Page 111 - Development of Functional Scaffolds for Bone Tissue Engineering Using 3D-Bioprinting of Cells and Biomaterials - Yasaman Zamani
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MC3T3-E1 pre-osteoblasts viability
A live/dead viability/cytotoxicity kit (L3224; Thermo Fisher Scientific, Waltham, MA, USA) was used to assess cell viability in the cell-seeded and the bioprinted scaffolds according to manufacturer’s instructions. Briefly, 16 h post cell seeding and cell printing, cell/scaffold constructs were washed in PBS, followed by incubation in 0.5 μl/ml calcein acetoxymetyl (Cal- AM) and 2 μl/ml ethidium homodimer-1 (EthD) in PBS at 37°C for 1.5 h. Concentration of Cal-AM and EthD stock solutions was 4 mM in anhydrous dimethyl sulfoxide (DMSO) and 2 mM in DMSO/H2O 1:4 (v/v), respectively. Cell/scaffold constructs were washed again in PBS and imaged using fluorescent microscopy (Leica Microsystems, Wetzlar, Germany). Live and dead cell quantification was performed in ImageJ v1.8 for windows.
MC3T3-E1 pre-osteoblasts proliferation
Proliferation was assessed by determining cell number in scaffolds at days 1, 7, 14, and 21, and by dividing these numbers to cell number in the scaffolds at day 1 using AlamarBlue® fluorescent assay, as described above under “cell retention”. At each time point, scaffolds were transferred to a new plate, AlamarBlue® was added to the scaffolds, and fluorescence was measured. Thereby, cells attached to the old plates were not included in the measurements. After performing the AlamarBlue® assay at each time point, 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.
Collagenous matrix deposition by MC3T3-E1 pre-osteoblasts
Picrosirius red stain kit (Chondrex, Inc., Redmond, WA, USA) was used to visualize collagen deposition on the scaffolds. After 21 days of culture, 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 Leica inverted microscope (Leica Microsystems, Wetzlar, Germany).
Alkaline phosphatase activity and protein assay
Alkaline phosphate (ALP) activity was measured to assess the osteoblastic phenotype of MC3T3- E1 pre-osteoblasts in the cell-seeded and the bioprinted PLGA/β-TCP scaffolds. At day 21 of cell culture on the scaffolds, cell/scaffold constructs were subjected to cell lysis. Cells were lysed with the CyQuant® lysis buffer (Molecular Probes/Invitrogen, Carlsbad, CA, USA) and freeze-thawed
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