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.