Page 36 - Development of Functional Scaffolds for Bone Tissue Engineering Using 3D-Bioprinting of Cells and Biomaterials - Yasaman Zamani

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 Nikon SMZ-10 stereo microscope (Nikon, Tokyo, Japan) and a Leica inverted microscope (Leica Microsystems, Wetzlar, Germany). For collagen quantification, picrosirius red stain was eluted from the constructs using 0.2 M NaOH/methanol (1:1, v/v) for 30 min under shaking. Hundred μl of this solution per well of a 96- well plate (Greiner, Bio-One, Alphen a/d Rijn, The Netherlands) was used to measure absorbance at 490 nm with a microplate reader (BioRad Laboratories Inc., Veenendaal, The Netherlands) [31]. Constructs were air-dried at room temperature for 24 h and weighed. Data were normalized to the weight of the dried construct and expressed as absorbance/g of construct. Constructs were assayed in triplicate.
Alkaline phosphatase activity and protein assay
Alkaline phosphate (ALP) activity was measured to assess the osteoblastic phenotype of MC3T3- E1 pre-osteoblasts in surface-modified and unmodified 3D-printed PCL scaffolds. At day 14 of cell culture on the scaffolds, one part (part 2 construct; volume: 171×10-3 cm3) of the cell/scaffold constructs was 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 [32]. The absorbance was read at 410 nm. ALP activity was expressed as nM/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. Constructs were assayed in triplicate.
Calcium deposition
Calcium deposition by MC3T3-E1 pre-osteoblasts on surface-modified and unmodified PCL scaffolds was analyzed at day 14. To determine calcium deposition, one part (part 3 construct; volume: 171×10-3 cm3) of the cell/scaffold constructs was washed with PBS and fixed in 4% formaldehyde. Fixed constructs were incubated with 40 mM alizarin red staining solution, pH 4.3, at room temperature for 30 min, and washed extensively with deionized water to remove the unreacted dye. Optical images were taken using a stereo microscope. For quantification of calcium deposition, constructs were immersed in 0.5 ml of 5% sodium dodecyl sulfate (SDS) in 0.5 N HCL at room temperature for 1 h under shaking. Hundred μl of this solution per well of a 96-well plate was used to measure absorbance at 405 nm with a microplate reader [33] (BioRad
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