2.1

Preparation of specimens

The experimental procedures included the fabrication of disc shaped control specimens made of commercial ceramic for margins (P) of MCRs (IPS Inline-Margin Ceramic System, Ivoclar, Schaan, Liechtenstein) as well as specimens of novel bioactive glass-dental ceramic composite material BP67 (BPc), containing 33.3 wt% of 58S bioactive glass and 66.7 wt% of dental ceramic (IPS Inline Margin, Ivoclar).

Novel BP67 composite material was synthesized by sol–gel method, as previously described . Briefly, the precursors of bioactive glass 58S (SiO ₂ 60 wt%, CaO 36 wt% and P ₂ O ₅ 4 wt%) distilled water, nitric acid, tetraethyoxysilane (TEOS), tri-ethyl-phosphate (TEP) and calcium nitrate tetra-hydrate (Ca(NO ₃ ) ₂ 4H ₂ O) have been mixed together. The TEOS to water ratio (R ratio) used was 1.39 . Dental porcelain was added in a ratio of 66.7%. After mixing of the components, the sol was sintered at 60 °C for 55 h, dried, and underwent firing cycle up to 700 °C in a programmable furnace (Nabertherm, Germany), so that the material remained at the stabilization temperature (700 °C) for 18 h. The resulting products were pulverized and sieved to powders of <40 μm.

For the fabrication of the specimens, the powders of BP67 and dental ceramic were mixed with modeling liquid (IPS Classic Margin Build-Up Liquid, Ivoclar), using the same liquid to powder ratio (0.335), transferred into silicone moulds and condensed using a vibrator, to accomplish excess liquid removal. The discs (d = 6 mm × 1.5 mm) were then removed and sintered in a ceramic oven (Programat P95, Ivoclar) at 930 °C with a rate of 60 °C/min under vacuum and according to manufacturers’ instructions for the conventional ceramic.

2.2

Preparation of 1.5 × SBF solution

Simulated body fluid concentrated 1.5-times (1.5×-SBF) was prepared by weighing and dissolving NaCl, NaHCO ₃ , KCl, K ₂ HPO ₄ ·3H ₂ O, MgCl ₂ ·6H ₂ O, CaCl ₂ , and NaSO ₄ in distilled water, as previously described . The pH of the solution was adjusted to 7.3–7.4 at a temperature of 36.5 °C in a water bath by the addition of 1.0 M hydrochloric acid (HCl) and tris-hydroxymethylaminomethane (CH ₂ OH) ₃ CNH _2.

2.3

Laser assisted biomimetic process—LAB

2.3.1

Experimental setup

The experiments were carried out using the 1064 nm line of a Q-switched Nd:YAG laser (Quantel YG980) emitting 8 ns duration pulses at 10 Hz maximum repetition rate. The pulse energy was adjustable from a maximum of 650 mJ down to almost zero.

The laser beam was directed through 50/50% beam splitter and a mirror and then focused by a 60 mm focal length lens onto the surface of the BP sample. The sample to be processed was immersed in 15 ml of 1.5 × SBF in a Plexiglas container, so that its surface was perpendicular to the incident laser beam. In turn, the container with sample and 1.5 × SBF liquid was mounted on a computer-controlled xy motorized transition stage, so that the sample’s irradiated area could be selected before each laser pulse. Laser energy was continuously monitored by a pyroelectric energy detector (gentec-EO), connected to a supervisory PC recording the energy of every single pulse. The focal area of the laser beam on the BP surface was controlled micro-metrically by adjusting the sample to lens distance.

2.3.2

Estimation of the ablation behavior of the material

In order to estimate the optimum value of laser energy density that would disrupt the energy state of the surface of material without causing structural damage, the samples were irradiated by single pulse, 10 pulses or 3000 pulses with a frequency of 10 Hz and at energy densities ranging from 1.19 to 4.10 J/cm ² . The calculated spot size was 1.52 mm. The samples were then carbon coated and examined by Scanning Electron Microscopy-SEM (JEOL J.S.M. 840A, Tokyo, Japan) in 50× magnifications.

