MATERIALS AND METHODS

Electrospraying apparatus

The experimental set-up is summarized in Fig. 1. All experiments were performed in a NANON 01 electrospinning machine (MECC; Fukuoka, Japan), thoroughly cleaned with 70 % (v/v) ethanol beforehand. The remaining used instruments were already sterile or autoclaved before use. Stainless-steel needles with varying internal diameters (ID) were connected to a high voltage power supply with the ability to supply up to 30 kV. The needles were attached to cell suspension-containing 5 mL plastic syringes. The samples were collected in culture medium containing-wells of 24-well plates having ring-shaped copper grounded electrodes on its surface (Fig. 1a).

Chondrocyte culture

An immortalized human chondrocyte cell line C28/I2 (kindly provided by Prof. Mary Goldring, Hospital for Special Surgery, New York and Harvard University) was used. Cells were maintained at 37 °C in a humidified atmosphere of 5% CO2 in air, in Dulbecco’s Modified Eagle Medium (DMEM)/Nutrient Mixture F-12 Ham 1:1 v/v (DMEM: Gibco, Life Technologies; F-12: Sigma-Aldrich) supplemented with 10% (v/v) non-heat-inactivated Fetal Bovine Serum (FBS; Gibco, Life Technologies), 1% (v/v) Penicillin/Streptomycin (P/S;Grisp). Medium refreshments were performed two times a week. Cells were harvested at pre-confluence using trypsin/EDTA solution (0.05%/0.02%, Sigma-Aldrich) for the electrospraying experiments.

Chondrocyte electrospraying

5 mL of Phosphate-Buffered Saline (PBS; Sigma Aldrich) supplemented with 2.5 µg/mL Amphotericin B was passed through the electrospraying apparatus. C28/I2 chondrocyte were split into three groups, each with 1×106 chondrocytes suspended in 300 µL of culture medium with 0.25 µg/mL Amphotericin B (Sigma-Aldrich): culture controls (CC), which were maintained in the laminar flow hood at room temperature during the electrospraying process; needle control (NC), where the cell suspensions were subjected to the mechanical stress of passing through the electrospraying apparatus; and electrosprayed samples (E), where the cell suspensions were pumped through the electrospraying apparatus and exposed to voltage. Several electrospraying parameters were tested (Fig. 1b): three needle gauges (NG) (25G – 0.26 mm ID, 27G – 0.2 mm ID and 30G – 0.159 mm ID, all with 15 mm length), two needle to collector distances (NCD) (5 and 10 cm), two applied voltages for each NG (applied voltages were selected based on the stability of the spray, i.e. lower and upper voltages of the stable cone-jet mode), and four flow rates (FR) (1, 2, 5 and 7 mL/h). A n = 5 was considered for each group and for each electrospraying parameter test.

Chondrocyte viability and morphology

Collected samples were then incubated for 24 hours, after which chondrocyte viability was assessed using resazurin reduction assay. Briefly, a resazurin solution (0.1 mg/mL; ACROS Organics) in PBS was added to culture medium at a final concentration of 10 % (v/v), and chondrocytes were incubated in this solution at 37 ºC for 4 h in the dark, after which 100 µl per well was transferred to a 96-well plate and absorbance at 570 and 600 nm was measured. The final absorbance values for each sample were calculated as the ratio Abs570/Abs600 nm minus the Abs570/Abs600 nm ratio of a negative control (culture medium). The absorbance values of CC were then taken as 100% and cell viability calculated as a percentage of these control values. Chondrocyte morphology was visualized in an inverted optic microscope (Euromex, CMEX-PRO 10MP; Netherlands).

Chondrocyte proliferative behavior

The proliferative ability of the electrosprayed C28/I2 chondrocytes subjected to different NG and NCD parameters was assessed. Briefly, 2x104 electrosprayed C28/I2 chondrocytes were seeded in 48-well plates and cultured over a 14-day culture period, where medium changes were also performed two times a week. At day 1, 7 and 14, chondrocyte viability and morphology were once more assessed as previously described.

Statistical analysis

All the quantitative data are expressed as mean ± standard deviation. Statistical significance was determined, using OriginLab, by performing as suited One-way analysis of variance (ANOVA), One-way ANOVA with repeated measures, and Two-way ANOVA, all followed by post hoc Tukey’s test. Significance was accepted at p -values inferior to 0.001, 0.01 and 0.05.