All statistical analyses were performed using GraphPad Prism 8 software. Samples were analyzed through ANOVA with Tukey post hoc and p < 0.05 was considered statically significant.
Experiments were conducted with NIH-3T3 cell lineage, from the Cell and Tissue Technology Laboratory, Brain Institute (BraIns) at PUCRS. Cells were cultivated in the following maintenance medium: Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal serum inactivated bovine (SFB), 100 U/mL penicillin/streptomycin and 100 µg/mL gentamicin, all purchased from the manufacturer GibcoTM (Gibco™, Life Technologies, California, USA).
Polymeric biomaterials preparation
Membranes of different film biomaterials were prepared in order to establish the most suitable for use in a 3D printed matrix. The biomaterials used were Policaprolactona (PCL), Poly Lactic-co-Glycolic Acid (PLGA), Polylactic acid (PLA) and Polypyrrole (PPY) in membrane. For the experiments, the membranes were used in the following configuration: PCL, blend of 70% of PCL and 30% of PLGA, PLA and PPY dispersed over the blend in the proportion of 10% m/m (15). For the experiments, film/membrane biomaterials were used. Briefly, the polymer matrices or their blends were prepared using the solvent evaporation methodology. The polymers were dispersed in chloroform and later placed in petri dishes. The solvent was evaporated for 24 h at room temperature (15). Blends containing conductive polymer were prepared from the addition of previously synthesized PPy nanofibers (10% w/w).
To evaluate film surfaces hydrophobicity, contact angle values were determined using a Phoenix 301 goniometer, SEO Company. The experiments were carried out with deionized water and determined soon after the contact of the drop with the material to be analyzed. Five drops of deionized water were applied and considered the medium value.
The molecular structure of the materials produced was determined by Fourier transform infrared spectroscopy (FT-IR) in order to identify the functional groups present in the samples. The infrared (IR) measurement was performed through absorption spectrum, between 400–4000 cm − 1, in Perkin-Elmer spectrometer. It was used materials without exposure as control.
To verify the cytotoxic potential of the materials, MTT protocol was performed through the extraction method following the norms of ISO 10993-5. Briefly, for the production of conditioned medium, the biomaterials were exposed, in the proportion of 3cm2/mL, to the medium used for the cultivation of cells in their maintenance phase for a period of 24 hours, 72 hours and 7 days at culture conditions stated above. Cells were plated at a density of 5 x 10 4 cells per well in a 96-well plate and cultured for 24 hours in maintenance medium. Afterwards, cells were exposed to this conditioned medium, for 24 h and then incubated with the MTT solution at a concentration of 5 mg/mL for 2 hours. Absorbance was measured at 570 nm in SpectraMax equipment, molecular devices, San Jose, California.
Direct cell adhesion
To evaluate the adhesion potential, cells were cultured directly on 3cm2 of the biomaterials, at a density of 5 x 104 cells for 24 hours. Afterwards, the biomaterials were fixed with 4% paraformaldehyde for 15 minutes, washed and incubated with nuclear staining, 4′,6'-diamino-2-phenyl-indole (DAPI), (10 µg/mL) for 15 minutes. Ten random fields were captured using a confocal microscope LSM 5 Exciter, Zeiss and the stained nuclei present on the matrices were quantified using Image Pro Plus® software version 6.0 (Media Cybernetics, Rockville, MD, USA)
Indirect cell adhesion
To assess the ability of the conditioned medium to influence cell adhesion, the biomaterials were exposed, at a rate of 3cm2/mL, to the same medium used for cell culture or to the conditioned medium for 24 hours, 72 hours and 7 days. Cell culture was performed in 2 moments, culture phase and adhesion phase, and then they were stained and the absorbance verified. Figure 1 demonstrates an schematic design of this experiment. In the culture phase, cells were cultured for 24 hours with maintenance or conditioned medium, at a density of 5 x 104 cells per well in 96-well plates, trypsinized and replated in the same concentrations to start adhesion phase. In the adhesion phase, the cells were exposed to the maintenance medium or to the conditioned medium for 4 hours. The cells were then washed with phosphate buffered saline, fixed with 4% paraformaldehyde for 15 minutes, washed with phosphate buffered saline, and incubated with cresyl violet for 15 min. After that, the absorbance was measured at 570 nm in the SpectraMax-M2 equipment, molecular devices, San Jose, California.