2.3.3

Irradiation of the specimens for different time periods

The LAB process was applied to BP67 sintered specimens. The sample-mirror distance was set at 32 mm and the calculated spot diameter was 2.82 mm. The laser beam was directed to the center of each sample, while it was immersed in 15 ml of 1.5 × SBF precursor liquid. Specimens were irradiated at laser fluence 1.52 J/cm ² for 30 min (BP_30), 1 h (BP_1) or 3 h (BP_3). Immediately after laser treatment, specimens were re-immersed in 1.5 × SBF and incubated at 60 °C for up to 7 days (d). Laser-untreated BP67 specimens incubated under the same conditions were used as control (BP_c). The solution was renewed after 6 h, 24 h and then every 2 days, and the solid to liquid ratio was preserved at 1 mg/1 ml. At the end of the incubation period, the samples were removed from the 1.5 × SBF solution, rinsed with acetone and distilled water and characterized by FTIR, XRD, SEM-EDS and micro-Raman techniques.

2.4

Surface characterization of the samples by means of different techniques

2.4.1

Fourier transformed infrared (FTIR) analysis

FTIR reflectance spectra were obtained by a FTIR Spectrometer (IFS113v, Bruker) in the MIR (4000–400 cm ⁻¹ ) region with a number of 64 scans per spectrum and a resolution 2 cm ⁻¹ .

2.4.2

X-ray diffraction (XRD) analysis

The XRD analysis was performed using a Philips PW1710 diffractometer with Ni-filtered CuKα radiation on the surface of the studied samples. The counting statistics were: start angle 5°, end angle 75° (2 θ ), step size 0.05° (2 θ ), time per step 1 s and scan speed 0.01°2 θ /s. The ICDD PDF-4+ (2009) database was used for the identification of the phases contained in the studied samples.

2.4.3

Micro Raman analysis

Raman measurements were conducted with a LabRAM HR (Horiba) or a T64000 (Horiba) spectrometer equipped with a Peltier and a liquid nitrogen cooled charge coupled device, respectively. Excitation was provided by a Cobolt Fandango™ diode pumped solid state laser at 515 nm with a power of ∼9 mW focused on the sample by a standard 10× objective at a spot diameter of ∼2.5 μm. Presented spectra were collected from an area of 50 × 50 μm ² , taking advantage of the DuoScan™ scanner. It allows fast scanning of the laser beam on the sample, resulting in more representative spectra and lower effective average power.

2.4.4

Scanning electron microcopy and energy dispersive X-ray microanalysis (SEM-EDS)

For topographical evaluation and analysis of surface elemental composition the specimens were carbon coated with an average thickness of 200 Å, using a vacuum evaporator JEOL-4X and were observed using a Scanning Electron Microscope (JEOL J.S.M. 840A, Tokyo, Japan) with associated EDS (Oxford INKA-300). All analyses were done with a 20 kV accelerating voltage and 0.4 mA probe. The images were taken in magnification ×1000. The cross-sections were prepared by embedding the specimens in epoxy resin (Epofix resin, Struers) under vacuum, followed by cutting using a water-cooled, diamond rotary saw. Sections were ground by hand using 400, 600, 800 and 1000 grit silicon carbide and polished in turn with diamond paste with a grain diameter of 6, 3 and 1 μm.

2.5

Investigation of changes in chemical composition of the 1.5 × SBF before and after laser irradiation

In order to evaluate changes in chemical composition of the 1.5 × SBF solution before and after laser irradiation for 3 h, Si, P, Al and Ca concentrations were analyzed in the same reacted solutions by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES), using a PerkinElmer 3100XL Axial Viewing Spectrometer, equipped with a cyclonic spray chamber. The sample introduction flow rate was 2 ml/min. Detection limits were as follows: Si ⁴⁺ (0.05 ppm), Al ³⁺ (0.01 ppm), Ca ²⁺ (0.005 ppm) and for P (0.03 ppm). Potential temperature changes of the 1.5 × SBF solution were monitored after 30 min, 1 h and 3 h of LAB process by a digital platinum resistance thermometer (Guildline 9540/B), while the pH of the 1.5 × SBF solution was measured before and after 3 h of laser irradiation (B&C PH125,2 pH/ORP meter), as well as during the process of liquid renewal.

