2.1

Cells and materials

Commercially available HUVECs pooled from 10 different healthy donors (Technoclone, Vienna, Austria) were used in the present study. HUVECs were cultured in endothelial cell medium (ECM, Technoclone, Austria) with 20% fetal bovine serum (FBS) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml fungizone, 2 mM l -glutamine, 5 U/ml heparin and 30–50 μg/ml endothelial cell growth supplement in culture flasks coated with 0.2% gelatine at 37 °C in a humidified atmosphere of 5% CO ₂ and 95% air. The HUVECs from the 4th to 7th passage in culture were used.

2.2

EMD separation

EMD fractions LMW and LMW-depleted were separated and purified by Institut Straumann using a modification of previously described methods . Briefly, EMD fractions LMW and LMW-depleted were extracted from porcine enamel matrix derivative via size exclusion high-performance liquid chromatography (Shodex KW 2003, Brechbühler AG, Switzerlandy) in 100 mM Na acetate pH 3.5 containing 100 mM NaCl. Lyophilized fractions were reconstituted in 0.1% acetic acid to produce a 10 mg/ml stock solution. Further dilutions of proteins (1–100 μg/ml) were performed into FBS-free ECM.

The working solution of 100 μg/ml EMD or EMD fractions contained 0.001% of acetic acid, which did not exert any significant effect on any parameter investigated in this study.

2.3

Cell proliferation/viability

For the proliferation/viability assay 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) dye was used . HUVECs were seeded in 24-well plates coated with 0.2% gelatine at a density of 5 × 10 ⁴ cells per well in 0.5 ml of ECM supplemented with 20% FBS. After 24 h, the medium in test wells was replaced by FBS-free ECM conditioned with EMD, LMW-depleted or LWM at concentrations of 1–100 μg/ml. Wells, stimulated with FBS-free ECM supplemented with 0.001% of acetic acid served as vehicle controls. After 24 h incubation, 100 μl of MTT solution (5 mg/ml in PBS) were added into each well and culture plates were incubated at 37 °C for 4 h. The medium was removed and 500 μl dimethylsulfoxide (DMSO) were added to each well, followed by 5 min incubation on a shaker. Finally, 100 μl of each cultured solution were transferred to a separate 96-well plate and the optical density (OD) was measured at 570 nm with an ELISA Reader (Molecular Devices, USA).

2.4

Apoptosis assay

HUVECs were stimulated with EMD and EMD fractions for 24 h in FCS free medium. Then the cells were detached with accutase (PAA, Austria) and washed twice with FACS buffer (PBS supplemented with 3% FCS and 0.9% NaN ₃ ). Cells were transferred to 5 ml FACS tubes and resuspended in 195 μl of FACS buffer. Afterwards, 5 μl of Annexin V-FITC (eBioscience, San Diego, USA) were added and cells were stained for 15 min at room temperature in the dark. Cells were then washed twice, resuspended in 190 μl of FACS buffer and 10 μl of propidium iodide (PI, eBioscience) were added into each tube. The proportion of annexin V- and PI-positive cells was measured immediately after staining by flow cytometry (FACSCalibur, Becton Dickinson, CA, USA) in FL1 and FL3 fluorescent channels, respectively. The cell counting was limited to 5000 events. In each experiment, three wells were used for each group. The apoptosis assay was performed in triplicate.

2.5

Chemotaxis assay

Cell migration was assessed in a 48-well microchemotaxis chamber (Neuroprobe, Gaithersburg, MD, USA) on a polycarbonate filter with 8 μm pore size as described previously . The chamber consisted of acrylic top and bottom plates, each containing 48 matched wells. Top and bottom plates were separated by a polycarbonate filter with 8 μm pore size (Neuroprobe, Gaithersburg, MD, USA). 26 μl of FBS-free medium containing EMD or EMD fractions (10 μg/ml) were filled in wells of the bottom plate. Wells filled with medium containing 0.0001% of acetic acid served as control. Subsequently, the bottom plate was covered with a filter and the top plate was applied so that each well corresponded to that of the bottom plate. A cell suspension containing 1 × 10 ⁴ cells in 50 μl FBS-free medium was added to each well of the top plate and the whole chamber was incubated at 37 °C in humidified air with 5% CO ₂ for 8 h. After incubation, cells on the upper surface of the filter were removed over the wiper blade and the filters were then fixed with methanol and stained with Hemacolor ^® for microscopy (Merck, Darmstadt, Germany). The cells migrated across the filter were counted under a light microscope at high-power magnification (×100) to measure transmigration in each well. Four fields were counted in each well and the total number was calculated. Four wells were used for each group; experiments were repeated in triplicate.

