Bioplotter

Each bioink of interest to the present study was loaded into an EnvisionTEC Bioplotter (Gladbeck, Germany) and heated to 37 °C for 30 min (to allow for temperature equilibration prior to printing). The scaffold 3D files were designed using Autodesk Fusion 360 (San Rafael, CA); the stereolithography files were then imported into the Bioplotter RP (Version 3.0.713.1406, EnvisionTEC). Each 3D model was sliced using Bioplotter RP to the desired slice thickness based on the print nozzle used (specifically, 160, 200, and 320 µm slice thickness corresponding to the 200, 250, and 400 µm tip diameter nozzle, respectively). Once sliced, the 3D models were imported to Visual Machines (Version 2.8.115; EnvisionTEC) to be printed. All scaffolds were printed at a bioink temperature of 37 °C, 0.5–0.7 bar pressure, and at a speed of 10–12 mm/s (actual speed based on the bioink composition). A 0.3 s preflow and 0.1 s post-flow were implemented, with the printing nozzle cleaned after every three printed layers. The printing stage was kept at a temperature of 27 °C. Upon completion of the printing process, each printed scaffold was allowed to thermally set at room temperature for 5 min before removal from the printing stage. After a period of at least 72 h, the 3D-printed scaffolds were sintered using a Hot Spot 110 furnace (Zircar Zirconia, Florida, NY) to 1240 °C for a cycle of 20 h.

Scaffold design and synthesis followed our previous methods to create 3D printed scaffolds with interconnecting microchannels43 (link)44 (link)66 (link)67 . Using 100 wt% poly-ε-caprolactone, PCL (Sigma-Aldrich Corp.,) with molecular weight ~65 KDa was 3D-printed using a bioplotter (EnvisionTec, Gladbeck, Germany). PCL pellets were first melt at 120 °C and then printed layer-by-layer as a cylinder (5 × 3 mm: diameter × height) using CAD software. Interlaid strands had diameters of 250–300 μm and interconnecting microchannels had a diameter of 400–500 μm. Scaffolds were sterilized in Ethylene Oxide (ETO) gas (Anprolene AN74i, Fisher, Pittsburgh, PA).

Additive manufacturing of the pre-gel suspension was conducted using a syringe based dispensing technique (Bioplotter, EnvisionTEC). Directly after preparation, the pre-gel was loaded into a syringe and left to settle for 24 h prior to printing. A diagram of the process can be observed in Figure 1. A dispersing needle with an inner diameter of 1 mm was used for printing. Line printing was used as a tool to evaluate the most suitable printing parameters to use for this material. The parameters with the linewidth most similar to the nozzle diameter (1 mm) was then chosen (this was 0.8 bar at a speed of 10 mm/s). Tensile samples were printed using two different dispersion orientations, longitudinal (toolpath along the testing direction) and transverse (toolpath perpendicular to the testing direction). Printing was conducted at room temperature, after printing, the material was UV-cured at λ-365 nm for 1 h at an intensity of 2200 μW/cm² with a distance of 20 mm between the samples and the UV source.