Abstract
Objectives
Glass ionomer cements (GICs) are a subject of research because of their inferior mechanical properties, despite their advantages such as fluoride release and direct bonding to bone and teeth. Recent research aims to improve the bioactivity of the GICs and thereby improve mechanical properties on the long term. In this study, two types of bioactive glasses (BAG) (45S5F and CF9) are combined with GICs to evaluate the physico-chemical properties and biocompatibility of the BAG-GIC combinations. The effect of the addition of Al 3+ to the BAG composition and the use of smaller BAG particles on the BAG-GIC properties was also investigated.
Materials and methods
Conventional aluminosilicate glass (ASG) and (modified) BAG were synthesized by the melt method. BAG-GIC were investigated on setting time, compressive strength and bioactivity. Surface changes were evaluated by Fourier transform infrared (FT-IR), scanning electron microscopy (SEM), EDS and PO 4 3− -and Ca 2+ uptake in SBF. Biocompatibility of selected BAG-GICs was determined by a direct toxicity assay.
Results
The addition of BAG improves the bioactivity of the GIC, which can be observed by the formation of an apatite (Ap) layer, especially in CF9-containing GICs. More BAG leads to more bioactivity but decreases strength. The addition of Al 3+ to the BAG composition improves strength, but decreases bioactivity. BAGs with smaller particle sizes have no effect on bioactivity and decrease strength. The formation of an Ap layer seems beneficial to the biocompatibility of the BAG-GICs.
Significance
Bioactive GICs may have several advantages over conventional GICs, such as remineralization of demineralized tissue, adhesion and proliferation of bone- and dental cells, allowing integration in surrounding tissue. CF9 BAG-GIC combinations containing maximum 10 mol% Al 3+ are most promising, when added in ≤20 wt% to a GIC.
1
Introduction
Glass ionomer cements (GICs) were invented in the early 70’s by Wilson and Kent . They are formed by an acid-base reaction between a polyalkenoic acid and a basic aluminosilicate glass (ASG) . The polyalkenoic acid is usually a polyacrylic acid, polyitaconic acid, polymaleic acid or a copolymer of the previous ones . The ASG contains Ca 2+ , Al 3+ and Si 4+ as essential components, linked to each other by bridging oxygens. Other ions, such as F − and PO 4 3− are usually added to the glass composition. Fluoride increases the compressive strength and decreases the setting time of the cement. This effect is ascribed to the formation of Al–F–Ca(n), F–Ca(n) and possibly Si–F–Ca(n) species, which increase the reactivity of the glass, so that less bonds have to be hydrolyzed during setting . PO 4 3− groups in the glass increase the working and setting time but decrease the compressive strength. These PO 4 3− groups, released during hydrolysis in the setting reaction, compete with the polyalkenoate groups of the PAA for the binding of cations . Water or an aqueous solution of tartaric acid is an essential component in the formation of a GIC to initiate the acid-base reaction . When polyalkenoic acid, ASG and water are mixed, a paste is formed in which the protons of the polyalkenoic acid degrade the ASG. The bonds in the glass-network are hydrolyzed and Ca 2+ , Al 3+ , F − and PO 4 3− are released. In a second reaction step, the released Ca 2+ and Al 3+ bind with the polyalkenoate groups, which form the strong matrix. A silica gel layer is formed around the remaining glass particles, which impedes further degradation .
GICs were initially pushed forward as a revolutionary white dental filling material to replace the less esthetic and more toxic amalgam fillings in restorative dentistry . Also in ENT surgery, GICs are more and more used, for example as otological implants . GICs have a lot of advantages such as direct bonding with teeth by the interaction of the natural apatite (Ap) with the carboxylate-groups of the PAA. Moreover, they have good biocompatibility properties, they release and take up fluoride and thus an antibacterial action and enhance remineralization of fluorapatite (FAp). In contrast to composites, their shrinkage upon setting is negligible . But despite their major benefits in comparison to other commonly used restorative materials, their mechanical properties are since the development of this product still subject of improvement .
Changing the composition of the ASG affects the mechanical properties of the GIC. Within certain limits, an increase of Al 3+ or F − content can improve strength, while PO 4 3− decreases the mechanical properties . Also the type, amount and molar mass of the PAA and the powder/liquid (P/L) ratio used to form the cement have an influence on the mechanical properties . Within the limits of workable cements, the highest P/L ratio leads to the best outcomes. As such, high viscosity GICs, with high concentrations of PAA and high molar mass, are now mostly used . Another way to increase packing density of a GIC is by adding nanoparticles to the ASG fraction. Nanoparticles have a higher specific surface area, and therefore release more ions within the same amount of added water, and thus react faster. Although nanoparticles indeed increase initial compressive strength, no improvement in strength can be seen on the long term .
