PVA coating
Polished steel plates or the rougher sides of aluminium foils, were cleaned with isopropanol, left to dry and then coated with a thin coat (ca. 0.03 ml/cm2) of a 100 mg/ml solution of poly (vinyl alcohol) (PVA Mw 30–70 kDa, Merck KGaA, Darmstadt, Germany) in water. The film was evenly distributed and left to dry at RT. When greater adhesion to the PVA film was desired, up to 11% (v/v) glycerin (Carl Roth, Karlsruhe, Germany) was added to the PVA solution before casting.
Electrospinning of PCL membranes
The PCL fiber membranes were produced by using a voltage difference of 9.5 kV, which was applied onto a 20 G needle (Microlance BD, New Jersey, USA). 1000 µl of the 24% w/v polycaprolactone (45 kDa, Sigma Aldrich, MO, USA) in 99% pure 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, abcr GmbH, Germany) solution were spun at a rate of 3 ml/h and a 17 cm distance between needle and collector. The grounded rotating drum collector (Ø 94 mm) was rotated at the speed of 1600 rpm for aligned and 100 rpm for random membranes. The membranes were spun onto an aluminium foil thinly coated with polyvinyl alcohol (PVA 30–70 kDa, Merck KGaA, Darmstadt, Germany) that was attached to the collector. The collected fiber membranes were submersed into a mixture of 70% ethanol (v/v) and soaked for ca. 1 min. The membranes were then washed thrice in H2O, dipped in 100% ethanol and dried.
Electrospun PCL/gelatin membranes: 8% w/v PCL (Mw 80 kDa Merck KGaA, Darmstadt, Germany) and gelatin 2% w/v (type A from porcine skin, Merck KGaA, Darmstadt, Germany), were dissolved in a solvent mixture composed of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)/formic acid (FA) (both from Sigma-Aldrich, HFIP:FA; 9:1 v/v). The solution was electrospun using a blunt 27G needle and the solution extruded at the speed of 0.3 ml/h, with a voltage of 15 kV applied to the needle. The grounded rotating drum collector (Ø 94 mm) was rotated at the speed of 100 rpm. The membranes were spun onto an aluminium foil thinly coated with polyvinyl alcohol (PVA 30–70 kDa, Merck KGaA, Darmstadt, Germany) that was attached to the collector.
Electrospinning of PLA membranes
A volume of 1.5 mL of a 2% Poly-L-Lactic Acid Mw 650 kDa (PLLA) (PL65 Purasorb, Netherlands) in HFIP solution was electrospun with a 27G nozzle, a voltage of 12 kV and a 15 cm distance from a rotating Ø 7 mm collector spinning at 100 rpm, to which, as previously mentioned, a PVA coated foil was attached. The collected fiber membranes were submersed into 70% ethanol (v/v) and soaked for ca. 1 min. The membranes were then washed thrice in 70% ethanol and dried.
Production of PCL MEW scaffolds
PCL box scaffolds were produced using melt electrowriting (MEW). The printing was conducted at a room temperature of 20 ºC with 40% humidity, a PCL (Purac PC12, Corbion, Amsterdam, the Netherlands) melt temperature of 95 ºC, a pressure of 1 bar and a print bed movement rate of 1000 mm/min onto a grounded steel build plate covered by a thin water-soluble poly (vinyl alcohol) (PVA, 30–70 kDa, Merck KGaA, Darmstadt, Germany) coating a 2.5 kV voltage difference applied over the 30 G needle (Nordson EFD, Ebensfeld, Germany), at a printing distance of 1.4 mm. Before application the PVA was dissolved using 70% ethanol, the scaffolds removed, washed in 70% ethanol and dried.
The thicker electrowritten PCL scaffolds were produced using similar parameters, except that a 25G needle (Nordson EFD, Ebensfeld, Germany), a pressure of 2 bar and a speed of 500 mm/s were used.
Laser cutting
The scaffolds were laser cut (Rayjet, Trotec, Plymouth USA) to the appropriate well size. The speed and intensity of the laser were varied to achieve a complete separation of the remaining scaffold.
