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.