Abstract
Objective
Bioactive glasses (BAG) form, in contrast to formerly used implant materials, a stable bond with tissues, especially bone, when implanted. Nowadays BAGs are often mixed with a cement/composite that hardens in situ to broaden its applications in dentistry or orthopedics. The bioactivity and biocompatibility of possible BAG candidates for BAG-cement/composite development were evaluated.
Methods
Two fluoride containing BAGs were tested: a Na + -containing (45S5F), based on the first commercial BAG, and a Na + -free BAG (CF9), with a higher Ca 2+ and PO 4 3− content. BAGs were tested on their bioactivity upon immersion in SBF for 7 days by evaluating the surface changes by FT-IR, SEM, EDS and PO 4 3− and Ca 2+ uptake and/or release from SBF. Moreover, the biocompatibility of the BAGs was investigated with a direct contact cell viability study with HFF cells and a cell adhesion study with MG-63 cells.
Results
The Na + -free BAG, CF9, showed the highest potential to bioactivate cements because of its high Ca 2+ -release and apatite (Ap) formation, as evidenced by SEM pictures and corresponding EDX patterns. FT-IR confirmed the formation of an Ap layer. Moreover CF9 had a higher biocompatibility than 45S5F.
Significance
For the bioactivation of GICs/composites in order to enhance bonding and remineralization of surrounding tissues, fluoride containing BAG may have advantages over other BAGs as a more stable fluorapatite can be formed. CF9 may be an excellent candidate therefore.
1
Introduction
Bioactive glass (BAG) was invented by Larry Hench in 1969 and was the first implant material that could form a stable interface or bond with tissues, such as bone or muscle. Until then implants were merely bioinert and evoked a non-wanted fibrous encapsulation of the material . The first 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 ® .
A bioactive material can be defined as a material that stimulates a beneficial response from the body, particularly bonding to host tissue . This bonding to for example bone is in BAG achieved by the formation of hydroxyapatite (HAp) as an interconnective layer on the dissolving glass and in the near environment of the glass in humid or aqueous environments . The surface reactions taking place upon the immersion of BAG in aqueous solutions are illustrated in Fig. 1 . In a first phase, due to hydrolysis, rapid cation exchange of Na + and/or Ca 2+ from the glass with H + from the solution occurs. In addition, phosphate is also released from the glass. The dissolution of these ions creates silanol bonds (Si OH) on the glass surface. Silanol groups condensate and repolymerization of the silica-rich layer occurs. Meanwhile, Ca 2+ and PO 4 3− migrate from the solution to the surface, forming a film rich in amorphous calciumphosphate (CaP) on the silica-rich layer . Hydroxyl groups and carbonate from solution are incorporated and the CaP-film crystallizes to hydroxyapatite (HAp) . This process can be mimicked by the immersion of a sample in simulated body fluid (SBF) and as such in vitro tests can predict the bioactivity of materials . However, some researchers are critical about this method, as both false positive as false negative results can occur. So after positive results in SBF, in vivo tests should be conducted to validate the results . The actual interconnection of the glass with bone namely occurs due to proteins, such as growth factors ( e.g. , bone morphogenic protein (BMP)), fibronectin and collagen that easily bind with the formed HAp and in this way attract for example mesenchymal stem cells (MSCs) and enhance them to differentiate .
Until now, 45S5 is the most used commercial BAG. This BAG regenerates bone better than commercially used HAp . It dissolves easier and therefore improves remineralization. A direct relationship between bioactivity (apatite formation) and glass network dissolution has been shown . This can be explained by the network connectivity (NC). NC is calculated as the relative amount of bridging oxygens per network forming element in the glass. A NC between 1.8 and 2.7 is described to be favorable to induce apatite (Ap) formation. A higher NC impedes the dissolution of the glass . Phosphate, present in the BAG is also favorable to induce apatite formation . But care should be taken when increasing the phosphate content as the NC also increases, except when simultaneously network balancing ions are added . Apart from the NC and phosphate content, the amount of Ca 2+ available to be released should also be taken into account. Substitution of Ca 2+ by Na + in the BAG leads to low NC and therefore has high reactivity, but decreases bioactivity by the low amount of Ca 2+ present in the BAG .