Nowadays, research groups are focusing on the bioactivation of GICs aiming to improve the long term mechanical properties . There are several definitions for bioactivity: first, it can be defined as the property of a material to bond with living tissue without the formation of a fibrous layer in vivo . Since GIC can bind chemically with enamel, dentin and bone by the interaction of the polyalkenoic acid component of GIC with the apatite component of these tissues, GIC can be considered bioactive. According to a second definition, a material is considered bioactive when it is able to form a layer of material inherent to the body, for example apatite, and in this way integrate with the body . According to this second definition, GICs are not yet bioactive. This last type of bioactivity can easily be monitored in vitro using a simulated body fluid (SBF) . Some researchers are critical about this method, as both false positive and false negative results can occur. So after positive results in SBF, in vitro cell tests and in vivo experiments should be conducted to validate the results . It is shown that the formation of a crystalline HAp layer is delayed in in vitro or in vivo conditions where proteins are able to co-adsorb on the surface of the cement . Cells can only adhere to a bioactive material, if the material can be resorbed in the body, apatite can be redeposited and proteins and growth factors can adhere to the remineralized material. But the adhesion of these proteins thus hamper further Ap growth. However, if apatite is formed in vitro , it is also believed to interact with living dental tissue in vivo as long as no toxic constituents are released . The amount of toxicity of course depends on the volume and the flow of surrounding tissue fluid in which ions are leached.
If GICs would have the potential to be bioactive, the possible applications of GICs would become much broader. Apatite will be able to integrate within dentin structures and may enhance the bonding with the implant by mechanical interlocking next to the chemical bonding and thus improve the (mechanical) properties of the material in the long term. Moreover, proteins and cells could be attracted and tissue regeneration could be accelerated significantly. The remineralization potential of bioactive GICs may also be of interest in ART as in this minimal invasive technique GIC may be placed in a cavity, still containing remnants of demineralized dentin. Also in shallow preparations, the remineralizing potential may be beneficial to improve the retention of the material
Bioactive glass (BAG) was developed in 1969 by Hench . Until then, implants were merely bioinert and evoked an undesirable fibrous encapsulation of the material. While this new material formed a stable bond or interface with tissues by the formation of an Ap layer . The first commercial bioactive glass consisted of 46.1 mol% SiO 2 , 24.4 mol% Na 2 O, 26.9 mol% CaO and 2.6 mol% P 2 O 5 and was called 45S5 or Bioglass ® . When this glass is incubated in aqueous conditions, an Ap layer is formed on the surface of these glasses. In a first phase, due to hydrolysis, rapid cation exchange of Na + and/or Ca 2+ with H + from the solution occurs. Phosphate is also leached from the glass. This creates silanol groups (Si–OH) on the glass surface. The pH of the solution increases and a silica-rich (cation-depleted) region forms near the glass surface. Soluble silica is lost in the form of Si(OH) 4 to the solution. These groups condensate near the glass surface and repolymerize in a silica-rich layer. Meanwhile, Ca 2+ and PO 4 3− migrate back from the solution to the surface, forming an amorphous calciumphosphate (CaP) film on the silica-rich layer. Hydroxide groups and carbonate are incorporated from the solution and CaP crystallizes to hydroxyapatite (HAp) .
Although bioactive glasses are very promising, their commercial success in restorative materials or implants is limited. Nowadays, they can be used in surgery as a bulk, but with the disadvantage that the scaffolds are mostly non-porous and brittle. Moreover, these bulk glasses show low degradability and hence not much ingrowth of cells and blood vessels. Another way to use BAG is as glass particles, often mixed with the patient’s own blood to form a putty . In this case however, nearly no initial strength exists. BAG in the form of an injectable paste could broaden the possible applications of this material, provided that the paste hardens in situ .
