DISCUSSION

In an attempt to fabricate homogeneous and functional TE constructs, several reports have explored bio-electrospraying as an alternative for conventional cell seeding techniques in electrospun scaffolds (Sampson et al., 2013; Stankus et al., 2006; Weidenbacher et al., 2017). Yet, for chondrocyte electrospraying to be effectively employed for cartilage TE, it is of the utmost importance to assess if chondrocytes are in any way adversely affected. So, the present work seeks to understand the influence of the electrospraying technology on chondrocyte viability and function, as well as the establishment of optimal operational parameters for maximum chondrocyte viability.
First, and since this technology is to be used for the precise and uniform cell placement in 3D architectures for TE constructs (Jayasinghe & Townsend-Nicholson, 2006; Stankus et al., 2006), jet stability should be achieved. Unlike previous reports (Hall et al., 2008; Jayasinghe et al., 2006; Odenwälder et al., 2007), it was possible to electrospray chondrocyte suspensions in a stable cone-jet mode, regardless of the NG and NCD. While it has been suggested that the cell suspension’s high conductivity and low viscosity can be responsible for spray instability (Odenwälder et al., 2007), it is also believed that the effect of the nozzle geometry – in this case NG, electrode configuration and FR have an important role in the achievement of a stable cone-jet mode (Morad et al., 2016). In fact, the combination of a smaller NG, a higher NCD and a higher FR in this instance might have been a defining factor.
Upon the establishment of a stable spray, chondrocyte viability was evaluated for the variation of each electrospraying operational parameter. From the three NG tested, only 30G had a detrimental effect on chondrocytes – NC. It is possible that chondrocyte shearing whilst passing through the needle, particularly using 2 mL/h, was the reason for this effect, which is consistent with previous reports (Ng et al., 2011; Ward et al., 2010). This chondrocyte mortality was exacerbated upon exposure to the electric field. A similar reduction on post-electrosprayed chondrocyte viability was observed for the 25G NG, while with 27G NG no significant harmful influence was observed. It is possible that the higher voltages required for spray stability on 25G needle had a somewhat adverse impact on the chondrocyte metabolism. As a matter of fact, increasing the system applied voltage systematically reduced chondrocyte viability. It has been suggested that high voltages, that generate strong electric fields, can induce pore formation and cell membrane damage, followed by an increased membrane permeabilization and, consequent cellular osmotic imbalance, ultimately resulting in cell death (Braghirolli et al., 2013; W. Chen et al., 1998; Sahoo et al., 2010). Moreover, beside electrical damages, strong electric fields can also incite thermal damage on the cells (W. Chen et al., 1998; Sahoo et al., 2010). Interestingly, this damage was not detected on the chondrocytes electrosprayed through a 27G needle, except when NCD was increased. In fact, a significant viability reduction was detected when chondrocytes were electrosprayed at 10 cm. Several reports have noticed a similar behaviour. Paletta et al perceived an increased evaporation rate of the cell-laden droplets at higher NCD, ultimately resulting in an increased salt concentration, and therefore, reduced cell survival (Paletta et al., 2011). On the other hand, several authors have attributed greater cell loss to higher NCDs (Braghirolli et al., 2013; van Aalst et al., 2008; Ward et al., 2010). Additionally, the higher voltages required at higher NCD to maintain the electric field strength might, as previously mentioned, generated a cascade of events that contributed to cell death.
Regarding FR, it was possible to narrow the optimal values for maximum chondrocyte viability from 2 to 5 mL/h, particularly using a 27G NG and 5 cm NCD. Above 5 mL/h, shear stresses played a significant role on chondrocyte mortality (Ng et al., 2011; Ward et al., 2010). Below 2 mL/h, it is believed that chondrocyte death was mainly due to the electrospraying duration. Indeed, electrospraying time using 1 mL/h was 18 minutes, while pumping at 2 and 5 mL/h only 9 and 4 minutes were necessary, respectively. Besides the longer high voltages’ submission time, the prolonged exposure to lower temperatures (∼25-27 ºC) and CO2 concentration (∼0.04%) may have contributed to chondrocyte death (Braghirolli et al., 2013; Paletta et al., 2011). Actually, Braghirolli et al performed an evaluation on electrosprayed cells with different electrospraying times and found that, while no differences were detected on cell viability, there were breaks in the DNA on the samples subjected to longer electrospraying periods (30 and 60 min), indicating that prolonged electrospraying periods of time might provoke cellular genotoxicity (Braghirolli et al., 2013). Several reports have suggested the inclusion of a polymeric hydrogel onto the cell suspension in order to increase its viscosity, and reduce the impact of high voltages, dehydration and environmental conditions (H. Chen et al., 2015; Jayasinghe et al., 2011; Stankus et al., 2006; van Aalst et al., 2008).
Regardless of the electrospraying parameter permutation, electrosprayed chondrocyte were still able to attach to the tissue culture polystyrene and present their typical rounded to polygonal morphology (Goldring et al., 1994). Moreover, the percentage of viable chondrocytes submitted to certain electrospraying (27G NG and 5 cm NCD) parameters remained high – above 70 %. It should also be emphasized that while other studies have reported higher post-electrosprayed cell viabilities (above 80 – 90 %) (Andreu et al., 2012; Braghirolli et al., 2013; Ng et al., 2011; Sahoo et al., 2010; Sampson et al., 2013), it is important to mention that most of these employed substantially bigger NG and smaller NCD, which according to the herein reported data should render high viabilities. Additionally, different electrospraying conditions, viability assay sensitivity and cell susceptibility to damage may also be responsible for the observed difference (Paletta et al., 2011; Sahoo et al., 2010).
Interestingly, despite the fact that short-time viability assays disclosed the detrimental effect of several electrospraying parameters, the long-term proliferation studies revealed that no obvious differences between each parameter permutation and the respective CC were found in terms of gross morphology and rate of growth to confluence, generating two hypothesis: chondrocyte reduced viability was predominantly caused by chondrocyte loss within the electrospraying chamber; or chondrocytes were able to recover over the 14-day period. In fact, previous reports have found evidence of cellular DNA repair were found after only 5 hours. These results further suggest bio-electrospraying under the optimal operational conditions allows not only the successful delivery of healthy chondrocytes, but also, when used in combination with polymer electrospinning, the development of highly cellularized nanofibrous scaffolds for cartilage TE. In this instance, an alternated chondrocyte electrospraying and polymer electrospinning approach – combining smaller NCD and higher NCD, respectively – may be the best solution for maximum chondrocyte survival coupled with maximum polymer solvent evaporation. This “cell layering” approach has been already reported with successful cell incorporation, although bio-electrospraying was not always employed in this instance (Canbolat et al., 2011; Stankus et al., 2006; Xu et al., 2012).
Despite the promising results here reported, further characterization should be performed particularly regarding gene expression patterns to validate the biological efficacy of bio-electrospraying for cartilage TE applications. Although the use of an immortalized cell line C28/I2 in this instance may have been advantageous in assessing the optimal electrospraying operational parameters in terms of speed and reproducibility (Goldring et al., 1994; Greco et al., 2011), it may also present distinct degrees of sensitivity to the process, suggesting that bio-electrospraying studies should also be performed using primary chondrocytes to validate the use of this technology.