phosphate content is necessary for crystallisation of orthophosphates such as apatites. Further, the sodium content of glass also influences the formed crystalline phases. Apatite is the predominant crystalline phase in low-sodium glasses (≤19.3 mol% Na2O), while the PO3− tetrahedra in high-sodium glasses are surrounded by 4 too much Na+ for apatite formation resulting in the formation of mixed sodium–calcium–fluoride–phosphates such as Na2Ca(PO4)F or Na2Ca4(PO4)3F [38]. By contrast, orthophosphate units in sodium-free glasses are charge balanced by Ca2+ only [16], resulting in the crystallisation of fluorapatite upon heat treatment. 6. Biocompatibility and cytotoxicity of fluoride-containing bioactive glasses The effect of F− ion release from FBG particles has been studied on MG-63 human osteoblast-like cells. At a concentration of ∼50 μg/mL, or F− ions equivalent to 1.5 mg/L (assuming complete glass dissolution), incorporation of as low as 5 mol% CaF2 into the glass is able to exert cytotoxic effects on MG-63 cells at 24 h (Fig. 9) [53]. Increased

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Fig. 9. Scheme suggesting a likely mechanism for fluoride toxicity. F−: fluoride ion; G6PD: glucose 6-phosphate dehydrogenase; PPP: pentose phosphate pathway; L-NMMA: N-monomethyl-L-arginine; SOD: superoxide dismutase; ROS: reactive oxygen species; P-eNOS: phosphorylated endothelial nitric oxide synthase; NO: nitric oxide; PTIO: 2-phenyl4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide; RBC: packed erythrocytes; LDH: lactate dehydrogenase; MDA: malonyldialdehyde [53].

lactate dehydrogenase (LDH) release into the extracellular medium, accumulation of intracellular malondialdehyde (MDA), release of reactive oxygen species (ROS), and inhibited pentose phosphate pathway (PPP) activity are observed in a dose-dependent fashion. At the same time, glucose 6-phosphate dehydrogenase (G6PD) activity decreases and the level of glutathione (GSH) diminishes [54]. In vitro tests on FBG discs have demonstrated higher numbers of adherent Saos-2 cells for low-fluoride glasses (1–4.75 mol%). Cells in direct contact with glass discs, and cells exposed to the dissolution products from the glass discs showed higher alkaline phosphatase (ALP) and interleukin-6 (IL-6) activity for glasses containing ∼18 mol% CaF2 [55].

several distinct advantages such as reduced pH rise upon dissolution as a function of total fluoride content (CaF2 + SrF2). Additionally, F− and Sr2+ release for caries inhibition, K+ release for nerve desensitisation, and Zn2+ release for antibacterial and anti-gingivitis effects are also envisioned. The combination of fluoride and strontium ions could potentially be of interest for anti-osteoporotic bone graft substitute applications. The multi-component nature of such glasses allows tailoring of the composition for specific clinical scenarios; e.g., rapid apatite formation is desirable for dentinal tubule occlusion, while controlled and relatively slower dissolution is critical to match glass degradation and bone apposition rates.

7.2. Oxynitride and oxyfluoronitride glasses 7. Recent developments 7.1. Multi-component glasses Novel multi-component (SiO2–P2O5–CaO–CaF2–SrO–SrF2–Na2O– K2O–ZnO) glass compositions have been demonstrated to form fluorapatite and release therapeutically active ions in physiological solutions (e.g., Tris–buffer) which makes these glasses attractive components for use in remineralising dentifrices, particularly for treating dentine hypersensitivity [56] (Fig. 10). Such compositions display

Oxynitride and oxyfluoronitride glasses based on the SiO2–CaO– Na2O–CaF2–Si3N4 system represent a recent advancement in fluoridecontaining bioactive glasses [57,58]. Nitrogen, present as SiO3N and SiO2N2 structural units, substitutes for oxygen in the silicate network. Nitrogen incorporation (oxynitride glasses) results in increased density and compactness of the glass network, and greater crosslinking of the silicate network as a direct consequence of the higher coordination number of nitrogen. The Tg, microhardness, Young's modulus, and the indentation fracture toughness all increase with nitrogen content. The

Fig. 10. Scanning electron microscopy images of dentine disc at 7 days in Tris–buffer with a multi-component glass powder F13 (13.62 mol% CaF2 + SrF2) showing (a) glass particles on dentin surface, scale bar: 100 μm, (inset shows dentin disc after etching with citric acid before storage in Tris–buffer, scale bar: 30 μm), and (b) dentinal tubules (white arrow) partially blocked by apatite crystals (black arrow), scale bar: 5 μm [56].

