Page 92 - Development of Functional Scaffolds for Bone Tissue Engineering Using 3D-Bioprinting of Cells and Biomaterials - Yasaman Zamani

were normalized to the weight of the dried constructs and expressed as absorbance/g of construct. Constructs were assayed in triplicate.
Alkaline phosphatase activity and protein assay
Alkaline phosphatase (ALP) activity was measured to assess the osteoblastic phenotype of MC3T3-E1 pre-osteoblasts in porous and 3D-printed PLGA/β-TCP scaffolds. At day 14 of cell culture on the scaffolds, one part (part 4 construct; volume: 0.118 cm3) of the 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 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 [17]. The absorbance was read at 410 nm. ALP activity was expressed as nmol/μg cellular protein. The total amount of protein was determined by using a BCA Protein Assay reagent Kit (PierceTM, Rockford, IL, USA), and the absorbance was read at 540 nm with a Synergy HT® spectrophotometer. ALP activity was also 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 (n=3). 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
Characterization of porous and 3D-printed PLGA/β-TCP scaffolds
The porous scaffolds had a pore size of 408 ± 90 μm and a porosity of 85 ± 5%. The 3D-printed scaffolds had a pore size of 315 ± 17 μm and a porosity of 39 ± 7% (Table 1, Fig. 1a). β-TCP particles were more homogeneously dispersed on the surface of the 3D-printed scaffolds than on the surface of the porous scaffolds (Fig. 1b). β-TCP aggregates were visible only on the surface of the porous scaffolds (Fig. 1b). The surface of the 3D-printed scaffolds was smoother (Ra: 22 ± 3 μm) and more regular compared with the highly rough surface of the porous scaffolds (Ra: 110 ± 15 μm) which had an irregular structure (p=0.0004, n=3; Fig. 2a). The 3D-printed scaffolds had a more hydrophilic surface (water contact angle: 76 ± 6°) compared with the porous scaffolds
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