2.6

Establishment of primary cell cultures

Human primary gingival fibroblast (hGF) and human primary periodontal ligament fibroblast (PDLF) cell cultures were established from free gingiva and periodontal ligament biopsies respectively of young healthy donors after routine third molar extraction. The research protocol was approved by the Ethical committee of the Institutional review board and all patients signed an informed consent form. The gingival tissue biopsies were minced into 1–2 mm fragments and placed in 25 cm ² culture flasks with Dulbecco modified Eagle medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and antibiotics/antimycotics (100 units/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml Amphotericin B). (all from Invitrogen, Karlsruhe, Germany) The flasks were incubated at 37 °C in humidified atmosphere of 5% CO ₂ . After reaching confluency, the cells were transferred to a larger 75 cm ² flask (passage 1), and then continuously subcultured for further experiments. Cells from passages 3–6 were used for all experiments.

For the establishment of PDLF cultures the teeth were washed with sterile PBS and disinfected with sodium hypochlorite and then placed into a falcon tube containing 0.25% trypsin/1 mM Ethylene Diamine Tetraacetic Acid-EDTA solution and kept in water bath at 37 °C for 45 min. Subsequently, complete culture medium (DMEM with 10% FBS and antibiotics/antimycotics) was added into the falcon tube to stop the trypsin action. The culture medium containing cells was transferred into a new centrifuge tube and centrifuged for 10 min at 115 × g . The supernatant was discarded and the cells were re-suspended in fresh medium and pipetted into 25 cm ² culture flasks. The flasks were incubated at 37 °C in humidified atmosphere of 5% CO ₂ . After reaching confluency, the cells were transferred to a 75 cm ² flask (passage 1), and then continuously subcultured for further experiments. Cells from passages 2–4 were used for all experiments.

SAOS-2 osteoblast-like cell line was obtained from the American Type Culture Collection (ATCC No. HTB 85). Cells were cultured at 37 °C in humidified atmosphere of 5% CO ₂ in a-Modification of Eagle’s Medium (aMEM) (Invitrogen), supplemented with 10% FBS and antibiotics/antimycotics, as described above.

2.7

Evaluation of viability by the MTT assay

For the evaluation of viability of cells seeded in direct contact with the experimental materials, the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed. Three groups of ceramic specimens (6 × 1.5 mm), prepared as described above were tested: (1) feldspathic dental ceramic (P), (2) bioactive glass ceramic composite (BP) and (3) laser-treated BP specimens coated with the developed HA layer (BP/HA specimens) (laser irradiated for 3 h and subsequent aging in 1.5 × SBF solution at 60 °C for 7 days). Prior to cell culture, sintered specimens were sterilized in an autoclave for 20 min at 2 bar pressure and then placed in each well of a 48-well plate (Corning, Cambridge, MA) and conditioned in 0.5 mL of culture medium for 24 h at 37 °C in an incubator. Cells (HGFs, PDLFs or SAOS-2) were seeded at a density of 2.5 × 10 ⁴ for HGFs and PDLFs and 5 × 10 ⁴ for SAOS-2 on the top of the experimental specimens. Cells grown on tissue culture plastic (C) were simultaneously evaluated as control.

After 24, 48 or 72 h, MTT (Sigma Aldrich, Taufkirchen, Germany) at final concentration of 0.5 mg/ml was added in each well. Afterwards, the plates were incubated at 37 °C for an additional 3 h. The purple formazan crystals that were formed were diluted by dimethyl-sulfoxide (DMSO) (Sigma Aldrich) for 1.5 h at 37 °C . The absorbance was measured against blank (DMSO) at a wavelength of 545 nm and a reference filter of 630 nm by a microplate reader (Epock, Biotek, Biotek instruments, Inc., Vermont, U.S.A). Experiments were performed in four replicates and repeated three times. The results were expressed as the averaged absorbance as % percentage of the control value.

2.8

SEM evaluation of the cell morphology

After 72 h of cell culturing, the specimens were washed twice with PBS, fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 0,1 M sucrose (pH 7.4), dehydrated in a graded series of alcohols (35%, 50%, 70%, 95%, and two changes of 100% ethanol) and air-dried in a hood for 20 min. Finally, the specimens were carbon coated and observed by SEM (JEOL J.S.M. 840A) in magnification ×1000.

2.9

Statistical analysis

Statistical analysis of the cell viability data was performed using two-way ANOVA analysis. Follow up comparisons between groups were carried out using Tukey’s post-hoc test. Analysis was performed with Prism 6.0 Software (GraphPad, CA, USA) and the level of statistical difference was 0.05 ( p < 0.05).