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inclusion of fluorine (oxyfluoronitride glasses) lowers the glass melting temperature (Tm) through a network disrupting effect, facilitating the dissolution of nitrogen into the molten glass. Fluorine inclusion therefore substantially increases the maximum nitrogen content, as a consequence of which, the maximum hardness and Young's modulus can be increased further [57]. The network disruption effect of non-bridging fluorine ions also lowers the T g and therefore lower viscosity melts can be obtained at lower temperatures, making processing easier [58]. Nitrogen inclusion also increases the Young's modulus, viscosity, and microhardness in the SiO2–CaO–Si3N4–Al2O3 system, resulting in significantly high Tm (∼1700 °C). To achieve sufficiently low viscosities for glass forming and drawing processes, fluorine substitution for oxygen in Ca–Si–Al–O–N glasses decreases both the Tg and the Tm [59] — the Tm being 150–800 °C lower than the equivalent SiO2–CaO–Si3N4– Al2O3 compositions. Incorporation of both nitrogen and fluorine in Ca– Si–Al–O glasses extends glass formation regions through combinations of lowered Tm (fluorine) and higher viscosities (nitrogen) [60]. The vastly different effects of nitrogen and fluorine on the Young's modulus and microhardness are due to the manner in which their substitution for oxygen affects the glass free volume and thus the fractional compactness (i.e., the atomic packing density), while the differences in the effects on Tg reflect their different effects on the glass crosslink density [61]. The substitution effects of fluorine and nitrogen for oxygen are independent, but seemingly additive with fluorine substitution [62]. 7.3. Mixed-alkali effect A strong tendency of certain bioactive glass compositions, e.g., Bioglass® 45S5, is to undergo crystallisation upon heat treatment, which severely limits their range of applications. Attempts at improving their processing by reducing their tendency to crystallise have included increasing their silica content and thus their network connectivity, or incorporating intermediate oxides, or even reducing their phosphate content — all of which reduce glass bioactivity. Therefore, bioactive glasses known for their good processing are considerably less bioactive. One study has investigated the optimisation of Bioglass® 45S5 processing whilst maintaining its network connectivity and its phosphate content so that the ability of the glasses to degrade in physiological conditions, release ions and subsequently form a surface apatite layer are not affected [63]. By increasing the calcium-to-alkali cation ratio, partially substituting potassium for sodium (thereby making use of the ‘mixedalkali effect’) and adding small amounts of fluoride (≤8 mol% CaF2), bioactive glasses can be obtained which can be processed more easily, to obtain sintered scaffolds or drawn fibres, while degrading readily and forming apatite in an aqueous solution within several hours. The addition of up to 8 mol% CaF2 was shown to exert no apparent effect on the processing window; however, it did affect the Tg and Tc, both of which decreased linearly with CaF2 content. It was demonstrated that the working range of bioactive glasses (i.e., their ability to be processed at high temperatures without spontaneous crystallisation) can be improved significantly by increasing their calcium-to-alkali ratio and partially replacing sodium by potassium [63].

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(CaF2 b 9.3 mol% and SrF2 b 4.5 mol%) glasses were obtained in an amorphous state, while high-fluoride content (CaF2 ≥ 9.3 mol% and SrF2 ≥ 4.5 mol%) compositions spontaneously crystallised to fluorapatite during quenching, whilst CaF2 and SrF2 were found in the glasses with 17.8 mol% CaF2 and 25.5 mol% SrF2, respectively. Upon heat treatment, fluorapatite crystallised from all glasses as the primary crystalline phase, and particularly in the glasses with N6 mol% CaF2, fluorapatite, cuspidine, and fluorite were the main crystalline phases following heat treatment. These sodium-free fluorapatite glass ceramics, although partially crystallised, are suggested to have enhanced bioactivity since the Ca10(PO4)6F2 and Sr10(PO4)6F2 phases may act as nuclei for further apatite formation, without resulting in a less reactive residual silicate glass since the glass network connectivity remained unaffected. The Q structure of these glasses was not observed to change upon crystallisation and the network connectivity remained fixed at 2.08 irrespective of how much apatite crystallisation occurred. Cuspidine and fluorite are phosphate free phases and are not believed to restrict bioactivity, particularly since the bioactive behaviour is a function of the amorphous structure having the Q2 speciation. These glass ceramics combine the beneficial effects of a bioactive amorphous phase with the presence of fluorapatite [64]. 7.5. Sol–gel derived glasses and glass-ceramics Part substitution of CaO by CaF2 in sol–gel derived glass-ceramics has also been shown to enhance the mechanical properties, e.g., flexural strength and microhardness, in addition to increase in density and the resulting decrease in porosity [65]. In comparison, part substitution of CaO by NH4F2 results in an increase in porosity and a resulting decrease in density and microhardness [66]. Interestingly, incorporation of NH4F2 lowers the density but increases the porosity to values close to those recorded for the fluoride-free Bioglass® 45S5, the flexural strength remains considerably higher. It appears that the incorporation of CaF2 and NH4F2 in combination may provide a good balance between strength and porosity (Table 4). Furthermore, in vitro cytotoxicity tests using the L-929 mouse fibroblast cell-line showed high relative growth rates for NH4F2 (94.7%) and CaF2 (93.2%) containing glasses. Apatite formation was detected after 7 days of soaking in SBF, while in vivo implantation demonstrated bonding at the interface between new bone and the glass-ceramic. 7.6. Fluorapatite-containing 3D scaffolds Melt-quenched fluorapatite-containing glass-ceramic (Fa-GC) powders in the SiO2–CaO–Na2O–K2O–P2O5–MgO–CaF2 system have been used as starting materials for ‘polymer foam replication’ [67] and ‘porogen bake-out’ [68] techniques, respectively, to produce 3D scaffolds for tissue engineering applications. In polymer foam replication, a polyurethane sponge is coated with Fa-GC powder (b32 μm) slurry, which is then heated at 700 °C to obtain an interpenetrating network of struts and pores. In the second technique, irregularly shaped, 50–150 μm polyethylene grains are mixed with Fa-GC particles (b106 μm), followed by heating at 800 °C to obtain a homogeneously porous scaffold, where