Although bioactive glasses are very promising, their commercial success is concentrated on incorporation in toothpaste as remineralizing agents. Their use in restorative applications has never reached its full potential. In surgery they can be used as a bulk scaffold, fitting large defects. But then the disadvantage is 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, 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 . For this reason, bioactive glasses have been combined with polymers such as PLA, PDLLA, PGA or chitosan, but compressive strength results are limited . Also, BAG has been incorporated in calcium phosphate cements (CPCs), resulting in good bioactivity but also increased degradability, which can hamper the mechanical properties of these cements . Recently, BAGs are also combined with resin based composites in order to minimize marginal leakage as it is shown that BAG included in these composites may act as an antibacterial and remineralizing agent .
Research groups have already shown that bioactive glasses can be incorporated in glass ionomer cement (GIC) formulations . GICs were invented in the 70s and are commonly used in restorative dentistry and more recently as medical applications for ENT surgery for bonding cochlear implants in place and repairing the occicular chain . GICs are formed by an acid–base reaction between a polyalkenoic acid and an aluminosilicate glass (ASG) . They bind directly with the apatite in dentin, enamel and bone . Due to the incorporation of fluoride in the glass network, GICs have the possibility to release fluoride, which leads to a continuous protection against caries by the formation of fluorapatite (FAp) and the anti-bacterial effect . Despite the advantages, their clinical use is relatively restricted because of their inferior mechanical properties. This drawback has already been tackled by the invention of resin modified GICs. The latter cements come however with higher sensitivity to moisture and contain toxic monomers . The mechanical properties of conventional GIC can also be enhanced by incorporating apatite particles in the GIC . As BAG form an apatite layer upon immersion, bioactivation of GIC by the incorporation of BAG could further improve the mechanical properties. The formation of an Ap layer in time on BAG containing GICs (BAG-GICs) may enhance the interaction of the cement with bone or pulpal cells and consequently mechanical interlocking may occur in addition to the normal chemical bonding of GICs to dental/bone tissue .
In order for a material to be biocompatible and allow cell growth, dissolution and precipitation reactions have to take place, preferably forming an interfacial hydroxyapatite layer on which biological molecules can be adsorbed . Since of as conventional GICs do not release high amounts of Ca 2+ and PO 4 3− and do not form apatite on their surfaces in SBF , they have no inherent bioactivity according to the previously discussed definition . Moreover, they even decrease the pH in the surrounding tissues, release F − and Al 3+ which can make them cytotoxic in certain applications, leading to a restricted use in dental applications and ENT surgery . Incorporation of Ap crystals, devitrification of ASG to Ap or incorporation of highly bioactive and biocompatible BAGs may thus overcome these problems .
However, the hypothesis that BAG-GICs improve mechanical properties seems to be false, at least on the short term, since research conducted by Yli-Urpo et al. showed the incorporation of BAG to decrease the mechanical properties of GICs. It has to be noted that these BAGs contained Na + , which may interfere with the efficient cross-linking of the polysalt matrix of the cement and increase the water sensitivity of the cement . Despite the fact that BAG-GICs do not have improved mechanical properties, they can be used for fixing orthopedic implants to bone or in atraumatic restorative therapy (ART) in dentistry. The remineralizing potential of BAG-GICs may 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 .
BAG containing fluoride may have several advantages over non-fluoride containing BAG in dental applications. When incorporated in composites, fluoride containing BAG may function as a single source for both calcium and fluoride ions in order to remineralize surrounding tissue, hence having a cariostatic effect. Moreover, the composite can be recharged with fluoride . In toothpaste, fluoride containing BAG may remineralize dentin by the formation of the more stable FAp rather than HAp . When incorporated in GICs, fluoride containing BAGs could also contribute to the fluoride release of GICs and lead to the formation of FAp. The surface reaction in F − containing BAGs is however slightly different from that of non-F − containing BAGs, which is illustrated in Fig. 1 . Now, NaF and CaF + are easily leached and less H + is necessary to leach out other ions, so the relative amount of OH − ions in the environment of the glass becomes lower . Due to the lower pH increase in comparison with BAG without F − , less SiO − groups are initially available to react with Ca 2+ . These BAGs therefore typically form CaCO 3 in a first step, followed by the (delayed) deposition of apatite contrary to BAG without F − . The rate of apatite deposition depends on the amount of Ca 2+ released . The apatite formed on these glasses is typically FAp, since the concentration of OH − is lower and F − is higher than when non-F − containing BAGs are used. However, the amount of fluoride added to BAG has to be limited to prevent CaF 2 depositions . A high PO 4 3− content in the glass prohibits the formation of CaF 2 and promotes the formation of FAp . Furthermore, low sodium containing bioactive glasses enhance the formation of FAp . This is due to their relatively higher Ca 2+ -content and release, the charge balancing of PO 4 3− only by Ca 2+ instead of Ca 2+ and Na + and their potential to crystallize to apatite upon annealing at high temperatures. Therefore, Ap is formed even before incubation in aqueous conditions .