Both BAG and GIC may benefit from their combination . Research groups however found that the addition of BAG decreases the mechanical strength of the cements markedly. This effect can be ascribed to the partial replacement of ASG by BAG in the total powder phase, so the relative amount of Al 3+ in the glass mixture decreases, and less strong bonds between the polyacrylic acid (PAA) and other released ions such as Ca 2+ , are formed . This is certainly the case when Na + is used as a network modifier in the glass. Therefore, it is assumed that the addition of Al 3+ to the BAG composition would be beneficial to the strength of BAG-GIC combinations. It is also already shown that nanoparticles can improve the initial mechanical properties of GICs . So when BAG is added as nanoparticles to a GIC formulation, the decrease in initial compressive strength may be less pronounced. Moreover, due to the smaller particle size, the reactivity of the BAG will be higher and thus the pH may increase more rapidly, which could be favorable to the development of a silicagel and Ap layer .
The aim of this study is thus threefold: first, it is investigated if the addition of 2 types of F − -containing BAGs can improve the bioactive properties of a conventional GIC. Secondly, the effect of the addition of Al 3+ to BAG on the compressive strength and bioactive properties of a BAG-GIC is investigated. It is hypothesized that a higher Al 3+ content will lead to improved compressive strength, but bioactive properties should still remain. Finally, the effect of adding BAG with smaller particle sizes is investigated. It is hypothesized that mechanical and bioactive properties, together with biocompatibility will increase.
2
Materials and methods
For each solution, deionized water was used (Millipore, Milli-Q academic, Bedford, MA, United States of America).
2.1
The glasses
The LG26 ASG with fluoride and two types of (modified) BAGs (45S5F and CF9 ) were synthesized by the melt method . Composition of the BAGs was modified by replacing Si 4+ by 10 and 20 mol% Al 3+ . The respective modified BAGs get the suffix −10Al or −20Al. Glass compositions and melting conditions are described in Table 1 . SiO 2 (Merck 7536, Darmstadt, Germany), Al 2 O 3 (Merck 1095), CaCO 3 (Merck 2066), CaF 2 (Mallinckrodt 4168, St. Louis, USA), P 2 O 5 (Fluka Chemika 79612, Buchs, Switzerland) and Na 2 CO 3 (Merck 6392) were used as precursors.
| ASG | 45S5F-0Al | 45S5F-10Al | 45S5F-20Al | CF9-0Al | CF9-10Al | CF9-20Al | |
|---|---|---|---|---|---|---|---|
| SiO 2 | 32.1 | 48 | 38 | 28 | 34.6 | 24.6 | 14.6 |
| Al 2 O 3 | 21.4 | 0 | 5 | 10 | 0 | 5 | 10 |
| Na 2 O | 0 | 25.4 | 25.4 | 25.4 | 0 | 0 | 0 |
| CaO | 21.4 | 14 | 14 | 14 | 50.38 | 50.38 | 50.38 |
| CaF 2 | 14.3 | 10 | 10 | 10 | 9.28 | 9.28 | 9.28 |
| P 2 O 5 | 10.7 | 2.6 | 2.6 | 2 | 5.74 | 5.74 | 5.74 |
| Temperature | 1450 | 1430 | 1430 | 1500 | 1430 | 1430 | 1430 |
| Time | 2 | 1.5 | 1.5 | 3 | 1 | 1 | 1.5 |
The glass was ground in a planetary mill (Pulverisette 6, Fritsch GmbH, Idar-Oberstein, Germany) and sieved (Vibratory Sieve-Shaker, Analysette 3 Fritsch GmbH) . The particle size was further reduced by grinding the macrogranular glass particles in isopropanol in the planetary mill using a zirconia mortar with zirconia beads (Ø 0.5 mm) at 600 rpm.
Particle size distribution of the glass particles was determined with laser diffraction (Mastersizer, Malvern, Worcestershire, UK). The homogeneity of the glasses was evaluated using XRD-patterns recorded for diffraction angles between 2° and 60° 2 θ with a PW1830 Philips diffractometer (PANalytical, Almelo, The Netherlands).
Reactivity of the glasses was measured by the addition of 0.025 g glass to 20 ml 0.01 M acetic acid and continuously monitoring the increase in pH. The rate by which the pH changes is a measure for reactivity. pH was measured with a pH electrode (Primatrode, Metrohm, Switzerland) .
