Page 112 - Development of Functional Scaffolds for Bone Tissue Engineering Using 3D-Bioprinting of Cells and Biomaterials - Yasaman Zamani

3 times to determine ALP activity and protein content. P-nitrophenyl-phosphate (Merck, Darmstadt, Germany) at pH 10.3 was used as substrate for ALP as described earlier [29]. The absorbance was read at 410 nm. ALP activity was expressed as μM/μg cellular protein. The amount of protein was determined by using a BCA Protein Assay reagent Kit (PierceTM, Rockford, lll, USA), and the absorbance was read at 540 nm with a Synergy HT® spectrophotometer. ALP activity was also stained and visualized using nitro blue tetrazolium chloride/5-bromo-4-chloro-3- indolyl phosphate (NBT/BCIP; Roche, Germany) following the standard protocols. Constructs were assayed in triplicate.
Statistical analysis
Data are expressed as mean ± standard deviation (SD). Differences in mean values were analyzed by unpaired two-tailed t test using GraphPad Prism® 7.0 (GraphPad Software Inc., La Jolla, CA, USA). Differences were considered significant if p<0.05.
RESULTS
MC3T3-E1 pre-osteoblasts retention in cell-seeded and bioprinted PLGA/β-TCP scaffolds
Cell retention, which is expressed as the percentage of cells attached to the scaffold to the total number of cells initially incorporated in the scaffolds, was measured 16 h after seeding and printing. Cell retention was 1.7-fold higher in the bioprinted scaffold (87 ± 3%) compared with the cell-seeded scaffold (51 ± 5%; Fig. 2a).
MC3T3-E1 pre-osteoblasts viability in cell-seeded and bioprinted PLGA/β-TCP scaffolds
Cell viability was measured 16 h after seeding and printing using live/dead staining. Cell viability was higher by 1.1-fold in the cell-seeded scaffold (87 ± 2%) compared with the bioprinted scaffold (78 ± 4%; Fig. 2b). Live cells appeared as green dots and dead cells appeared as red dots in the fluorescent microscopy images (Fig. 2c). Lower cellular density was observed on the cell-seeded scaffold compared with the bioprinted scaffold by visual inspection.
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