Page 35 - Development of Functional Scaffolds for Bone Tissue Engineering Using 3D-Bioprinting of Cells and Biomaterials - Yasaman Zamani

Seeding efficiency
Twelve hours after cell seeding, cell/scaffold constructs in a 24-well culture plate (“old plate”) were washed twice with PBS, and transferred to a new 24-well culture plate. Seeding efficiency was assessed by determining the number of cells attached to the wells of the old plate as well as the number of cells attached to the scaffolds, using AlamarBlue® fluorescent assay (Invitrogen, Frederick, MD, USA), according to the manufacturer’s instructions. We found a linear relationship between AlamarBlue® fluorescence and cell number (data not shown). Ten percent AlamarBlue® solution in fresh osteogenic medium was added to the wells of the old plate and to each scaffold until it completely covered the top of the scaffolds. Scaffolds and old plate were incubated in AlamarBlue® solution for 4 h in a humidified incubator with 5% CO2 at 37°C. The solution was harvested from the scaffolds and the old plate, and the fluorescence measured at 530 nm with a Synergy HT® spectrophotometer. Scaffolds were washed twice with PBS, and incubated in a humidified incubator with 5% CO2 at 37°C. Seeding efficiency was calculated according to the following equation [15]:
Seeding efficiency (%) = number of cells attached to scaffold × 100 number of cells attached to scaffold + number of cells attached to plate
Scaffolds were assayed in triplicate.
MC3T3-E1 proliferation
Proliferation was assessed by determining cell number in 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”. We found a linear relationship between AlamarBlue® fluorescence and cell number (data not shown). At each time point, scaffolds were transferred to a new plate, alamar blue was added to the scaffolds, and fluorescence was measured. Therefore, cells attached to the old plates were not included in the measurements. After performing the AlamarBlue® assay at each day, 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
Picrosirius red stain kit (Chondrex, Inc., Redmond, WA, USA) was used to visualize and quantify total collagen deposition and to obtain an indication of cell distribution inside scaffolds [30]. After 14 days of culture, one part (part 1 construct; volume: 171×10-3 cm3) of the cell/scaffold constructs
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