2.6

Cell motility measurements by time lapse microscopy

HUVECs were harvested by accutase and stained with Cell Tracker Orange CMRA (Molecular Probes, Invitrogen, UK) according to the manufacture’s instructions. After staining and wash-out steps, cells were seeded in 4-well plates pre-coated with 0.2% gelatine at a density of 2 × 10 ⁴ cells pro well in 0.8 ml of ECM. In each experiment, one well was supplemented with 0.001% acetic acid and was used as a vehicle control, whereas other three wells contained EMD, LMW-depleted, or LMW at a concentration of 25 μg/ml. Then cells were incubated in an individually designed 37 °C temperature-controlled incubation chamber supplied with 95% air and 5% CO ₂ , which was attached to an upright fluorescence microscope (Nikon Eclipse E 800 M microscope; Nikon Instruments Europe B.V, Badhoevedorp, Netherlands). Fluorescently labeled cells were observed dynamically and photographed with a digital imaging system (Photometrics ^® Cascade 512F, Germany) every 30 min for 120 h with the aid of Lucia imaging analysis software (NIS-Elements AR, Nikon). Cell motility was analyzed using tracking module by manual tracking mode. For each experiment 15 randomly selected cells pro well/group were tracked in the time period from 12 h until 24 h after seeding. Cell motility was described by average migration speed.

2.7

Measurements of gene expression levels by quantitative real time PCR

The effect of EMD fractions on mRNA expression levels of E-selectin, ICAM-1, Flt-1, KDR, ang-2, and vWF were determined by qPCR similarly to the method described previously , taking the GAPDH encoding gene as internal reference. HUVECs were seeded in 24-well plates similar to MTT experiments and stimulated in FBS-free ECM with EMD, LMW-depleted fraction, or LWM fraction at concentrations of 10 and 100 μg/ml. Some wells were stimulated with FBS-free ECM supplemented with 0.001% of acetic acid and served as vehicle control. Bovine serum albumin (BSA) was diluted in FBS-free ECM and filtered through 0.22 μm filter to serve as a non-specific control. Isolation of total cellular mRNA and transcription into cDNA was performed using the TaqMan ^® Gene Expression Cells-to-CT™ kit (Ambion/Applied Biosystems, CA, USA) according to manufacture’s instructions). Real-time PCR was performed on an Applied Biosystems Step One Plus real-time PCR system (Applied Biosystem, CA, USA) using Taqman ^® gene expression assays with the following ID numbers (all from Applied Biosystems, CA, USA): E-selectin, Hs00174057_m1; ICAM-1, Hs00164932_m1; Flt-1, Hs01052961; KDR-1, Hs00911700_m1; ang-2, Hs01048043_m1; vWF, Hs00169795_m1; GAPDH, Hs99999905_m1). Duplicate PCR reactions were prepared and the point at which the PCR product was first detected above a fixed threshold (termed cycle threshold, C _t ), was determined. Changes in the expression of target genes were calculated using 2 ^−ΔΔCt method, where ΔΔC _t = (C _t ^target − C _t ^GAPDH ) _sample − (C _t ^target − C _t ^GAPDH ) _{vehicle control} , taking samples treated with 0.001% of acetic acid as a vehicle control.