2.2
The cements
Glass mixtures were prepared by combining 10, 20 and/or 30 wt% of each type of BAG ( Table 1 ) with respectively 90, 80 and/or 70 wt% ASG depending on the consistency and workability of the corresponding cements. The BAG-GIC powder was formulated by mixing freeze-dried polyacrylic acid (versicol E7, Allied Colloids, Bradford, England) with the glass mixture in a glass:polyacid ratio of 5:1. The cement was prepared by mixing the BAG-GIC powder with a 10 wt% tartaric acid solution by hand in a P/L ratio of 4. 30-CF9-0Al and 30-CF9-10Al had to be mixed in a P/L ratio of 3 to obtain workable cements . Cements made with the BAGs described in Table 1 get the prefix 10-, 20- or 30- when respectively 10, 20 or 30 wt% BAG is combined with ASG to form the GIC. Cements with 45S5F glass with smaller particle sizes get the suffix “n”. Cements made of 100% melt glass were used as a reference.
2.2.1
Characterization of the physico-mechanical properties
Setting time at 22 °C was determined with an indentator (Ø 1 mm, 400 g) .
Compressive strength was determined with a universal testing machine (LRX plus, Lloyd Instruments, Bognor Regis, UK). For that purpose, 18 cement cylinders (Ø 6 mm, H 12 mm) were prepared for each glass combination ( Table 1 ) using a split stainless steel mold. For the 45S5F type of BAGs, another batch of 18 cylinders per glass combination was made, but now BAGs with smaller particle sizes were used. All cylinders were matured for 24 h in 85% relative humidity (RH) at 37 °C. 6 of the 18 cylinders were directly tested, 6 were subjected to an immersion in 25 ml simulated body fluid (SBF) and 6 were immersed in 25 ml H 2 O each for 28 days before testing. Cylinders were loaded at a rate of 1 mm/min.
2.2.2
Characterization of the bioactive properties
SBF was prepared with NaCl (VWR Prolabo 27810.295), NaHCO 3 (Merck 6329), KCl (Merck 4936), K 2 HPO 4 (Merck 5104), MgCl 2 ·6H 2 O (Merck 5833), CaCl 2 (Merck 2391), Na 2 SO 4 (Merck 6647), and Tris(hydroxymethyl)aminomethane (VWR Prolabo 103156X). These components were dissolved at room temperature as described in the protocol of Kokubo and Takadama . pH was adjusted to 7.4 with a 1 M HCl solution.
Cement discs were prepared (Section 2 ) (Ø 5 mm, H 1 mm) and stored at 37 °C, 85% RH for 1 h. 2 discs of each cement composition were subsequently stored in a plastic container with 25 ml SBF at 37 °C for 28 days. One batch was made where SBF was not changed for FT-IR analysis and SEM. In another batch SBF was renewed and collected every 2 days. The collected SBF was used to determine the phosphate- and calcium uptake by the cement-discs. PO 4 3− was determined with a differential spectrophotometric method using a Pye Unicam PU 8670 VIS/NIR spectrophotometer (Philips Scientific Equipment, Brussels, Belgium). Ca 2+ was determined with atomic absorption spectrometry (AAS) (Varian SpectrAA-30, Agilent Technologies, Santa Clara, USA) with an air-acetylene flame . The cumulative phosphate and calcium uptake from SBF (in %) was calculated.
FT-IR spectra of crushed cements were recorded before and after 28 days of incubation in SBF using a Spectrum One spectrometer (PerkinElmer Instruments, U.S.) for wavelengths between 4000 and 400 cm −1 . In particular the formation of CaP was investigated. Therefore the height of the double phosphate peak at around 561 cm −1 was measured in absorbance with Spectrum v5.0.1. software (PerkinElmer Instruments, U.S.) In order to be able to compare the height of the peaks, identical amounts of crushed cements were mixed with KBr.
Scanning electron microscopic (SEM) images were taken of the surface with a scanning electron microscope (FEI, Quanta FEG, Hillsboro, OR, USA). The samples were investigated in particular for the presence of a CaP layer after 28 days of incubation in SBF. The cements, only matured 24 h in RH, were used as control. EDX (FEI, Quanta FEG with EDAX silicon-drift detector, Hillsboro, OR, USA) was further conducted on specific structures, to determine their atomic composition.
2.2.3
Characterization of the biocompatibility
Cement discs (Ø: 5 mm, h: 1 mm) of the 8 selected BAG-GIC and the reference cement were sterilized for 1 h at each side under a UV-light source. Biocompatibility was investigated with and without SBF incubation.