7.4. Alkali-free glasses and glass ceramics Novel sodium-free bioactive glasses have been developed, incorporating CaO/CaF2 or SrO/SrF2 [64]. The addition of fluoride (CaF2 and SrF2 content) into bioactive glasses leads to a reduction in Tg and the first crystallisation peak temperature (Tp1). However, in the asquenched state, increasing CaF2/SrF2 content was shown to be associated with an increased crystallisation tendency, where Ca10(PO4)6F2 (fluorapatite), Ca4Si2O7F2 (cuspidine) and CaF2 (fluorite) crystallise in sequence for the Ca-containing system, and Sr10(PO4)6F2, SrSiO3 and SrF2 for the Sr-containing system. Low-fluoride content

Table 4 The effect of CaF2 and NH4F2 incorporation on the mechanical properties of Bioglass® 45S5 ([65,66]). Glass

Density (g·cm−3)

45S5 1.63 45S5-CaF2 3.88 45S5-NH4F2 1.80

Porosity (%)

Flexural strength (MPa)

Microhardness (HV)

51.16 29.53 47.01

3.97 26.74 13.79

15.05 110.46 22.40

[65] [65,66] [66]

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together with their ability to release fluoride locally makes FBGs interesting candidates for various clinical applications, such as the treatment of dentine hypersensitivity by occlusion of dentinal tubules through formation of apatite and released ions such as fluoride, strontium, and potassium [56] in the treatment of dentin hypersensitivity. The incorporation of fluoride ions into bioactive glasses is also advantageous from the perspective of enhancing the mechanical properties and improving the processing parameters, e.g., Tg and Tc, without compromising the bioactivity. A large body of published literature exists documenting the development and characterisation of fluoride-containing bioactive glasses. But despite the beneficial effects of F− ions for various clinical situations, not nearly enough compositions are evaluated in vivo, which would be critical in order to confirm not only the attractive features of this class of bone-bonding biomaterials, but also to identify their adverse effects on the local physiological environment – most notably the risk of skeletal and dental fluorosis. Acknowledgements This work was supported by the Swedish Research Council (grant K2015-52X-09495-28-4), the BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, the Region Västra Götaland, the Hjalmar Svensson Foundation, the Materials Science Area of Advance at Chalmers and the Department of Biomaterials, University of Gothenburg. A special thanks to Prof. Dr. Delia S. Brauer, Prof. Robert G. Hill, and Dr. Karin A. Hing for all their support and guidance. References Fig. 11. 3D scaffold prepared by the porogen bake-out technique. Fa-GC with 70% volumetric fraction of polyethylene grains [68].

most pores may be within 100–300 μm (Fig. 11). In both techniques, the thermal treatment is intended to completely burn out the polymeric template and achieve sintering and partial crystallisation of the glassceramic. 8. Concluding remarks The physiological pH is widely regarded as being pH 7.4. However, acidic pH conditions are encountered under several ‘normal’ conditions, for example at healing wound sites or in the oral environment. While infection and bacterial growth at a fracture site may produce persistent acidic local pH [69,70], the pH of uninfected tissue too is lower than normal serum pH during early healing [71]. Moreover, in the oral environment, the pH fluctuates to below 5.5 due to caries activity [29], or during consumption of acidic beverages. As part of the normal bone remodelling cycle, osteoclasts reduce the local pH to 4.5 in order to demineralise and resorb bone tissue [72]. Local acidosis affects physiological mechanisms, for instance, bone nodule mineralisation [30], and upregulation of vascular endothelial growth factor (VEGF) expression from osteoblasts [73]. Despite this, in vitro tests on BGs and other biomaterials are commonly performed in test solutions such as SBF or cell culture media, at pH 7.4. Acidic pH conditions prolong the inflammatory process and delay tissue healing and therefore BG compositions designed to perform well in such conditions could find novel applications in orthopaedics [74]. By virtue of its ability to promote the formation of osteocalcin and enhance the alkaline phosphatase activity of human osteoblasts, fluorine is considered one of the most effective in vivo bone anabolic factors [75]. In low concentrations, fluoride ions increase bone mass and mineral density [76], and improve the resistance of the apatite structure to acid attack [33]. Moreover, fluoride ions have well documented antibacterial properties [77]. FBGs have been shown to enhance and control osteoblast proliferation, differentiation and mineralisation [55]. And,

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