Both BAG and GIC may thus benefit from their combination. But the bioactivity of BAG is lower when combined with GIC compared to pure BAG . The aim of this study is to evaluate two types of fluoride containing BAGs on their bioactive properties and biocompatibility in order to choose the most appropriate candidate for the formulation of BAG-GICs/composites . A BAG (45S5F), based on the commercial Na + containing Bioglass ® and a BAG without sodium, but with a higher Ca 2+ and PO 4 3− content (CF9) .
2
Materials and methods
2.1
Physical properties
Both types of bioactive glasses (45S5F and CF9 ) were synthesized by the melt method as described before for ASG . Glass compositions and exact melting conditions are described in Table 1 . SiO 2 (Merck 7536), CaCO 3 (Merck 2066), CaF 2 (Mallinckrodt 4168), P 2 O 5 (Fluka Chemika 79612) and Na 2 CO 3 (Merck 6392) were used as precursors.
| 45S5F | CF9 | |
|---|---|---|
| SiO 2 | 47.71 | 34.60 |
| Na 2 O | 25.19 | / |
| CaO | 14.20 | 50.38 |
| CaF 2 | 10.20 | 9.28 |
| P 2 O 5 | 2.69 | 5.74 |
| Temperature | 1430 | 1430 |
| Time | 1.5 | 1 |
The glass was ground in a planetary mill (Pulverisette 6, Fritsch GmbH, Idar-Oberstein, Germany) and sieved (Vibratory Sieve-Shaker, Analysette 3 Fritsch GmbH, Idar-Oberstein, Germany) as described before .
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
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.
250 mg of the glass powders was pressed into discs (Ø: 10 mm, h: 1 mm) under vacuum and a 10 ton weight. Of each type of BAG, individual discs were incubated in 25 ml SBF or H 2 O for 1, 3 or 7 days. At every interval time the discs were removed from the SBF/H 2 O, rinsed in water and let to dry in a vacuum dessicator. Their relative increase in mass was measured. SBF and H 2 O was collected at every interval time.
The collected SBF and H 2 O was analyzed for PO 4 3− and Ca 2+ . PO 4 3− content was determined with a differential spectrophotometric method using a Pye Unicam PU 8670 VIS/NIR spectrophotometer (Philips Scientific Equipment, Brussels, Belgium). Ca 2+ content was determined with atomic absorption spectrometry (AAS) (Varian SpectrAA-30, Agilent Technologies, Santa Clara, USA) with an air-acetylene flame . The amount of phosphate and calcium, already present in SBF before the incubation of the discs was subtracted from the results. The phosphate and calcium uptake or release in SBF or H 2 O at every renewal point was calculated per unit of mass of the respective disc (in mg/g).
FT-IR spectra of the initial BAG disc, the discs after every incubation period in SBF and the precipitations on the polypropylene tubes (PP) were recorded using a Spectrum One spectrometer (PerkinElmer Instruments, U.S.) for wavelengths between 4000 and 400 cm −1 with a resolution of 1 cm −1 . In particular the formation of calcium phosphate was investigated. Where Ap was formed, CaCO 3 was added as an internal standard and the absorbance ratio of the phosphate (561 cm −1 ) to carbonate peak (714 cm −1 ) was monitored with Spectrum v5.0.1. software (PerkinElmer Instruments, U.S.). These absorption bands are characteristic for PO 4 3− /CO 3 2− and isolated from other absorption peaks.