2.8

Measurements of cell surface protein expression by flow cytometry

The expression of adhesion molecules ICAM-1 and E-selectin as well as VEGF receptors Flt-1 and KDR on the cell surface of HUVECs was measured by fluorescence flow cytometry . For the measurements of ICAM-1 and E-selectin expression, cells were stained with one of the following monoclonal antibodies conjugated with phycoerythrin (all eBioscience, San Diego, CA, USA): mouse anti-human ICAM-1 antibody, mouse anti-human E-selectin antibody, and isotype control antibody. Surface expression of different proteins was analyzed using a flow cytometer (FACScan, Becton Dickinson, San Jose, CA, USA). Cell counting was limited by 50,000 events and the mean fluorescence intensities values were determined for each sample. The expression of ICAM-1 and E-selectin for each sample was quantified using Cell Quest software (Becton Dickinson, San Jose, CA, USA) based on mean fluorescence intensity values of cells stained with ICAM-1 and E-selectin antibodies . Unspecific staining was assessed by measuring cells stained with the isotype control antibody. For the measurements of Flt-1 and KDR expression cells were stained with primary rabbit polyclonal andibodies and subsequently with secondary goat anti-rabbit antibody conjugated with phycoerythrin (all Santa Cruz Biotechnology, Dallas, Texas, USA). The percentage of Flt-1- and KDR-positive cells was analyzed by Cell Quest software (Becton Dickinson, San Jose, CA, USA).

2.9

ELISA analysis

Commercially available ELISA kits were used for measurements of vWF (Novateinbio, Woburn, MA, USA),ang-2 (RayBiotech, Inc., Norcross GA, USA), and VEGF (BosterBio, Pleasanton, USA) in the conditioned media. For the measurement of vWF and ang-2 the samples were diluted 1:20 and 1:10, respectively. For the measurements of VEGF samples were not diluted.

2.10

Statistical analysis

The normal distribution of all data was tested with the Kolmogorov–Smirnov test. For normally distributed data, the statistical differences between different groups were analyzed by one-way analysis of variance (ANOVA) for repeated measures followed by post-hoc t -test. For non-normally distributed data, the statistical differences between groups were analyzed by Friedman test and pairwise comparison was performed using Wilcoxon test for paired variables. All statistical analysis was performed using statistical program SPSS 19.0 (SPSS, Chicago, IL, USA). Data are expressed as mean ± S.E.M. Differences were considered to be statistically significant at p < 0.05.

2.1

Cells and materials

Commercially available HUVECs pooled from 10 different healthy donors (Technoclone, Vienna, Austria) were used in the present study. HUVECs were cultured in endothelial cell medium (ECM, Technoclone, Austria) with 20% fetal bovine serum (FBS) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 0.25 μg/ml fungizone, 2 mM l -glutamine, 5 U/ml heparin and 30–50 μg/ml endothelial cell growth supplement in culture flasks coated with 0.2% gelatine at 37 °C in a humidified atmosphere of 5% CO ₂ and 95% air. The HUVECs from the 4th to 7th passage in culture were used.

2.2

EMD separation

EMD fractions LMW and LMW-depleted were separated and purified by Institut Straumann using a modification of previously described methods . Briefly, EMD fractions LMW and LMW-depleted were extracted from porcine enamel matrix derivative via size exclusion high-performance liquid chromatography (Shodex KW 2003, Brechbühler AG, Switzerlandy) in 100 mM Na acetate pH 3.5 containing 100 mM NaCl. Lyophilized fractions were reconstituted in 0.1% acetic acid to produce a 10 mg/ml stock solution. Further dilutions of proteins (1–100 μg/ml) were performed into FBS-free ECM.

The working solution of 100 μg/ml EMD or EMD fractions contained 0.001% of acetic acid, which did not exert any significant effect on any parameter investigated in this study.

2.3

Cell proliferation/viability

For the proliferation/viability assay 3,4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT) dye was used . HUVECs were seeded in 24-well plates coated with 0.2% gelatine at a density of 5 × 10 ⁴ cells per well in 0.5 ml of ECM supplemented with 20% FBS. After 24 h, the medium in test wells was replaced by FBS-free ECM conditioned with EMD, LMW-depleted or LWM at concentrations of 1–100 μg/ml. Wells, stimulated with FBS-free ECM supplemented with 0.001% of acetic acid served as vehicle controls. After 24 h incubation, 100 μl of MTT solution (5 mg/ml in PBS) were added into each well and culture plates were incubated at 37 °C for 4 h. The medium was removed and 500 μl dimethylsulfoxide (DMSO) were added to each well, followed by 5 min incubation on a shaker. Finally, 100 μl of each cultured solution were transferred to a separate 96-well plate and the optical density (OD) was measured at 570 nm with an ELISA Reader (Molecular Devices, USA).