FDM printing onto scaffolds
The scaffolds were reinforced with polylactic acid (PLA, Form Futura, Amsterdam, Netherlands), polyvinyl alcohol (PVA) ( Form Futura, Amsterdam, Netherlands), or polycaprolactone (PCL) (Facilan Ortho, 3D4makers, Haarlem, The Netherlands) using a 0.4 mm nozzle, a layer height of 0.28 mm, a print speed of 5–20 mm/s, a 10 mm retraction distance and 80 mm/s retraction speed, without a heated print bed and the nozzle temperatures of 180 ⁰C, 190 ⁰C and 130 ⁰C for the different polymers respectively. Thermoplastic polyurethane (TPU) (FilaFlex 60A, Recreus, Elda, Spain) was printed using the same conditions, except that the nozzle temperature was 210 ºC and the retraction was disabled.
Tensile testing
The testing of the different reinforcement rings and scaffold was performed with a universal testing machine (Z010, Zwick Roell, Ulm, Germany) with a 100 N load cell. The samples were stretched with a velocity of 10 mm/min mounted between two clamps. The upper force limit was set to 95 N. The force dependent on the stretch was measured and evaluated.
Hanging test wet/dry
The hanging test was performed using a 3D printed construct into the beak of which the edges of the mesh were fastened. A background showing the different angles from 0° to 90° in steps of 10° was placed. The scaffolds were tested dry and wet, whereby the wet scaffolds were wetted by letting these absorb the liquid they could take up. A photograph was taken at a perpendicular height to the mesh. Triplicates were performed for each experiment and the angle of the hanging scaffold determined.
Projector calibration
To calibrate the projector (YABER V5, YABER, Austin, USA) to the build plate of the FDM printer (modified Ender 3 V2, Creality, Shenzen China), a calibration print was conducted, upon which the projection was calibrated. Thereafter the calibrated projection was used to calibrate the laser cut substrates to the correct position, where the printed strut would then be deposited.
FTIR measurements
The samples were measured dry using a Nicolet iS10 with smart iTR diamond ATR (attenuated total reflectance, Thermo Fisher Scientific, Waltham, USA).
Scanning electron microscopy (SEM)
The samples were analyzed using a SEM device (Crossbeam CB 340 SEM, Carl Zeiss).
Ethanol drying procedure
The already fixed cell samples were transferred to PBS and thoroughly washed. Afterwards these were incubated in 70%, 90% and 100% ethanol, twice for 10 min for each respective step. Thereafter the samples were incubated in hexamethyldisilazan (HMDS) (Merck KGaA, Darmstadt, Germany) twice for 10 min and subsequently left to dry.
U87 culture and seeding
(U-87 MG, ATCC HTB-14, LGC Standards GmbH, Germany) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (41966-029, Gibco, MA, USA) supplemented with 10% FCS (10270-106 Life Technologies, MA, USA) and 10,000 U/mL pen/strep (15140-122 Life Technologies, MA, USA). Cells were split twice per week.
Scaffold were sterilized with 70% ethanol and were placed 15–30 min under UV light. Afterwards scaffolds were washed three times with ddH2O and once with PBS. 3 cm dishes with four 93 mm2 inner rings (627170, Greiner, Greiner Bio-One, Kremsmünster, Austria) were used to place the scaffolds and add 50 µl of full media. Finally, scaffolds were incubated for 30 min at 37°C with 5% CO2, thereafter the cells were added at concentration of 10000 cells/well and further incubated.
Ethical statement: Experiments were approved by the local veterinary authority (Veterinäramt der Stadt Würzburg, Germany) and the Ethics Committee of Animal Experiments, i.e., Regierung von Unterfranken, Würzburg, Germany (license no.: FBVVL 568/200-324/13).
Astrocytes isolation and culture: CD-1 pups (P0-P1) were used to isolate primary astrocytes. After extracting the brains, cortices were dissected and collected in ice-cold phosphate-buffered saline (PBS). Following a brief homogenization and filtration through a 70 µm cell strainer (542070, Greiner Bio-One, Kremsmünster, Austria), cells were centrifuged (10 min, 1400 rpm), resuspended and seeded in 6 cm dishes with 5 mL of DMEM supplemented with 10% fetal calf serum, 2 × 10−3 m GlutaMAX, 1 × 10−3 m sodium pyruvate, and 50 U/mL penicillin/streptomycin (15140-122 Life Technologies, MA, USA). Astrocytes grew under standard conditions at 37 °C with 5% CO2. Cells were washed with PBS and medium was exchanged 3–4 days after seeding. After seven days, cells were detached and counted. 150,000 astrocytes in suspension were pipetted on top of each scaffold. An O-metal ring was used to fix the scaffolds. Afterward 3 mL of supplemented DMEM medium were added.