Scanning electron microscopic (SEM) images were taken of the surface with a scanning electron microscope (JEOL JSM-5600, USA). The samples were investigated in particular for the presence of calcium phosphate depositions after 7 days of incubation in SBF. The discs, only matured 24 h in RH, were used as control.
Depositions were further investigated on their composition by EDS on a JEOL JSM-5600 instrument equipped with an electron microprobe JED2300 and an EDS detector for elemental analysis. Prior to analysis, all samples were coated with a thin conductive layer of carbon (ca 15 nm) by flash evaporation or a plasma magnetron sputter-coated layer of gold (ca 20 nm). Both deposition methods were used as C-content cannot be measured on carbon-flash evaporated samples and P-content cannot be measured on Au-sputtered samples due to signal overlap as the resolution is 140 eV. Samples are analyzed in mapping mode on three random spots of 200 μm × 250 μm or by spot analysis where large (≳10 μm diameter) structures were observed. The elemental composition is determined using the “Phi-Rho-Z Quantitative Analysis Software” purchased from JEOL.
2.3
Biocompatibility studies
BAG discs (Ø: 10 mm, h: 1 mm) were sterilized for 30 min at each side under a UV-light source.
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% foetal calf serum (Gibco Invitrogen), 0.5 vol% penicillin–streptomycin (Gibco Invitrogen) and 0.5 vol% sodium pyruvate at 37 °C in a humidified atmosphere containing 5% CO 2 . MG-63 cells (osteoblast-like cells, derived from human osteosarcoma cell line) were cultured in Dulbecco’s Minimal Essential Medium (DMEM) l -glutamax (Gibco Invitrogen) supplemented with 10% fetal calf serum (Gibco Invitrogen) and 1 vol% sodium pyruvate at 37 °C in a humidified atmosphere containing 5% CO 2 .
2.3.2
Direct contact assay
HFF cells were seeded in 24-well tissue culture dishes at a density of 100 000 cells/ml/well. After 24 h, the glass 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 taken as a positive control.
2.3.3
Cell adhesion
The discs were placed in 24-well suspension culture plates and incubated for 5 days in 1 ml serum-free DMEM prior to cell seeding in order to remove potential impurities . MG-63 cells were seeded at a density of 100 000 cells/ml culture medium/disc and incubated. Cell adhesion and viability was evaluated after 24 h with the MTT assay.
2.3.4
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 −1 ) (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.
2.3.5
Phase contrast microscopy
Cells and debris from the glass pellet in the direct contact assay were evaluated by phase contrast microscopy (Type U-RFL-T, XCellence Pro software, Olympus, Belgium).
2.3.6
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.
2.4
Statistical evaluation
Statistical analysis of the EDX results and the MTT test were performed with ANOVA and a post hoc Tamhane T2/Bonferroni test. The level of significance was set at 0.05.
2
Materials and methods
2.1
Physical properties
Both types of bioactive glasses (45S5F and CF9 ) were synthesized by the melt method as described before for ASG . Glass compositions and exact melting conditions are described in Table 1 . SiO 2 (Merck 7536), CaCO 3 (Merck 2066), CaF 2 (Mallinckrodt 4168), P 2 O 5 (Fluka Chemika 79612) and Na 2 CO 3 (Merck 6392) were used as precursors.
| 45S5F | CF9 | |
|---|---|---|
| SiO 2 | 47.71 | 34.60 |
| Na 2 O | 25.19 | / |
| CaO | 14.20 | 50.38 |
| CaF 2 | 10.20 | 9.28 |
| P 2 O 5 | 2.69 | 5.74 |
| Temperature | 1430 | 1430 |
| Time | 1.5 | 1 |
The glass was ground in a planetary mill (Pulverisette 6, Fritsch GmbH, Idar-Oberstein, Germany) and sieved (Vibratory Sieve-Shaker, Analysette 3 Fritsch GmbH, Idar-Oberstein, Germany) as described before .
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
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.
250 mg of the glass powders was pressed into discs (Ø: 10 mm, h: 1 mm) under vacuum and a 10 ton weight. Of each type of BAG, individual discs were incubated in 25 ml SBF or H 2 O for 1, 3 or 7 days. At every interval time the discs were removed from the SBF/H 2 O, rinsed in water and let to dry in a vacuum dessicator. Their relative increase in mass was measured. SBF and H 2 O was collected at every interval time.
