Page 89 - Development of Functional Scaffolds for Bone Tissue Engineering Using 3D-Bioprinting of Cells and Biomaterials - Yasaman Zamani

Scaffolds characterization
Scanning electron microscopy (SEM) was used to study surface morphology, dispersion of β-TCP particles, and pore size of porous and 3D-printed scaffolds. Scaffolds were coated with a layer of gold using an Edwards Sputter Coater S150B (Edwards, Burgess Hill, UK) and observed using a Zeiss EVO LS-15 scanning electron microscope (Zeiss, Oberkochen, Germany) with an accelerating voltage of 10 kV. Pore size measurements (n=5), were carried out randomly from the three replicates of scaffolds and data were expressed as mean ± SD. Surface hydrophilicity was evaluate by measuring the water contact angle at the surface of porous and 3D-printed scaffolds using a USB digital microscope (Veho, Hampshire, UK). Surface roughness was measured using a surface profilometer (Fanavari Kahroba Co., Tehran, Iran). To determine the porosity of the scaffolds, a liquid displacement method was used. Ethanol was used as the displacement liquid since it can easily penetrate the scaffold pores without inducing shrinkage or swelling [14]. Scaffolds were immersed in a known volume of ethanol (V1) for 20 min under vacuum. The total volume of ethanol and ethanol-impregnated scaffolds was recorded as V2. The ethanol-impregnated scaffolds were removed and the residual ethanol volume was recorded as V3. The porosity of the scaffolds (p) was calculated using the following equation:
(𝑉𝑉𝑉𝑉 − 𝑉𝑉𝑉𝑉 ) 𝑝𝑝𝑝𝑝=13
(𝑉𝑉𝑉𝑉2 − 𝑉𝑉𝑉𝑉3)
Scaffolds compressive strength was assessed using a universal testing machine (Instron 6022, Instron Limited, High Wycombe, UK) with a load cell of 1 kN. The cross head speed was set to 1 mm/min. Values of load F (N) and time (sec) were recorded during the test. Stress values (MPa) were determined by dividing the load (N) by the cross sectional area of each specimen (mm2). Strain values were determined by dividing displacement values (mm) by the initial height of the specimen. Compressive strength was determined by 1% offset of the first linear section of the stress-strain curves.
Cell culture and seeding onto the scaffolds
MC3T3-E1 pre-osteoblasts were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown and maintained in 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. Upon reaching ~75% confluency, cells were detached using 0.25% trypsin (Gibco, Invitrogen, Waltham, MA, USA) and 0.1% EDTA (Merck, Darmstadt, Germany) in phosphate-buffered saline (PBS) at 37°C. Cells were then re-suspended
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