2.2.3.1
Cell culture
Human foreskin fibroblast (HFF) cells were cultured in Dulbecco’s Minimal Essential Medium (DMEM) L-glutamax (Gibco Invitrogen) supplemented with 10% fetal bovine serum (Gibco Invitrogen), 0.5 vol% penicillin–streptomycin (Gibco Invitrogen) and 0.5 vol% sodium pyruvate (Gibco Invitrogen) at 37 °C in a humidified atmosphere containing 5% CO 2 .
2.2.3.2
Direct contact assay
The discs were placed in 24-well suspension culture plates and incubated for 24 h in 1 ml serum-free DMEM prior to cell seeding in order to remove potential impurities. HFF cells were seeded in 24-well tissue culture dishes at a density of 40,000 cells/ml/well. After 24 h of culture, the cement discs were placed on the monolayer and incubated at 37 °C (5% CO 2 /95% air). The viability of the HFF monolayer was evaluated after 24 h with the live/dead assay and MTT assay. Cells cultured on tissue culture polystyrene were used as a positive control.
2.2.3.3
Live/dead assay
To visualize cell viability (direct contact assay), the cells were evaluated after live/dead staining. After rinsing the tissue culture dishes, the supernatant was replaced by 1 ml of phosphate buffered saline supplemented with 2 μl calcein AM (1 mg/ml) (Anaspec, USA) and 2 μl propidium iodide (PI) (1 mg/ml) (Sigma–Aldrich). The cultures were incubated for 10 min at room temperature, washed twice with phosphate buffered saline (PBS) and evaluated by fluorescence microscopy (Type U-RFL-T, XCellence Pro software, Olympus, Belgium) . The fluorescence of calcein AM and propidium iodide was monitored at 460–495 nm excitation/550 nm emission and 545–580 nm excitation/610 nm emission respectively.
2.2.3.4
MTT assay
The colorimetric MTT assay, using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Merck, Promega) was performed to quantify the viability of cells in contact with the discs. The tetrazolium component is reduced in living cells by mitochondrial dehydrogenase into a purple formazan product, which can be solubilized by the addition of a lysis buffer and measured by spectrophotometry.
Cell culture medium was aspirated and replaced by 0.5 ml (0.5 mg/ml) MTT solution and incubated for 4 h at 37 °C and 5% CO 2 . The MTT reagent was removed and replaced by 0.5 ml lysis buffer (0.1% Triton X-100 in isopropanol/0.04 N HCl) for 30 min. The dissolved formazan solution (200 μl) was transferred into a 96-well plate and measured spectrophotometrically at 580 nm (Universal microplate reader EL800, Biotek Instruments). Triplicate measurements were performed.
2.3
Statistical evaluation
Statistical analysis of the results was performed with ANOVA and a post hoc Bonferroni test. Where variances were not equal, a Tamhane T2 post hoc test was used. The level of significance was set at 0.05.
2
Materials and methods
For each solution, deionized water was used (Millipore, Milli-Q academic, Bedford, MA, United States of America).
2.1
The glasses
The LG26 ASG with fluoride and two types of (modified) BAGs (45S5F and CF9 ) were synthesized by the melt method . Composition of the BAGs was modified by replacing Si 4+ by 10 and 20 mol% Al 3+ . The respective modified BAGs get the suffix −10Al or −20Al. Glass compositions and melting conditions are described in Table 1 . SiO 2 (Merck 7536, Darmstadt, Germany), Al 2 O 3 (Merck 1095), CaCO 3 (Merck 2066), CaF 2 (Mallinckrodt 4168, St. Louis, USA), P 2 O 5 (Fluka Chemika 79612, Buchs, Switzerland) and Na 2 CO 3 (Merck 6392) were used as precursors.
| ASG | 45S5F-0Al | 45S5F-10Al | 45S5F-20Al | CF9-0Al | CF9-10Al | CF9-20Al | |
|---|---|---|---|---|---|---|---|
| SiO 2 | 32.1 | 48 | 38 | 28 | 34.6 | 24.6 | 14.6 |
| Al 2 O 3 | 21.4 | 0 | 5 | 10 | 0 | 5 | 10 |
| Na 2 O | 0 | 25.4 | 25.4 | 25.4 | 0 | 0 | 0 |
| CaO | 21.4 | 14 | 14 | 14 | 50.38 | 50.38 | 50.38 |
| CaF 2 | 14.3 | 10 | 10 | 10 | 9.28 | 9.28 | 9.28 |
| P 2 O 5 | 10.7 | 2.6 | 2.6 | 2 | 5.74 | 5.74 | 5.74 |
| Temperature | 1450 | 1430 | 1430 | 1500 | 1430 | 1430 | 1430 |
| Time | 2 | 1.5 | 1.5 | 3 | 1 | 1 | 1.5 |
The glass was ground in a planetary mill (Pulverisette 6, Fritsch GmbH, Idar-Oberstein, Germany) and sieved (Vibratory Sieve-Shaker, Analysette 3 Fritsch GmbH) . The particle size was further reduced by grinding the macrogranular glass particles in isopropanol in the planetary mill using a zirconia mortar with zirconia beads (Ø 0.5 mm) at 600 rpm.
