8 mm and the layer thickness was 0.225 mm. A 0�C90�� selleck chem inhibitor configuration was used for scaffold architecture where fibers were deposited with 90�� orientation steps between successive layers. Deposition of electrospun fibers onto 3DF scaffolds PA was dissolved in a mixture of chloroform- hexafluoroisopropanol (HFIP) (78�C22%v/v) respectively. A 28% (w/v) PEOT/PBT solution in chloroform- HFIP were prepared and stirred overnight at room temperature. The solution was loaded into a syringe and the flow rate was controlled using a syringe pump (KDS 100, KD Scientific). The other end of the syringe was connected to a needle, on which a positive high voltage was applied using a high voltage generator (Gamma High Voltage Research Inc.). A metallic sheet of stainless steel was the collector (ground).
An electrostatic field was formed between the needle and the collector when the generator was turned on. The polymer solution was pushed through the syringe to the tip of the needle. When the electrostatic field strength overcame the surface tension of the liquid drop at the tip of the needle, the drop was stretched into fibers and deposited onto the 3DF scaffold placed on the collector. A flow rate of 15 mL/h, distance of 15 cm and voltage of 12 kV were used for the spinning and fibers were deposited for 30 sec on the collector. This process was repeated at 0.9 mm, 2.025 mm and 2.925mm of 3DF scaffold height to create 3DF-ESP scaffolds. For the in vitro experiments, configuration 1 ESP layer per 4 3DF layers was selected. Temperature and humidity were monitored during the process and ranged between 19.
6?20.4��C and 33?42% respectively. Preparation of biomimetic Ca-P coatings on scaffolds 3DF and 3DF-ESP scaffolds were punched to cylinders (5 mm diameter, 4 mm height) and coated with Ca-P using a two-step coating process. In the first step, a five times concentrated simulated body fluid solution (SBF �� 5) was prepared by dissolving reagent grade NaCl (40 g), CaCl2.2H2O (1.84 g), MgCl2.6H2O (1.52 g), NaHCO3 (1.76 g) and Na2HPO4.2H2O (0.89 g) salts in 1l of demineralised water at 37��C under CO2 gas bubbling. The CO2 source was then removed from the solution and the 3DF and 3DF-ESP scaffolds were immersed in the solution in a partly open vial and left to coat for 24 h under continuous gentle stirring at 37��C. This process was previously shown to result in a formation of thin, amorphous Ca-P layer.
In the second step, scaffolds pre-coated in step 1 were immersed in a calcium phosphate solution (CPS) at physiological pH of 7.4 and temperature of 37��C for 24 h to deposit a crystalline Drug_discovery layer onto previously formed amorphous Ca-P layer. CPS was prepared by dissolving NaCl (8 g), CaCl2.2H2O (0.59 g), Na2HPO4.H2O (0.36 g) and Tris (6.05 g) in MilliQ water and the pH of the solution was adjusted to 7.4 with 1M HCl. The coated scaffolds were thoroughly washed in MilliQ water and dried overnight at 50��C.