2.4

Apoptosis assay

HUVECs were stimulated with EMD and EMD fractions for 24 h in FCS free medium. Then the cells were detached with accutase (PAA, Austria) and washed twice with FACS buffer (PBS supplemented with 3% FCS and 0.9% NaN ₃ ). Cells were transferred to 5 ml FACS tubes and resuspended in 195 μl of FACS buffer. Afterwards, 5 μl of Annexin V-FITC (eBioscience, San Diego, USA) were added and cells were stained for 15 min at room temperature in the dark. Cells were then washed twice, resuspended in 190 μl of FACS buffer and 10 μl of propidium iodide (PI, eBioscience) were added into each tube. The proportion of annexin V- and PI-positive cells was measured immediately after staining by flow cytometry (FACSCalibur, Becton Dickinson, CA, USA) in FL1 and FL3 fluorescent channels, respectively. The cell counting was limited to 5000 events. In each experiment, three wells were used for each group. The apoptosis assay was performed in triplicate.

2.5

Chemotaxis assay

Cell migration was assessed in a 48-well microchemotaxis chamber (Neuroprobe, Gaithersburg, MD, USA) on a polycarbonate filter with 8 μm pore size as described previously . The chamber consisted of acrylic top and bottom plates, each containing 48 matched wells. Top and bottom plates were separated by a polycarbonate filter with 8 μm pore size (Neuroprobe, Gaithersburg, MD, USA). 26 μl of FBS-free medium containing EMD or EMD fractions (10 μg/ml) were filled in wells of the bottom plate. Wells filled with medium containing 0.0001% of acetic acid served as control. Subsequently, the bottom plate was covered with a filter and the top plate was applied so that each well corresponded to that of the bottom plate. A cell suspension containing 1 × 10 ⁴ cells in 50 μl FBS-free medium was added to each well of the top plate and the whole chamber was incubated at 37 °C in humidified air with 5% CO ₂ for 8 h. After incubation, cells on the upper surface of the filter were removed over the wiper blade and the filters were then fixed with methanol and stained with Hemacolor ^® for microscopy (Merck, Darmstadt, Germany). The cells migrated across the filter were counted under a light microscope at high-power magnification (×100) to measure transmigration in each well. Four fields were counted in each well and the total number was calculated. Four wells were used for each group; experiments were repeated in triplicate.

2.6

Cell motility measurements by time lapse microscopy

HUVECs were harvested by accutase and stained with Cell Tracker Orange CMRA (Molecular Probes, Invitrogen, UK) according to the manufacture’s instructions. After staining and wash-out steps, cells were seeded in 4-well plates pre-coated with 0.2% gelatine at a density of 2 × 10 ⁴ cells pro well in 0.8 ml of ECM. In each experiment, one well was supplemented with 0.001% acetic acid and was used as a vehicle control, whereas other three wells contained EMD, LMW-depleted, or LMW at a concentration of 25 μg/ml. Then cells were incubated in an individually designed 37 °C temperature-controlled incubation chamber supplied with 95% air and 5% CO ₂ , which was attached to an upright fluorescence microscope (Nikon Eclipse E 800 M microscope; Nikon Instruments Europe B.V, Badhoevedorp, Netherlands). Fluorescently labeled cells were observed dynamically and photographed with a digital imaging system (Photometrics ^® Cascade 512F, Germany) every 30 min for 120 h with the aid of Lucia imaging analysis software (NIS-Elements AR, Nikon). Cell motility was analyzed using tracking module by manual tracking mode. For each experiment 15 randomly selected cells pro well/group were tracked in the time period from 12 h until 24 h after seeding. Cell motility was described by average migration speed.