2.1

Preparation of specimens

The experimental procedures included the fabrication of disc shaped control specimens made of commercial ceramic for margins (P) of MCRs (IPS Inline-Margin Ceramic System, Ivoclar, Schaan, Liechtenstein) as well as specimens of novel bioactive glass-dental ceramic composite material BP67 (BPc), containing 33.3 wt% of 58S bioactive glass and 66.7 wt% of dental ceramic (IPS Inline Margin, Ivoclar).

Novel BP67 composite material was synthesized by sol–gel method, as previously described . Briefly, the precursors of bioactive glass 58S (SiO ₂ 60 wt%, CaO 36 wt% and P ₂ O ₅ 4 wt%) distilled water, nitric acid, tetraethyoxysilane (TEOS), tri-ethyl-phosphate (TEP) and calcium nitrate tetra-hydrate (Ca(NO ₃ ) ₂ 4H ₂ O) have been mixed together. The TEOS to water ratio (R ratio) used was 1.39 . Dental porcelain was added in a ratio of 66.7%. After mixing of the components, the sol was sintered at 60 °C for 55 h, dried, and underwent firing cycle up to 700 °C in a programmable furnace (Nabertherm, Germany), so that the material remained at the stabilization temperature (700 °C) for 18 h. The resulting products were pulverized and sieved to powders of <40 μm.

For the fabrication of the specimens, the powders of BP67 and dental ceramic were mixed with modeling liquid (IPS Classic Margin Build-Up Liquid, Ivoclar), using the same liquid to powder ratio (0.335), transferred into silicone moulds and condensed using a vibrator, to accomplish excess liquid removal. The discs (d = 6 mm × 1.5 mm) were then removed and sintered in a ceramic oven (Programat P95, Ivoclar) at 930 °C with a rate of 60 °C/min under vacuum and according to manufacturers’ instructions for the conventional ceramic.

2.2

Preparation of 1.5 × SBF solution

Simulated body fluid concentrated 1.5-times (1.5×-SBF) was prepared by weighing and dissolving NaCl, NaHCO ₃ , KCl, K ₂ HPO ₄ ·3H ₂ O, MgCl ₂ ·6H ₂ O, CaCl ₂ , and NaSO ₄ in distilled water, as previously described . The pH of the solution was adjusted to 7.3–7.4 at a temperature of 36.5 °C in a water bath by the addition of 1.0 M hydrochloric acid (HCl) and tris-hydroxymethylaminomethane (CH ₂ OH) ₃ CNH _2.

2.3

Laser assisted biomimetic process—LAB

2.3.1

Experimental setup

The experiments were carried out using the 1064 nm line of a Q-switched Nd:YAG laser (Quantel YG980) emitting 8 ns duration pulses at 10 Hz maximum repetition rate. The pulse energy was adjustable from a maximum of 650 mJ down to almost zero.

The laser beam was directed through 50/50% beam splitter and a mirror and then focused by a 60 mm focal length lens onto the surface of the BP sample. The sample to be processed was immersed in 15 ml of 1.5 × SBF in a Plexiglas container, so that its surface was perpendicular to the incident laser beam. In turn, the container with sample and 1.5 × SBF liquid was mounted on a computer-controlled xy motorized transition stage, so that the sample’s irradiated area could be selected before each laser pulse. Laser energy was continuously monitored by a pyroelectric energy detector (gentec-EO), connected to a supervisory PC recording the energy of every single pulse. The focal area of the laser beam on the BP surface was controlled micro-metrically by adjusting the sample to lens distance.

2.3.2

Estimation of the ablation behavior of the material

In order to estimate the optimum value of laser energy density that would disrupt the energy state of the surface of material without causing structural damage, the samples were irradiated by single pulse, 10 pulses or 3000 pulses with a frequency of 10 Hz and at energy densities ranging from 1.19 to 4.10 J/cm ² . The calculated spot size was 1.52 mm. The samples were then carbon coated and examined by Scanning Electron Microscopy-SEM (JEOL J.S.M. 840A, Tokyo, Japan) in 50× magnifications.

