2.1. Development and Characterization of Bioactive Glass Microparticles
To prepare the glasses, we first selected a base composition that (a) was bioactive and (b) belonged to the SiO2-CaO-Na2O-P2O5 system. The mixture of raw materials was prepared in the necessary ratio to obtain the desired composition in a 45S5-type base glass containing (in wt %) 45% of SiO2, 24.5% Na2O, 24.5% CaO, and 6% P2O5, where Na2O was partially substituted by 5% Li2O (45S5.5Li). The mixtures were merged in a platinum crucible at 1500 °C using a Carbolite electric oven, keeping the same temperature for 3 h to allow the merger of the components and the homogenization of the glass. The melted glass was placed on graphite plates so as to induce fast cooling and thus prevent its crystallization. To verify the non-crystallization of the material, we carried out X-ray diffraction (XRD) using a Philips PW 1830 with Co tube, using Kα radiation of this element at 40 kV.
Considering the fast in vitro release of ions from the 45S5 bioactive glass when using particles smaller than or equal to 5 µm [
10], in the present study, the glass blocks obtained were ground and reduced to powders of micrometric particles (<5 µm) and were then examined by scanning electronic microscopy (SEM, JEOL JSM 6480 LV) to evaluate their homogeneity and size.
2.3. Cell Culture
For the in vitro assays, HUVECs, kindly donated by Dr. M. Schattner (Academia Nacional de Medicina, Buenos Aires, Argentina), were used. HUVECs were grown in T75 flasks with M199 medium and 10% fetal bovine serum (FBS) and 50 µg mL−1 of gentamicin supplemented with 2 ng mL−1 of basic fibroblast growth factor (bFGF), and were kept in an incubator gassed with 5% CO2 in air at 37 °C. Then, the cells were washed with PBS and incubated at 37 °C for 5 min with 1 mL of a dilution of 0.017% w/v of trypsin and 0.18 mmol EDTA in PBS. Immediately after, 10 mL of M199 medium was added to dilute the trypsin and then centrifuged at 1000 rpm for 5 min. The supernatant was discarded and the cells were suspended in the appropriate volume of medium and counted in a Neubauer chamber to be used in the various assays detailed below:
Proliferation Assay: As reported in previous studies [
10], HUVECs were sown in 96-well plates at a density of 5000 cells per well in 100 µL of M199 medium containing 10% of FBS and 50 µg mL
−1 of gentamicin. At 24 h, 50 µL of M199 medium supplemented with the ionic dissolution products (M199 + 45S5; M199 + 45S5.5Li) or M199 medium enriched with 0.20 mmol of LiCl or NaCl (using the latter as control of the effect of osmolarity) was added in sextuplicate.
The proliferative response of HUVECs treated with the ionic dissolution products, LiCl and NaCl was also studied in the presence of quercetin (10 µM, dissolved in DMSO), an inhibitor of the Wnt/β-catenin canonical pathway.
As a positive control, we used M199 medium with bFGF, whereas for the negative control we used M199 medium without the ionic products or growth factors. At 24 h, 25 µL per well of the same medium, containing methyl-[3H]-thymidine at a final concentration of 2.5 µCi mL
−1, was added and the incubation was continued for 24 additional hours. The assay was stopped by adding 50 µL of 6 M guanidinium chloride. Complete lysis of the cells was achieved through three cycles of freezing at −70 °C and thawing. Cellular DNA was collected in Whatman GFC filters with a Cell Harvester (Cell Harvester 8, Nunc, Rochester, NY, USA), fixed with 96% ethanol, and air-dried. The incorporated radioactivity was determined as described in Haro Durand et al. [
10].
Migration Assay: To evaluate migration, we followed the method previously described [
10]. HUVECs were seeded to confluence in 96-well culture plates overnight to ensure their adherence to the surface of the wells. Then, a gap was generated by means of a probe tip. After generating the gap, the cells were washed with PBS three times to remove detached cells and cell debris, and then were cultured for 8 h in M199 medium enriched with the ionic dissolution products (M199 + 45S5; M199 + 45S5.5Li) or 2 ng mL
−1 bFGF (M199 + bFGF). As the negative control, we used M199 medium without the ionic dissolution products or bFGF. The effect of M199 medium enriched with 0.20 mmol LiCl or NaCl was also evaluated.
Photographic images were taken immediately after generating the gap (time zero) and after 8 h. Cell migration was quantified by means of an Image J image analyzer (NIH, Bethesda, MD, USA). The mean gap area was expressed as the recovery percentage (% R) of five wells, treated in the same way, using the following equation:
where
A0 is the area of the gap at time 0 and
At is the area of the gap at 8 h.