Particle size distribution of the glass particles was determined with laser diffraction (Mastersizer, Malvern, Worcestershire, UK). The homogeneity of the glasses was evaluated using XRD-patterns recorded for diffraction angles between 2° and 60° 2 θ with a PW1830 Philips diffractometer (PANalytical, Almelo, The Netherlands).
Reactivity of the glasses was measured by the addition of 0.025 g glass to 20 ml 0.01 M acetic acid and continuously monitoring the increase in pH. The rate by which the pH changes is a measure for reactivity. pH was measured with a pH electrode (Primatrode, Metrohm, Switzerland) .
2.2
The cements
Glass mixtures were prepared by combining 10, 20 and/or 30 wt% of each type of BAG ( Table 1 ) with respectively 90, 80 and/or 70 wt% ASG depending on the consistency and workability of the corresponding cements. The BAG-GIC powder was formulated by mixing freeze-dried polyacrylic acid (versicol E7, Allied Colloids, Bradford, England) with the glass mixture in a glass:polyacid ratio of 5:1. The cement was prepared by mixing the BAG-GIC powder with a 10 wt% tartaric acid solution by hand in a P/L ratio of 4. 30-CF9-0Al and 30-CF9-10Al had to be mixed in a P/L ratio of 3 to obtain workable cements . Cements made with the BAGs described in Table 1 get the prefix 10-, 20- or 30- when respectively 10, 20 or 30 wt% BAG is combined with ASG to form the GIC. Cements with 45S5F glass with smaller particle sizes get the suffix “n”. Cements made of 100% melt glass were used as a reference.
2.2.1
Characterization of the physico-mechanical properties
Setting time at 22 °C was determined with an indentator (Ø 1 mm, 400 g) .
Compressive strength was determined with a universal testing machine (LRX plus, Lloyd Instruments, Bognor Regis, UK). For that purpose, 18 cement cylinders (Ø 6 mm, H 12 mm) were prepared for each glass combination ( Table 1 ) using a split stainless steel mold. For the 45S5F type of BAGs, another batch of 18 cylinders per glass combination was made, but now BAGs with smaller particle sizes were used. All cylinders were matured for 24 h in 85% relative humidity (RH) at 37 °C. 6 of the 18 cylinders were directly tested, 6 were subjected to an immersion in 25 ml simulated body fluid (SBF) and 6 were immersed in 25 ml H 2 O each for 28 days before testing. Cylinders were loaded at a rate of 1 mm/min.
2.2.2
Characterization of the bioactive properties
SBF was prepared with NaCl (VWR Prolabo 27810.295), NaHCO 3 (Merck 6329), KCl (Merck 4936), K 2 HPO 4 (Merck 5104), MgCl 2 ·6H 2 O (Merck 5833), CaCl 2 (Merck 2391), Na 2 SO 4 (Merck 6647), and Tris(hydroxymethyl)aminomethane (VWR Prolabo 103156X). These components were dissolved at room temperature as described in the protocol of Kokubo and Takadama . pH was adjusted to 7.4 with a 1 M HCl solution.
Cement discs were prepared (Section 2 ) (Ø 5 mm, H 1 mm) and stored at 37 °C, 85% RH for 1 h. 2 discs of each cement composition were subsequently stored in a plastic container with 25 ml SBF at 37 °C for 28 days. One batch was made where SBF was not changed for FT-IR analysis and SEM. In another batch SBF was renewed and collected every 2 days. The collected SBF was used to determine the phosphate- and calcium uptake by the cement-discs. PO 4 3− was determined with a differential spectrophotometric method using a Pye Unicam PU 8670 VIS/NIR spectrophotometer (Philips Scientific Equipment, Brussels, Belgium). Ca 2+ was determined with atomic absorption spectrometry (AAS) (Varian SpectrAA-30, Agilent Technologies, Santa Clara, USA) with an air-acetylene flame . The cumulative phosphate and calcium uptake from SBF (in %) was calculated.