2.3.3

Irradiation of the specimens for different time periods

The LAB process was applied to BP67 sintered specimens. The sample-mirror distance was set at 32 mm and the calculated spot diameter was 2.82 mm. The laser beam was directed to the center of each sample, while it was immersed in 15 ml of 1.5 × SBF precursor liquid. Specimens were irradiated at laser fluence 1.52 J/cm ² for 30 min (BP_30), 1 h (BP_1) or 3 h (BP_3). Immediately after laser treatment, specimens were re-immersed in 1.5 × SBF and incubated at 60 °C for up to 7 days (d). Laser-untreated BP67 specimens incubated under the same conditions were used as control (BP_c). The solution was renewed after 6 h, 24 h and then every 2 days, and the solid to liquid ratio was preserved at 1 mg/1 ml. At the end of the incubation period, the samples were removed from the 1.5 × SBF solution, rinsed with acetone and distilled water and characterized by FTIR, XRD, SEM-EDS and micro-Raman techniques.

2.4

Surface characterization of the samples by means of different techniques

2.4.1

Fourier transformed infrared (FTIR) analysis

FTIR reflectance spectra were obtained by a FTIR Spectrometer (IFS113v, Bruker) in the MIR (4000–400 cm ⁻¹ ) region with a number of 64 scans per spectrum and a resolution 2 cm ⁻¹ .

2.4.2

X-ray diffraction (XRD) analysis

The XRD analysis was performed using a Philips PW1710 diffractometer with Ni-filtered CuKα radiation on the surface of the studied samples. The counting statistics were: start angle 5°, end angle 75° (2 θ ), step size 0.05° (2 θ ), time per step 1 s and scan speed 0.01°2 θ /s. The ICDD PDF-4+ (2009) database was used for the identification of the phases contained in the studied samples.

2.4.3

Micro Raman analysis

Raman measurements were conducted with a LabRAM HR (Horiba) or a T64000 (Horiba) spectrometer equipped with a Peltier and a liquid nitrogen cooled charge coupled device, respectively. Excitation was provided by a Cobolt Fandango™ diode pumped solid state laser at 515 nm with a power of ∼9 mW focused on the sample by a standard 10× objective at a spot diameter of ∼2.5 μm. Presented spectra were collected from an area of 50 × 50 μm ² , taking advantage of the DuoScan™ scanner. It allows fast scanning of the laser beam on the sample, resulting in more representative spectra and lower effective average power.

2.4.4

Scanning electron microcopy and energy dispersive X-ray microanalysis (SEM-EDS)

For topographical evaluation and analysis of surface elemental composition the specimens were carbon coated with an average thickness of 200 Å, using a vacuum evaporator JEOL-4X and were observed using a Scanning Electron Microscope (JEOL J.S.M. 840A, Tokyo, Japan) with associated EDS (Oxford INKA-300). All analyses were done with a 20 kV accelerating voltage and 0.4 mA probe. The images were taken in magnification ×1000. The cross-sections were prepared by embedding the specimens in epoxy resin (Epofix resin, Struers) under vacuum, followed by cutting using a water-cooled, diamond rotary saw. Sections were ground by hand using 400, 600, 800 and 1000 grit silicon carbide and polished in turn with diamond paste with a grain diameter of 6, 3 and 1 μm.

2.5

Investigation of changes in chemical composition of the 1.5 × SBF before and after laser irradiation

In order to evaluate changes in chemical composition of the 1.5 × SBF solution before and after laser irradiation for 3 h, Si, P, Al and Ca concentrations were analyzed in the same reacted solutions by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES), using a PerkinElmer 3100XL Axial Viewing Spectrometer, equipped with a cyclonic spray chamber. The sample introduction flow rate was 2 ml/min. Detection limits were as follows: Si ⁴⁺ (0.05 ppm), Al ³⁺ (0.01 ppm), Ca ²⁺ (0.005 ppm) and for P (0.03 ppm). Potential temperature changes of the 1.5 × SBF solution were monitored after 30 min, 1 h and 3 h of LAB process by a digital platinum resistance thermometer (Guildline 9540/B), while the pH of the 1.5 × SBF solution was measured before and after 3 h of laser irradiation (B&C PH125,2 pH/ORP meter), as well as during the process of liquid renewal.