Transmigration Assay: HUVECs were re-seeded in T75 flasks to obtain the desired number of cells for the experiment, and were then raised with EDTA and counted in a Neubauer chamber prior to centrifugation. At the beginning of the assay, 500 µL of M199 medium enriched with the ionic dissolution products of the bioactive glasses (M199 + 45S5; M199 + 45S5.5Li), M199 medium with 2 ng mL−1 bFGF (M199 + bFGF) as the positive control, and M199 medium without ionic dissolution products or bFGF as the negative control were placed, in triplicate, in each well of a transwell plate (Corning, New York, NY, USA). The effect of M199 medium enriched with 0.20 mmol of LiCl or NaCl was also studied. Then, the transwell inserts were placed and 20,000 cells were seeded in M199 medium (200 µL) in the upper compartment of each insert. After 8 h of incubation, the inserts were removed and placed in Crystal Violet for 10 min for fixation and staining of the cells. The inserts were then washed with water, and the residual cells which had not migrated through the microporous membrane (transmigration) were removed with a cotton swab. Cells were analyzed by means of an inverted optical microscope, and photos of two different areas were taken and the number of cells which had transmigrated to the lower side of the membranes was quantified.
Tubulogenesis Assay in Matrigel™: For the assay of tubulogenesis in Matrigel™, 60 µL of Matrigel™ (BD Biosciences, San Jose, CA, USA) was placed into each well of a 96-well plate and was left to solidify for 30 min at 37 °C before seeding the cells. HUVECs were trypsinized and raised, centrifuged, and counted in a Neubauer chamber. A total of 10,000 cells were seeded in each well onto the surface of the Matrigel™ in 150 µL of M199 medium supplemented with ionic dissolution products (M199 + 45S5; M199 + 45S5.5Li). The effect of M199 medium enriched with 0.20 mmol LiCl or NaCl was also evaluated. The assay was conducted in quadruplicate using M199 medium with bFGF as the positive control and M199 medium without ionic dissolution products or growth factor as the negative control.
The assay was monitored every hour for 8 h. At the end of the assay, the cells were evaluated by means of an inverted optical microscope and photographs of two fields per well were taken and the number of fully enclosed tubes present within each photographed area was quantified.
2.4. Enzyme Linked Immunosorbent Assay (ELISA)
An ELISA was conducted to measure the levels of secretion of proangiogenic cytokines, including bFGF, VEGF, IGF1, and TGF-β, among others. HUVECs were seeded to confluence in six-well culture plates. After reaching the cell monolayer, the cells were stimulated with M199 medium enriched with the ionic dissolution products (M199 + 45S5; M199 + 45S5.5Li), M199 medium with 2 ng mL−1 bFGF (M199 + bFGF) as the positive control, and M199 medium without ionic dissolution products or bFGF as the negative control. After 48 h, the supernatant was extracted and the assay continued according to the protocol of the commercial ELISA (EA-1011, Signosis, Holy Clear, CA, USA).
2.5. Determination of the Levels of β-Catenin by Western Blot
HUVECs were seeded to confluence in six-well culture plates. After reaching the monolayer, the cells were stimulated for 24 h with the ionic dissolution products of the bioactive glass 45S5.5Li (M199 + 45S5.5Li). The effect of the M199 medium enriched with 0.20 mmol of LiCl or NaCl was also studied. M199 medium without ionic dissolution products or bFGF was used as the negative control. At 24 h post-stimulation, the cells were washed with PBS three times to stop the reaction and were frozen at −20 °C. After 24 h, the cells were thawed and homogenized in 200 µL of RIPA buffer (50 mmol Tris-HCl, pH 7.4, 1% NP-40, 0.5% Na deoxycholate, 0.1% SDS, 150 mmol NaCl, 2 mmol EDTA, 2 mmol PMSF, and 50 mmol NaF), sonicated, and centrifuged for 15 min at 12,500 rpm. Then, 30 µg of total protein, determined by Bradford, was heated in seeding buffer (50 mmol Tris-HCl, pH 6.6; 2% SDS, 10% glycerol, and 0.05% bromophenol blue), for 5 min at 100 °C, and seeded in a 10% mini-polyacrylamide gel. Electrophoresis was performed as previously described [
10]. The nitrocellulose membranes were blocked with 5% skim milk powder prepared in TBS (Tris 25 mmol, 150 mmol NaCl, 2 mmol KCl, pH 7.4), supplemented with 0.1% Tween-20 (TBS-T) for 1 h, washed with TBS-T three times, and incubated overnight with primary monoclonal anti-active β-catenin antibody, produced in mouse (1:500) (Millipore™ (Upstate™) clone: 8E7, 50-171-753, Waltham, MA, USA) or with a polyclonal anti-GAPDH IgG antibody produced in rabbit as a loading control (1:2500, Santa Cruz Biotechnology, sc-25778, Dallas, TX, USA). This incubation step was performed in TBS-T with 5% BSA at 4 °C. Then, the membranes were washed with TBS-T and detection was carried out by incubating for 1 h with the corresponding secondary antibodies: anti-mouse antibody produced in horse (1:2000, Vector PI-2000, Burlingame, CA, USA) and anti-rabbit antibody produced in goat (1:2500, Cell Signaling, #7074, Danvers, MA, USA) conjugated to peroxidase in TBS-T with 5% skim milk powder at room temperature. Subsequently, the membranes were washed and incubated for 1 min with a chemiluminescent peroxidase substrate solution (ECL™, Amersham Biosciences, GE Healthcare UK Ltd., Chalfont, Buckinghamshire, UK). The results were visualized by autoradiography of the chemiluminescent reaction.