Page 33 - Development of Functional Scaffolds for Bone Tissue Engineering Using 3D-Bioprinting of Cells and Biomaterials - Yasaman Zamani

containing 10 ml NaOH solution with moderate shaking to ensure uniform contact between NaOH solution and the surface of PCL strands everywhere in the scaffold. Scaffolds were subsequently washed extensively with deionized water until the pH of the washing water was neutral. Finally, the scaffolds were air-dried and stored at room temperature until further experimentation. Scaffolds were UV sterilized for 30 min, and immersed in 70% ethanol for 1 h prior to cell seeding [25-27]. Scaffolds were treated in triplicate.
RGD-immobilization on the surface
For RGD immobilization, scaffolds were immersed in 10% w/v solution of 1,6-hexanediamine in isopropanol at 37°C for 1 h to create amine groups on the surface, followed by washing in deionized water. The aminated PCL scaffolds were then washed 3 times with activation buffer (0.1 M phosphate buffer containing 0.15 M NaCl, pH 7.2). Subsequently, 600 μl of hetero- bifunctional crosslinker sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) was pipetted on the aminated PCL scaffolds and incubated at room temperature for 1 h. Scaffolds were then washed with conjugation buffer (activation buffer with 0.1 M EDTA, pH 7). Finally, 600 μl RGD at a concentration of 0.125 mg/ml of conjugation buffer was added to each scaffold and incubated overnight at 4°C with moderate shaking [28]. Scaffolds were then washed with deionized water, air-dried, and stored at room temperature until further experimentation. Scaffolds were UV sterilized for 30 min, and immersed in 70% ethanol for 1 h prior to cell seeding. Scaffolds were treated in triplicate.
Scaffold characterization
The surface composition of the scaffolds was studied using a Shimadzu FTIR-8400s spectrophotometer (Shimadzu, Kyoto, Japan). Scanning electron microscopy (SEM) was used to study the surface morphology and measure strand and pore dimensions. 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. Five measurements in total were carried out randomly from the three replicates of scaffolds and data were expressed as mean ± SD. To study cell morphology on different PCL scaffolds using SEM, cell seeded scaffolds were first fixed using 4% glutaraldehyde and subsequently dehydrated in graded ethanol series (50, 70, 80, 90, 100%). The amount of RGD peptide on the scaffold surface was quantified using Fluorescein isothiocyanate (FITC, Molecular Probes, Eugen, OR). PCL scaffolds with or without immobilized RGD on the surface were immersed in FITC solution (2 mg/ml in DMF) at room temperature for
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