Adipose-derived stem cell (ASCs) culture and seeding: Cells were centrifuged (5 min, 1200 rpm), resuspended and 15.000 cells were seeded on the electrospun membranes and on control glass slides in well plates with 5 mL of DMEM F-12 (1:1) supplemented with 200 mM GlutaMAX, 100 U/mL penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA), 10% fetal calf serum, basic fibroblasts growth factor (FGF) and 50 µg/mL ascorbic acid (Sigma-Aldrich, Germany). ASC grew under standard conditions at 37 °C with 5% CO2 atmosphere for three days. Medium was exchanged one day after seeding and every day after.
Immunocytochemistry: For adipocytes the random electrospun PCL membranes were removed from the reactor by wetting the sides of the membrane outside of the main chamber with PBS Astrocyte/Adipocyte were washed once with PBS (pH 7.4) and fixed for 20 min with a 2% paraformaldehyde (PFA) solution or 3.7% gluteraldehyde. Following fixation, astrocytes were permeabilized and blocked with 5% normal goat serum (NGS) with 0.2 % Triton-X 100 in PBS for 30 min. Adipocytes were treated with 0.1% TritonX-100 in PBS for 5 minutes and blocked with 5% BSA in PBS for 30 min at room temperature. Astrocytes were incubated with ActinGreen™ 488 ReadyProbes™ Reagent (R37110 Invitrogen, Carlsbad, CA) in blocking solution for 1 hour. Finally, scaffolds were mounted with ProLong Glass Antifade Mountant containing Hoechst 33 342 (Thermo Fisher Scientific, Waltham, MA) on glass slides. Adipocytes were washed with PBS and incubated with primary antibody anti-vincullin (1:50; V4505 Sigma Aldrich, Germany) for 1 hour followed by secondary antibody incubation goat anti-rabbit-Cy3 (1:500, 111-165-003 Dianova, Hamburg, Germany). In the same step ActinGreen™ 488 readyProbes™ (1:50 R37110 Invitrogen, Carlsbad, CA) reagent staining was included. Cells were stained with DAPI (1:5000, D3571 Invitrogen, Canada) for 10 min and mounted on glass slides with Mowiol 4-88 (81381-50G Sigma Aldrich, Germany). The embedding was done in a FDM 3D printed chamber, slightly higher than the reinforced scaffold, and glued to a glass slide using nail lacquer, whilst the top was also sealed with glass and lacquer. This was done to easily embed the whole scaffolds and remove all bubbles.
Live Dead staining of U87 cells and primary astrocytes: The staining was performed at day 1 and day 7 post-seeding at 21 °C for 20 min with Calcein-AM (2×10−6 M, green/living cells; Thermo Fisher Scientific, Waltham, MA) and Ethidium Homodimer (2×10−6 M, red/dead cells; Sigma-Aldrich, St. Louis, MO) diluted in PBS and incubated for 20 min.
Confocal Microscopy and Image Acquisition: Samples were imaged using an inverted Olympus IX81 microscope equipped with an Olympus FV1000 confocal laser scanning system, a FVD10 SPD spectral detector, and diode lasers of 405 nm (DAPI), 473 nm (Alexa488) and 559 nm (Cy3) (Olympus, Tokyo, Japan). All images shown were acquired using an Olympus UPLSAPO 10× (air, numerical aperture 0.4) or Olympus UPLFLN 40x (oil, numerical aperture: 1.3) and were processed using ImageJ/Fiji 1 and Imaris 7.7.2 (Oxford Instrumentals, Abingdon, UK). For cell viability z-stacks of about 2–3.52 µm step size throughout each sample were acquired. Imaris was used for 3D reconstruction, video generation and reconstruction of the z-stack images to quantitatively analyze live and dead cell numbers The Spots function was used to determine the live/dead ratio. 5 image stacks per experimental condition were analyzed (N = 3). Dynamic range adjustments and projections were done with ImageJ/Fiji Software.
2.4. Statistical analysis
GraphPad Prism 8.3.0 (Graphpad Software, San Diego, CA, USA) was used to calculate mean values, standard deviation (SD), standard error of the mean (SEM), and values for statistical significance. Statistical significance was estimated *p < 0.05 using two-way ANOVA.