FT-IR spectra of crushed cements were recorded before and after 28 days of incubation in SBF using a Spectrum One spectrometer (PerkinElmer Instruments, U.S.) for wavelengths between 4000 and 400 cm −1 . In particular the formation of CaP was investigated. Therefore the height of the double phosphate peak at around 561 cm −1 was measured in absorbance with Spectrum v5.0.1. software (PerkinElmer Instruments, U.S.) In order to be able to compare the height of the peaks, identical amounts of crushed cements were mixed with KBr.
Scanning electron microscopic (SEM) images were taken of the surface with a scanning electron microscope (FEI, Quanta FEG, Hillsboro, OR, USA). The samples were investigated in particular for the presence of a CaP layer after 28 days of incubation in SBF. The cements, only matured 24 h in RH, were used as control. EDX (FEI, Quanta FEG with EDAX silicon-drift detector, Hillsboro, OR, USA) was further conducted on specific structures, to determine their atomic composition.
2.2.3
Characterization of the biocompatibility
Cement discs (Ø: 5 mm, h: 1 mm) of the 8 selected BAG-GIC and the reference cement were sterilized for 1 h at each side under a UV-light source. Biocompatibility was investigated with and without SBF incubation.
2.2.3.1
Cell culture
Human foreskin fibroblast (HFF) cells were cultured in Dulbecco’s Minimal Essential Medium (DMEM) L-glutamax (Gibco Invitrogen) supplemented with 10% fetal bovine serum (Gibco Invitrogen), 0.5 vol% penicillin–streptomycin (Gibco Invitrogen) and 0.5 vol% sodium pyruvate (Gibco Invitrogen) at 37 °C in a humidified atmosphere containing 5% CO 2 .
2.2.3.2
Direct contact assay
The discs were placed in 24-well suspension culture plates and incubated for 24 h in 1 ml serum-free DMEM prior to cell seeding in order to remove potential impurities. HFF cells were seeded in 24-well tissue culture dishes at a density of 40,000 cells/ml/well. After 24 h of culture, the cement discs were placed on the monolayer and incubated at 37 °C (5% CO 2 /95% air). The viability of the HFF monolayer was evaluated after 24 h with the live/dead assay and MTT assay. Cells cultured on tissue culture polystyrene were used as a positive control.
2.2.3.3
Live/dead assay
To visualize cell viability (direct contact assay), the cells were evaluated after live/dead staining. After rinsing the tissue culture dishes, the supernatant was replaced by 1 ml of phosphate buffered saline supplemented with 2 μl calcein AM (1 mg/ml) (Anaspec, USA) and 2 μl propidium iodide (PI) (1 mg/ml) (Sigma–Aldrich). The cultures were incubated for 10 min at room temperature, washed twice with phosphate buffered saline (PBS) and evaluated by fluorescence microscopy (Type U-RFL-T, XCellence Pro software, Olympus, Belgium) . The fluorescence of calcein AM and propidium iodide was monitored at 460–495 nm excitation/550 nm emission and 545–580 nm excitation/610 nm emission respectively.
2.2.3.4
MTT assay
The colorimetric MTT assay, using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Merck, Promega) was performed to quantify the viability of cells in contact with the discs. The tetrazolium component is reduced in living cells by mitochondrial dehydrogenase into a purple formazan product, which can be solubilized by the addition of a lysis buffer and measured by spectrophotometry.
Cell culture medium was aspirated and replaced by 0.5 ml (0.5 mg/ml) MTT solution and incubated for 4 h at 37 °C and 5% CO 2 . The MTT reagent was removed and replaced by 0.5 ml lysis buffer (0.1% Triton X-100 in isopropanol/0.04 N HCl) for 30 min. The dissolved formazan solution (200 μl) was transferred into a 96-well plate and measured spectrophotometrically at 580 nm (Universal microplate reader EL800, Biotek Instruments). Triplicate measurements were performed.
2.3
Statistical evaluation
Statistical analysis of the results was performed with ANOVA and a post hoc Bonferroni test. Where variances were not equal, a Tamhane T2 post hoc test was used. The level of significance was set at 0.05.
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