Page 34 - Development of Functional Scaffolds for Bone Tissue Engineering Using 3D-Bioprinting of Cells and Biomaterials - Yasaman Zamani
P. 34

6 h and then washed three times with PBS to remove unreacted FITC. The fluorescence intensity was measured using a Synergy HT® spectrophotometer (490 nm excitation and 520 nm emission). A standard calibration curve was obtained from the intensity of known concentrations of BSA (Life technologies, Carlsbad, CA) [29].
Cell culture and seeding onto the scaffolds
MC3T3-E1 pre-osteoblasts were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown and maintained in α-Minimum Essential Medium (α-MEM; Gibco, Life Technologies, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; BioWest SAS, Nuaille, France) and 1% PSF (antibiotic antimycotic solution, Sigma-Aldrich®, St. Louis, MO, USA) in a humidified incubator with 5% CO2 in air at 37°C. After reaching ~75% confluency, cells were detached using 0.25% trypsin (Gibco, Invitrogen, Waltham, MA, USA) and 0.1% ethylenediaminetetraacetic acid (Merck, Darmstadt, Germany) in phosphate-buffered saline (PBS) at 37°C. Cells were then re-suspended in α-MEM with 10% FBS, 1% PSF, 50 μg/ml ascorbic acid, and 10 mM ß-glycerophosphate (osteogenic medium). Five 40 μl drops of the cell suspension were carefully spread all over the scaffolds surface 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. For determination of the optimum NaOH concentration for surface modification that enhances cell proliferation and matrix production, cells were cultured up to 8 days on unmodified control, 1 M NaOH-treated, 3 M NaOH-treated, and 5 M NaOH-treated 3D- printed PCL scaffolds. Thereafter, cell proliferation was determined as described below. Moreover, cell extracellular matrix deposition was visualized by picrosirius red staining (see below). For further experimentation, cells were cultured up to 14 days on unmodified control, 24 h NaOH-treated, 72 h NaOH-treated, and RGD-immobilized scaffolds 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 3 equal parts (volume: 171×10-3 cm3) since cell distribution pattern from top to bottom of scaffolds was relatively homogeneous (see also under “Collagenous matrix deposition”). Left parts of all scaffold groups (part 1 construct) were compared with respect to collagenous matrix deposition, middle parts of all scaffold groups (part 2 construct) were compared with respect to ALP activity, and right parts of all scaffold groups (part 3 construct) were compared with respect to calcium deposition, 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 groups.
32































































































   32   33   34   35   36