Page 97 - Magnesium-based supports for stem cell therapy of vascular disease - Mónica Echeverry Rendón

PLASMA ELECTROLYTIC OXIDIZED MAGNESIUM ADVERSELY INFLUENCES VASCULAR CELLS TYPES BUT NOT MESENCHYMAL CELLS AND MONOCYTES
were cultured in 96 wells plates at a concentration of 50,000 cells/cm2, until confluence. Next, medium was removed and replaced with 100μl of twofold serial dilutions of materials’ leachables. Cells were incubated at 37°C, 5% CO2 and 98% humidity for 48 h. Then, 20μl per well of 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT, Sigma-Aldrich, St. Louis, Missouri, USA) at 5mg/mL was added per well. Cells were incubated for 3h. After that, medium was removed and 100 μl of dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, Missouri, USA) was added to dissolve the deposited crystals. Absorbance was measured at 570nm wavelength in a spectrophotometer (Biorad). The conversion of MTT of treated cells was normalized to untreated (control) cells and plotted as fraction (%) of mitochondrial activity. Measurements were performed in triplicate and repeated at least three times independently. Mean and deviation standard were calculated and plotted using GraphPad-Prism7 (GraphPad, CA, USA).
2.5 Cell-material interaction
Surface-coated materials and controls were seeded with 100μl ASC at 100,000 per cm2 in individual wells of 12 wells plates. After 30 min, 1ml of medium was carefully added and cells were incubated at 37°C for 48h. Next, medium was removed and samples washed twice with PBS and fixed with 2% paraformaldehyde in PBS for 30 min. Cytoskeleton was visualized by staining with Phalloidin-iFluor 647 Reagent – CytoPainter (1:500, Abcam, Cambridge, U.K) and counterstaining of nuclei with DAPI (1:5000, Sigma-Aldrich, D9542, St. Louis, Missouri, USA,). Fluoromicrographs were taken with a TissueFAXs scanning microscope (TissueGnostics GmbH, Vienna, Austria).
2.6 Western Blot Analysis
Proliferation and apoptosis of cells cultured in contact with materials’ leachables from surface-coated and control magnesium sections was investigated with Western blotting. Cells were lysed in RIPA buffer and the protein con- centration determined. Proteins (20μg/lane) were separated in SDS-PAGE Gradient Gels (4-20%) (BioRad, California, USA). Next, proteins were transferred onto a nitrocellulose membrane (BioRad, California, USA). The membrane was blocked with 5% non-fat milk for 2 h, followed by overnight incubation at 4°C with primary antibody, respectively directed against PARP (1:500, Santa Cruz, sc-7150, California, USA), caspase 3 (1:100, Cell Signaling, 9665S, Danvers, Massachusetts, USA), PCNA (1:2000, Cell signaling, 2586S, Danvers, Massachusetts, USA) or β-actin (1:2000, Cell Signaling, 4967L, Danvers, Massachusetts, USA). Following incubation with HRP-conjugated goat anti-mouse IgG (Dako, P0447, California, USA) and HRP-conjugated rabbit anti-goat IgG (Cell Signaling Technology, 7054S, Danvers, Massachusetts, USA) at room temperature for 2 h, proteins were visualized using alkaline phosphatase method.
2.7 Wound healing – scratch – assay
For the wound healing assay, the materials’ leachables were used. In addition, conditioned medium (ASC-Cme) was collected from cultures of ASC (seeded at 100,000 per cm2) on surface-coated materials and c.p Mg controls after 48h. Sentinel cells used for the assay were PK84 fibroblasts seeded at 10,000 per cm2 in 24 wells tissue plates and used upon reaching confluency. A straight scratch was made across the center of the well with a 200 μl pipette tip simulating a wound. Wells were washed with PBS to remove detached cells and fragments. Next, 0.5 ml of materi- als’ leachables or 0.5 ml of ASC-Cme were added and the cellular responses monitored for 24h. Micrographs were taken every 8h with a Leica DMRXA inverted microscope and processed with Leica Software of Leica Microsystems (Wetzlar, Germany). The relative wound size at each time point was analyzed using ImageJ 1.48v (Wayne Rasband National Institutes of Health, USA), values were normalized to the initial wound size. Experiments were performed in triplicate and done three times independently.
2.8 Cell differentiation of ASC
Adipogenic, osteogenic and myogenic differentiation capacity of confluent monolayers of ASC (passage 3 to 7) was initiated with differentiation medium (see below) that was changed twice per week for two weeks. Then, cells were fixed and their cell types assessed by staining. For adipogenic differentiation, ASC were treated with 0.1μM of
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