2.7

Measurements of gene expression levels by quantitative real time PCR

The effect of EMD fractions on mRNA expression levels of E-selectin, ICAM-1, Flt-1, KDR, ang-2, and vWF were determined by qPCR similarly to the method described previously , taking the GAPDH encoding gene as internal reference. HUVECs were seeded in 24-well plates similar to MTT experiments and stimulated in FBS-free ECM with EMD, LMW-depleted fraction, or LWM fraction at concentrations of 10 and 100 μg/ml. Some wells were stimulated with FBS-free ECM supplemented with 0.001% of acetic acid and served as vehicle control. Bovine serum albumin (BSA) was diluted in FBS-free ECM and filtered through 0.22 μm filter to serve as a non-specific control. Isolation of total cellular mRNA and transcription into cDNA was performed using the TaqMan ^® Gene Expression Cells-to-CT™ kit (Ambion/Applied Biosystems, CA, USA) according to manufacture’s instructions). Real-time PCR was performed on an Applied Biosystems Step One Plus real-time PCR system (Applied Biosystem, CA, USA) using Taqman ^® gene expression assays with the following ID numbers (all from Applied Biosystems, CA, USA): E-selectin, Hs00174057_m1; ICAM-1, Hs00164932_m1; Flt-1, Hs01052961; KDR-1, Hs00911700_m1; ang-2, Hs01048043_m1; vWF, Hs00169795_m1; GAPDH, Hs99999905_m1). Duplicate PCR reactions were prepared and the point at which the PCR product was first detected above a fixed threshold (termed cycle threshold, C _t ), was determined. Changes in the expression of target genes were calculated using 2 ^−ΔΔCt method, where ΔΔC _t = (C _t ^target − C _t ^GAPDH ) _sample − (C _t ^target − C _t ^GAPDH ) _{vehicle control} , taking samples treated with 0.001% of acetic acid as a vehicle control.

2.8

Measurements of cell surface protein expression by flow cytometry

The expression of adhesion molecules ICAM-1 and E-selectin as well as VEGF receptors Flt-1 and KDR on the cell surface of HUVECs was measured by fluorescence flow cytometry . For the measurements of ICAM-1 and E-selectin expression, cells were stained with one of the following monoclonal antibodies conjugated with phycoerythrin (all eBioscience, San Diego, CA, USA): mouse anti-human ICAM-1 antibody, mouse anti-human E-selectin antibody, and isotype control antibody. Surface expression of different proteins was analyzed using a flow cytometer (FACScan, Becton Dickinson, San Jose, CA, USA). Cell counting was limited by 50,000 events and the mean fluorescence intensities values were determined for each sample. The expression of ICAM-1 and E-selectin for each sample was quantified using Cell Quest software (Becton Dickinson, San Jose, CA, USA) based on mean fluorescence intensity values of cells stained with ICAM-1 and E-selectin antibodies . Unspecific staining was assessed by measuring cells stained with the isotype control antibody. For the measurements of Flt-1 and KDR expression cells were stained with primary rabbit polyclonal andibodies and subsequently with secondary goat anti-rabbit antibody conjugated with phycoerythrin (all Santa Cruz Biotechnology, Dallas, Texas, USA). The percentage of Flt-1- and KDR-positive cells was analyzed by Cell Quest software (Becton Dickinson, San Jose, CA, USA).

2.9

ELISA analysis

Commercially available ELISA kits were used for measurements of vWF (Novateinbio, Woburn, MA, USA),ang-2 (RayBiotech, Inc., Norcross GA, USA), and VEGF (BosterBio, Pleasanton, USA) in the conditioned media. For the measurement of vWF and ang-2 the samples were diluted 1:20 and 1:10, respectively. For the measurements of VEGF samples were not diluted.

2.10

Statistical analysis

The normal distribution of all data was tested with the Kolmogorov–Smirnov test. For normally distributed data, the statistical differences between different groups were analyzed by one-way analysis of variance (ANOVA) for repeated measures followed by post-hoc t -test. For non-normally distributed data, the statistical differences between groups were analyzed by Friedman test and pairwise comparison was performed using Wilcoxon test for paired variables. All statistical analysis was performed using statistical program SPSS 19.0 (SPSS, Chicago, IL, USA). Data are expressed as mean ± S.E.M. Differences were considered to be statistically significant at p < 0.05.