Page 109 - Development of Functional Scaffolds for Bone Tissue Engineering Using 3D-Bioprinting of Cells and Biomaterials - Yasaman Zamani

MC3T3-E1 pre-osteoblasts seeding on the 3D-printed PLGA/β-TCP 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 β-glycerophosphate (osteogenic medium) and seeded on the scaffolds at a density of 5×105 cells/cm3 scaffold and incubated in a humidified incubator with 5% CO2 in air at 37°C.
MC3T3-E1 pre-osteoblasts encapsulation in alginate
Sodium alginate powder (Sigma-Aldrich) was sterilized under UV for 20 min and dissolved in culture medium. The alginate solution was vortexed at high speed and subsequently kept at room temperature for at least 1 h to minimize bubbles in the solution. To encapsulate cells in alginate, cultured pre-osteoblasts were slowly suspended at a density of 4×106 cells/ml in 6 wt% alginate solution and transferred into a 10 ml sterile syringe. Every 0.5 ml of this cell suspension was used to print 4 scaffolds and therefore, 5×105 cells were printed in each scaffold.
3D-bioprinting of MC3T3-E1 pre-osteoblasts in PLGA/β-TCP scaffolds
Scaffolds were designed using BioCADTM software (RegenHU, Villaz-St-Pierre, Switzerland). PLGA/β-TCP pellets were melted at 110°C in the heating tank and extruded through the pre- heated thermoplastic polymer nozzle at 0.4 MPa (4 Bar). In the first layer, PLGA/β-TCP struts were deposited in parallel and then, cells encapsulated in alginate were deposited between the PLGA/β-TCP struts by passing through a 21-gauge (21G) needle. The subsequent layers were deposited with the same pattern but with a deposition angle of 90° until the required height was obtained. When the structure was complete, scaffolds were immersed in 102 mM CaCl2 for 10 min to crosslink the alginate. Afterwards, osteogenic medium was added to the scaffolds in 24- well culture plates. Scaffolds were incubated in a humidified incubator with 5% CO2 in air at 37°C. The fabrication process of the cell-seeded and the bioprinted scaffolds is schematically illustrated in figure 1.
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