Magnetic Resonance (MR)
Structural magnetic resonance images were acquired on a General Electric (GE) 3 T Scanner with a 3D sagittal gradient acquisition by sequence echo (MP-RAGE). Repeat time of approximately 2300 ms, echo time of approximately 3 ms and inversion time of approximately 900 ms, and voxel dimensions were approximately 1.20 × 1.015 × 1.015 mm.
Scanning electron microscopy
The morphology and surface of the samples were analyzed using the scanning electron microscope (SEM), PHILIPS model XL30 with a resolution of 3.5 nm and a range of magnifications of 500 times for the films of polymeric biomaterials and up to 20.000 times for the hydrogels, with accelerating voltage of 20 kV. Samples were metallized with gold in the Sputter Coater equipment (Balzers SCD050). In the case of hydrogels with cells, samples were fixated with 4% paraformaldehyde for 24 hours, dehydrated with acetone prior to metallization protocol.
Polymer 3D printing
First for the hippocampal digital project model for the hippocampus, it was used Computer Aided Design (CAD) using the software CREO Parametric 3.0 in format *.stl for print. Data and measurements were extracted from the magnetic resonance imaging. For the 3D printing of the PLA mold, Fusion deposit modeling (FDM) technologies were used in Sethi S3 3D printer with simplify3D software. Print settings used as follows:
• Nozzle diameter: 0.4 mm
• Layer height: 0.18 mm
• Extrusion multiplier: 0.95 mm (extrudes 0.95 mm of filament for every 1 mm the extruder travels)
Table = 60°C
Nozzle = 220°C
• Maximum speed (advance): 12 mm/s
Filling = 8.4 mm/s
Perimeter = 10.2 mm/s
• Cooling: 0% (0 PWM)
Other layers settings:
• Nozzle diameter: 0.4 mm
• Layer height: 0.12 mm
• Extrusion multiplier: 0.94 mm
Table = 60°C
Nozzle = 220°C
• Maximum speed (advance): 30 mm/s
Filling = 21 mm/s
Perimeter = 25.5 mm/s
• Cooling: 60% (153 PWM) – from the third layer
3 layers closed at the base (equivalent to 0.42 mm thickness)
5 layers closed on top (equivalent to 1 mm thickness)
4 layers (turns) closed on the perimeter (equivalent to 1.6 mm thickness
For the hippocampus structural model it was used StandardInk™ and PlayInk™ from TissueLabs™ Sagl, Manno, Switzerland. For Methacrylate decellularized pig brain hydrogel it was used MatriXpectm GelMA also from TissueLabs™ Sagl, Manno, Switzerland. All bioinks were used following the manufacturer's instructions. The decellularized hydrogel composition by the manufacturer is as follows: 10% gelatin methacrylate; type I collagen < 1.5%; <0.3% elastin; 0.25% lithium trimethylbenzophosphinate; heparan sulfate < 0.2% chondroitin sulfate < 0.2% fibronectin < 0.1%.
BioInk 3D Model
3D printer was used through Repetier software, Hot-World GmbH & Co. In the software, the 5x5x1 mm cubic object was sliced with the Slic3r software (Slic3r, online open source company). The 22G extruder was placed 0.1 mm from the print platform. The object was printed at 0.07 mm3/s, in three layers. For crosslinking, the object was left at 405 nm UV for 240 seconds, as recommended by the manufacturer. Parameters are below:
• Width of extrusion of external perimeters = 0.45 mm (0.06 mm3/s)
• Width of perimeter extrusion = 0.50 mm (0.07 mm3/s)
• Width of fill extrusion = 0.50 mm (0.07 mm3/s)
• Width of solid fill extrusion = 0.50 mm (0.07 mm3/s)
• Top fill extrusion width = 0.50 mm (0.07 mm3/s)