Centrifuge 5420

Both 3D spheres and 2D cultures of SCAPs were cultivated in 6-well plates for another four days, and 5 × 10⁵ cells were used in both groups. The supernatants were collected and centrifuged at 1500 rpm (Centrifuge 5420; Eppendorf, Hamburg, Germany). BDNF and nerve growth factor-beta (NGF-β) were measured by the ELISA kits according to the manufacturer’s instructions (ab212166 and ab193760, Abcam).

The compounds quercetin, lutein, and zeaxanthin used in the analytical studies were purchased from Extrasynthese (France) and were of analytical grade. The samples of courgette were treated according to the drying section and were sliced and mortar ground. Then, approximately 100–300 mg of each sample was transferred to a glass vial (10 mL), and 5 mL of extraction solvent was added; the vial was then closed tightly, and the sample was sonicated in a water bath at 40 °C for 15 min (Bransonic^® cpx). Five consecutive extraction solvents were used: (1) an ethyl acetate:hexane mixture (1:1 v/v), (2) ethyl acetate, (3) an ethyl acetate:methanol mixture (1:1, v/v), and (4 and 5) methanol; combined organic extracts were evaporated on a rotary evaporator to dryness, and the samples were dissolved in 2 mL of methanol, aided by sonication, and centrifuged for 1 min at 20,000 rpm (Eppendorf Centrifuge 5420). Then, 100 µL of the supernatant was transferred to an amber vial with 900 µL of methanol, which was closed and stored at −80 ℃ for analysis, and the rest of the supernatant (about 1.5 mL) was stored in an Eppendorf vial (2.0 mL) at –80 ℃ for further analysis. All the samples were prepared in triplicate.

OraGRAFT^® Demineralized Cortical Particulate (1.2 cm³; IDs: 1817033-3066; 1817033-3079; 1817003-3080; 1817033-3086; and 1817033-3094) and OraGRAFT^® Prime consisting of moldable fibers of DBM (1.0 cm³; IDs: 1910583-3057; 1910583-3060; 1910583-3064; 1910583-3067; and 1910583-3069; LifeNet Health Europe GmbH, Vienna, Austria) were submerged in 4.8 mL and 8.0 mL, respectively, of serum-free Dulbecco’s Modified Eagle Medium (DMEM) for 72 h (Sigma Aldrich, St. Louis, MO, USA) and left under continuous shaking overnight at room temperature. Supernatants of each respective OraGRAFT^® were obtained upon centrifugation at 21,000 g for 10 min (Centrifuge 5420, Eppendorf, Hamburg, Germany). The supernatant of OraGRAFT^® Demineralized Cortical Particulate (SC) and OraGRAFT^® Prime consisting of moldable fibers (SF) were collected. The pelleted remaining allografts were resuspended in 1N HCl and left shaking for 72 h at room temperature. Acid lysates from each respective OraGRAFT^® (LC and LF) were collected through one centrifugation step. Their pH was then neutralized. All samples were stored in aliquots at –20 °C.