RESULTS

Establishment of electrospray stability

Electrospray stability, identified here by the presence of the Taylor cone in the spray, as depicted in Fig. 2a, was assessed through a range of applied voltages, and the results are displayed in Fig. 2b. Stable cone-jet modes at 5 cm NCD were observed for 25G between 12 and 13 kV, for 27G between 9 and 11 kV, and for 30G between 8 and 12 kV. At 10 cm, higher voltages were necessary to obtain a stable cone-jet mode (p < 0.001), for all NG; indeed, for 25G this mode was observed between 15.5 and 16.5 kV, for 27G between 12 and 15 kV, and for 30G between 12 and 16 kV. Below the lower voltage of each range, no spray was produced, only droplets fell from the needle. Above the upper voltage value of each range reported, the spray was irregular and unstable, causing discontinuous jetting. A statistically significant difference was found between the voltage range of the 25 G needle with the other NG for both NCD (p < 0.001 at 5cm andp < 0.01 at 10 cm). Also, wider stability voltage ranges were obtained with decreasing NG.

Impact of electrospraying parameters on chondrocytes viability

Electrospraying process and needle gauge

A statistically significant difference on the percentage of viable chondrocytes between CC and E groups was found for 25G (76 ± 18 %;p < 0.05) and for 30G (35 ± 22 %; p < 0.001) (Fig. 3a). Moreover, using a 30G needle, a reduction of the E cells’ number was also observed in the NC in comparison with CC (68 ± 10 %; p < 0.05). Still, no differences were observed on the viability of the CC and NC groups when 25G (98 ± 5 %) and 27G needles (99 ± 2 %) were used (Fig. 3a). Regarding chondrocyte morphology, no visible differences were detected between CC and NC groups’ viability. Additionally, the apparent number of visible chondrocytes on the micrographs was consistent with the viability results (Fig. 3b).

Applied voltage

Increasing the applied voltage at 5 cm – within the stable cone-jet mode – generated a considerable reduction of the viable E chondrocytes’ percentage, when 25G (from 88 ± 12 to 60 ± 11 %; p < 0.001) and 30G (from 53 ± 15 to 17 ± 12 %, p < 0.001) needles were used (Fig. 4a). On the contrary, no statistically significant differences were observed on chondrocyte viability using a 27G NG (from 89 ± 11 % to 81 ± 16 %). These results are corroborated by the apparent number of chondrocytes visible on the micrographs, which was considerably lower using a 30G needle (Fig. 4b). At 10 cm, a similar behavior was observed for the 30G needle (28 ± 2 to 16 ± 4 %, p< 0.05; Fig. S1), while for the 25G (50 ± 8 to 44 ± 5 %) and 27G (48 ± 9 to 39 ± 11 %) needles, no significant differences were found (Fig. S1).

Needle to collector distance

A higher NCD (10 cm) substantially lowered the number of viable chondrocytes when 25G (from 76 ± 18 to 44 ± 3 %; p < 0.05) and 27G (from 85 ± 14 to 44 ± 11 %; p < 0.001) were employed, while for 30G NG group no statistically significant differences were found between the tested NCD (Fig. 5a). Furthermore, post-electrosprayed samples possessed significantly lower percentage of viable chondrocytes when the 30G was used (35 ± 22 % at 5 cm, p< 0.001; and 21 ± 7 % at 10 cm, p < 0.01, Fig. 5a). These results are in agreement with the lower number of chondrocytes visible in the micrographs of the samples electrosprayed at 10 cm and with 30G (Fig.5b).

Flow rate

FR’s impact on E C28/I2 chondrocytes was also assessed for a constant NG (27G) and NCD (5 cm) (Fig. 6). 2 (86 ± 6 %) and 5 (91 ± 8 %) mL/h allowed substantially higher number of viable post-electrosprayed chondrocytes, whereas 1 mL/h resulted in extensive chondrocyte death (4 ± 2 %, p < 0.001; Fig. 6a), which is also consistent with the fewer chondrocytes exhibited in the micrographs (Fig. 6b). Likewise, 7 mL/h also generated a substantial reduction on the percentage of viable chondrocytes (66 ± 10 %; p < 0.01).

Influence of the electrospraying parameters on chondrocyte long-term proliferative behavior

The proliferative behavior of the electrosprayed C28/I2 chondrocytes was then assessed over a culture period of 14 days, where a significant increase on the percentage of viable post-electrosprayed chondrocytes was observed over time for all the NG and NCD combinations (p< 0.001; Fig. 7a). This behavior was also detected on the chondrocyte micrographs, where substantially more cells were found with increasing culture time (Fig. 7b and Fig. S2). After 1 day of culture, significant differences were found between the NCD employed (p< 0.05). At day 7, statistically significant differences were found on viable chondrocyte percentage between 25 and 27 NG for both NCD tested (p < 0.05). Yet, by the end of the culture period no significant differences were observed between the number of the viable E chondrocytes subjected to all the parameters permutation (Fig. 7a). Furthermore, the morphological studies were indistinguishable between the CC and E samples, regardless of the NG and NCD combination (Fig. 7b and Fig. S2).