The collected SBF and H 2 O was analyzed for PO 4 3− and Ca 2+ . PO 4 3− content was determined with a differential spectrophotometric method using a Pye Unicam PU 8670 VIS/NIR spectrophotometer (Philips Scientific Equipment, Brussels, Belgium). Ca 2+ content was determined with atomic absorption spectrometry (AAS) (Varian SpectrAA-30, Agilent Technologies, Santa Clara, USA) with an air-acetylene flame . The amount of phosphate and calcium, already present in SBF before the incubation of the discs was subtracted from the results. The phosphate and calcium uptake or release in SBF or H 2 O at every renewal point was calculated per unit of mass of the respective disc (in mg/g).
FT-IR spectra of the initial BAG disc, the discs after every incubation period in SBF and the precipitations on the polypropylene tubes (PP) were recorded using a Spectrum One spectrometer (PerkinElmer Instruments, U.S.) for wavelengths between 4000 and 400 cm −1 with a resolution of 1 cm −1 . In particular the formation of calcium phosphate was investigated. Where Ap was formed, CaCO 3 was added as an internal standard and the absorbance ratio of the phosphate (561 cm −1 ) to carbonate peak (714 cm −1 ) was monitored with Spectrum v5.0.1. software (PerkinElmer Instruments, U.S.). These absorption bands are characteristic for PO 4 3− /CO 3 2− and isolated from other absorption peaks.
Scanning electron microscopic (SEM) images were taken of the surface with a scanning electron microscope (JEOL JSM-5600, USA). The samples were investigated in particular for the presence of calcium phosphate depositions after 7 days of incubation in SBF. The discs, only matured 24 h in RH, were used as control.
Depositions were further investigated on their composition by EDS on a JEOL JSM-5600 instrument equipped with an electron microprobe JED2300 and an EDS detector for elemental analysis. Prior to analysis, all samples were coated with a thin conductive layer of carbon (ca 15 nm) by flash evaporation or a plasma magnetron sputter-coated layer of gold (ca 20 nm). Both deposition methods were used as C-content cannot be measured on carbon-flash evaporated samples and P-content cannot be measured on Au-sputtered samples due to signal overlap as the resolution is 140 eV. Samples are analyzed in mapping mode on three random spots of 200 μm × 250 μm or by spot analysis where large (≳10 μm diameter) structures were observed. The elemental composition is determined using the “Phi-Rho-Z Quantitative Analysis Software” purchased from JEOL.
2.3
Biocompatibility studies
BAG discs (Ø: 10 mm, h: 1 mm) were sterilized for 30 min at each side under a UV-light source.
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% foetal calf serum (Gibco Invitrogen), 0.5 vol% penicillin–streptomycin (Gibco Invitrogen) and 0.5 vol% sodium pyruvate at 37 °C in a humidified atmosphere containing 5% CO 2 . MG-63 cells (osteoblast-like cells, derived from human osteosarcoma cell line) were cultured in Dulbecco’s Minimal Essential Medium (DMEM) l -glutamax (Gibco Invitrogen) supplemented with 10% fetal calf serum (Gibco Invitrogen) and 1 vol% sodium pyruvate at 37 °C in a humidified atmosphere containing 5% CO 2 .
2.3.2
Direct contact assay
HFF cells were seeded in 24-well tissue culture dishes at a density of 100 000 cells/ml/well. After 24 h, the glass 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 taken as a positive control.
2.3.3
Cell adhesion
The discs were placed in 24-well suspension culture plates and incubated for 5 days in 1 ml serum-free DMEM prior to cell seeding in order to remove potential impurities . MG-63 cells were seeded at a density of 100 000 cells/ml culture medium/disc and incubated. Cell adhesion and viability was evaluated after 24 h with the MTT assay.
2.3.4
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 −1 ) (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.
2.3.5
Phase contrast microscopy
Cells and debris from the glass pellet in the direct contact assay were evaluated by phase contrast microscopy (Type U-RFL-T, XCellence Pro software, Olympus, Belgium).
2.3.6
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.
2.4
Statistical evaluation
Statistical analysis of the EDX results and the MTT test were performed with ANOVA and a post hoc Tamhane T2/Bonferroni test. The level of significance was set at 0.05.
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