Page 91 - Development of Functional Scaffolds for Bone Tissue Engineering Using 3D-Bioprinting of Cells and Biomaterials - Yasaman Zamani

Cell proliferation
Proliferation was assessed by determining cell number in the scaffolds at days 3, 7, and 14, and by dividing these numbers to cell number in the scaffolds at day 1 using AlamarBlue® fluorescent assay, as described above under “seeding efficiency”. At each time point, scaffolds were transferred to a new plate, 10% AlamarBlue® was added to the scaffolds, and fluorescence was measured. Cells attached to the old plates were not included in the measurements. After performing the AlamarBlue® assay, scaffolds were washed twice with PBS, and incubated in osteogenic medium in a humidified incubator with 5% CO2 at 37°C. Scaffolds were assayed in triplicate.
Cell morphology and dispersion
To study cell morphology on porous and 3D-printed scaffolds, one part (part 1 construct; volume: 0.118 cm3) of the cell/scaffold constructs were fixed using 4% glutaraldehyde and subsequently dehydrated in graded ethanol series (50, 70, 80, 90, and 100%). After drying, the fixed constructs were coated with a layer of gold using an Edwards Sputter Coater S150B (Edwards, Burgess Hill, UK) and observed using a Zeiss EVO LS-15 scanning electron microscope (Zeiss, Oberkochen, Germany) with an accelerating voltage of 10 kV. To determine cell dispersion in porous and 3D- printed scaffolds, another part (part 2 construct; volume: 0.118 cm3) of the cell/scaffold constructs were washed thoroughly with PBS and fixed in 4% formaldehyde. Cells in the fixed constructs were fluorescently stained by using 4’,6-diamidino-2-phenylindole (DAPI), and visualized using a Leica inverted microscope (Leica Microsystems, Wetzlar, Germany).
Collagenous matrix deposition by cells
Picrosirius red stain kit (Chondrex, Inc., Redmond, WA, USA) was used to visualize and quantify total collagen deposition by the pre-osteoblasts. After 14 days of culture, one part (part 3 construct; volume: 0.118 cm3) of the cell/scaffold constructs were washed thoroughly with PBS and fixed in 4% formaldehyde. Fixed constructs were stained for 2 h with picrosirius red at room temperature. Then, constructs were washed twice with acidified water (5 ml acetic acid/L distilled water) and visualized using a Nikon SMZ-10 stereo microscope (Nikon, Tokyo, Japan). For collagen quantification, picrosirius red stain was eluted from the constructs using 0.2 M NaOH/methanol (1:1, v/v) for 30 min under shaking. Hundred μl of this solution per well of a 96- well plate (Greiner, Bio-One, Alphen aan den Rijn, The Netherlands) was used to measure absorbance at 490 nm with a microplate reader (BioRad Laboratories Inc., Veenendaal, The Netherlands) [16]. Constructs were air-dried at room temperature for 24 h and weighed. Data
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