Page 90 - Development of Functional Scaffolds for Bone Tissue Engineering Using 3D-Bioprinting of Cells and Biomaterials - Yasaman Zamani

in α-MEM with 10% FBS, 1% PSF, 50 μg/ml ascorbic acid, and 10 mM β-glycerophosphate (osteogenic medium). Five 40 μl drops of the cell suspension were carefully spread all over the surface of both porous and 3D-printed PLGA/β-TCP scaffolds at 5×105 cells/cm3 scaffold in 24- well culture plates. Attachment was allowed for 30 min, and osteogenic medium was added to cell/scaffold constructs. The cell-seeded scaffolds were cultured up to 14 days in a humidified incubator with 5% CO2 in air at 37°C. Seeding efficiency and cell proliferation were determined as described below. At day 14, scaffolds were collected and cut longitudinally into 4 equal parts (volume: 0.118 cm3). One part (part 1 construct) was used to assess cell morphology, one part (part 2 construct) was used to assess cell dispersion, one part (part 3 construct) was used to assess collagenous matrix deposition, and one part (part 4 construct) was used to assess ALP activity, as described below. Our experimental set up was in such a way that we always used parts from the same location in the scaffolds for comparison between the scaffolds.
Cell seeding efficiency
Sixteen hours after cell seeding, cell/scaffold constructs in a 24-well culture plate (“old plate”) were washed twice with PBS, and transferred to a fresh 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. 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, and incubated in AlamarBlue® solution for 4 h in a humidified incubator with 5% CO2 at 37°C. The solutions were harvested from the scaffolds and the old plate, and fluorescence was measured at 530 nm with a Synergy HT® spectrophotometer. Scaffolds were washed twice with PBS, and culture was continued in a humidified incubator with 5% CO2 at 37°C in fresh osteogenic medium. 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. We found a linear relationship between AlamarBlue® fluorescence and cell number (data not shown).
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