2.6

Establishment of primary cell cultures

Human primary gingival fibroblast (hGF) and human primary periodontal ligament fibroblast (PDLF) cell cultures were established from free gingiva and periodontal ligament biopsies respectively of young healthy donors after routine third molar extraction. The research protocol was approved by the Ethical committee of the Institutional review board and all patients signed an informed consent form. The gingival tissue biopsies were minced into 1–2 mm fragments and placed in 25 cm ² culture flasks with Dulbecco modified Eagle medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and antibiotics/antimycotics (100 units/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml Amphotericin B). (all from Invitrogen, Karlsruhe, Germany) The flasks were incubated at 37 °C in humidified atmosphere of 5% CO ₂ . After reaching confluency, the cells were transferred to a larger 75 cm ² flask (passage 1), and then continuously subcultured for further experiments. Cells from passages 3–6 were used for all experiments.

For the establishment of PDLF cultures the teeth were washed with sterile PBS and disinfected with sodium hypochlorite and then placed into a falcon tube containing 0.25% trypsin/1 mM Ethylene Diamine Tetraacetic Acid-EDTA solution and kept in water bath at 37 °C for 45 min. Subsequently, complete culture medium (DMEM with 10% FBS and antibiotics/antimycotics) was added into the falcon tube to stop the trypsin action. The culture medium containing cells was transferred into a new centrifuge tube and centrifuged for 10 min at 115 × g . The supernatant was discarded and the cells were re-suspended in fresh medium and pipetted into 25 cm ² culture flasks. The flasks were incubated at 37 °C in humidified atmosphere of 5% CO ₂ . After reaching confluency, the cells were transferred to a 75 cm ² flask (passage 1), and then continuously subcultured for further experiments. Cells from passages 2–4 were used for all experiments.

SAOS-2 osteoblast-like cell line was obtained from the American Type Culture Collection (ATCC No. HTB 85). Cells were cultured at 37 °C in humidified atmosphere of 5% CO ₂ in a-Modification of Eagle’s Medium (aMEM) (Invitrogen), supplemented with 10% FBS and antibiotics/antimycotics, as described above.

2.7

Evaluation of viability by the MTT assay

For the evaluation of viability of cells seeded in direct contact with the experimental materials, the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed. Three groups of ceramic specimens (6 × 1.5 mm), prepared as described above were tested: (1) feldspathic dental ceramic (P), (2) bioactive glass ceramic composite (BP) and (3) laser-treated BP specimens coated with the developed HA layer (BP/HA specimens) (laser irradiated for 3 h and subsequent aging in 1.5 × SBF solution at 60 °C for 7 days). Prior to cell culture, sintered specimens were sterilized in an autoclave for 20 min at 2 bar pressure and then placed in each well of a 48-well plate (Corning, Cambridge, MA) and conditioned in 0.5 mL of culture medium for 24 h at 37 °C in an incubator. Cells (HGFs, PDLFs or SAOS-2) were seeded at a density of 2.5 × 10 ⁴ for HGFs and PDLFs and 5 × 10 ⁴ for SAOS-2 on the top of the experimental specimens. Cells grown on tissue culture plastic (C) were simultaneously evaluated as control.

After 24, 48 or 72 h, MTT (Sigma Aldrich, Taufkirchen, Germany) at final concentration of 0.5 mg/ml was added in each well. Afterwards, the plates were incubated at 37 °C for an additional 3 h. The purple formazan crystals that were formed were diluted by dimethyl-sulfoxide (DMSO) (Sigma Aldrich) for 1.5 h at 37 °C . The absorbance was measured against blank (DMSO) at a wavelength of 545 nm and a reference filter of 630 nm by a microplate reader (Epock, Biotek, Biotek instruments, Inc., Vermont, U.S.A). Experiments were performed in four replicates and repeated three times. The results were expressed as the averaged absorbance as % percentage of the control value.

2.8

SEM evaluation of the cell morphology

After 72 h of cell culturing, the specimens were washed twice with PBS, fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 0,1 M sucrose (pH 7.4), dehydrated in a graded series of alcohols (35%, 50%, 70%, 95%, and two changes of 100% ethanol) and air-dried in a hood for 20 min. Finally, the specimens were carbon coated and observed by SEM (JEOL J.S.M. 840A) in magnification ×1000.

2.9

Statistical analysis

Statistical analysis of the cell viability data was performed using two-way ANOVA analysis. Follow up comparisons between groups were carried out using Tukey’s post-hoc test. Analysis was performed with Prism 6.0 Software (GraphPad, CA, USA) and the level of statistical difference was 0.05 ( p < 0.05).