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\begin{document}

\title{Bio-electrospraying assessment towards in situ chondrocyte-laden
electrospun scaffold fabrication}



\author[1]{Ângela Semitela}%
\author[1]{Gonçalo Ramalho}%
\author[2]{Cátia Sousa}%
\author[2]{Alexandrina Mendes}%
\author[1]{Paula Marques}%
\author[1]{António Completo}%
\affil[1]{University of Aveiro}%
\affil[2]{University of Coimbra}%


\vspace{-1em}



  
  \date{June 14, 2024}


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\selectlanguage{english}
\begin{abstract}
Electrospinning has been widely used for the development of fibrous
scaffolds for cartilage tissue engineering (TE), however their small
pores significantly limit cell infiltration throughout the scaffolds,
particularly in three-dimensional (3D) designs. In this endeavor, a
direct incorporation of chondrocytes into the fibers mesh during the
electrospinning presents itself as a promising solution by use of
bio-electrospraying. Yet, for this technology to be effectively employed
for cartilage TE, it is necessary to assess if chondrocytes are in any
way adversely affected. So, in this work, several electrospraying
experiments were performed by adjusting various operational parameters
to evaluate their influence on chondrocyte viability and function. A
high percentage of post-electrosprayed chondrocytes remained viable upon
the exposure of an external electric field generated by low needle to
collector distances and low applied voltages. No obvious differences
were found with non-electrosprayed chondrocytes in terms of viability,
morphology and proliferation. The data reported here further suggest
that bio-electrospraying under the optimal operational conditions might
be a promising alternative to the existent cell seeding techniques,
promoting not only cells safe delivery to the scaffold, but also the
development of highly cellularized and uniform tissue constructs for
cartilage repair.%
\end{abstract}%



\sloppy


\textbf{KEYWORDS}

Cartilage tissue engineering, Electrospraying, Chondrocyte, Needle
gauge, Operational parameters

\section*{INTRODUCTION}

{\label{introduction}}

Tissue engineering (TE) strategies have been actively seeking for an
optimal approach for the development of suitable articular cartilage
tissue replacements, given that the current treatment options do not
constitute a feasible long-term solution (Correa \& Lietman, 2017).
Considerable efforts have been made to improve scaffolds design --
choice of material and fabrication technique, topography and
three-dimensional (3D) anisotropic design -- for functional cartilage
tissue formation support, as well as effective cell incorporation and
subsequent interaction of host cells within the construct
(Camarero-Espinosa et al., 2016; Griffith \& Swartz, 2006).
Electrospinning, for instance, has been widely employed for the
fabrication of fibrous scaffolds for cartilage TE, not only due to its
simplicity and versatility, but also the ECM-mimicking nanofibers
produced, known to trigger a suitable chondrocyte response (Gir\selectlanguage{ngerman}ão et
al., 2018; Jin et al., 2018; Jun et al., 2018; McCullen et al., 2012;
Reboredo et al., 2016; Steele et al., 2014). Still, the pores generated
by electrospinning are usually too small to allow effective cell
migration into the inner regions of the scaffold, particularly in 3D
designs, resulting in poor and time dependent cellular infiltration, and
ultimately, in the production of non-functional tissue constructs (Bueno
et al., 2007; Griffon et al., 2011; Rnjak-Kovacina \& Weiss, 2011;
Villalona et al., 2010). In this regard, a logical conclusion would be
to directly incorporate the cells into the fibers mesh during scaffolds
production in order to fabricate functional and homogeneous tissue
constructs, by overcoming the challenges of cell infiltration through
small pores by literally surrounding the cells with the fiber matrix as
it is produced. Indeed, there are reports of successful development of
cell-laden scaffolds by combining fiber electrospinning with cell
electrospraying (Canbolat et al., 2011; H. Chen et al., 2015; Paletta et
al., 2011; Stankus et al., 2006). Cell electrospraying, or
bio-electrospraying, a concept first introduced in 2005 by
Jayasinghe\emph{et al} , enables the deposition of living cells onto
specific targets by exposing the cell suspension to an external high
intensity electric field (Jayasinghe et al., 2006; Jayasinghe \&
Townsend-Nicholson, 2006). The principle underlying electrospraying
involves the application of voltage on a capillary holding the flow of
liquid media, resulting in the ejection of a liquid microjet of charged
droplets onto an oppositely charged collector. Moreover, when an
electric potential difference threshold between the capillary and the
collector is achieved, a stable conical liquid meniscus is formed --
Taylor cone (Hartman et al., 1999; Kavadiya \& Biswas, 2018; Morad et
al., 2016; Rosell-Llompart et al., 2018). Concerning cell
electrospraying, the establishment of this stable cone-jet is crucial
for the control of the precise cell placement, and it requires certain
operational conditions, such as a particular flow rate, surface tension,
conductivity and voltage (Hartman et al., 1999). Still, it is necessary
to understand how the exposure to the electric field, as well as shear
stress of passing through the cell electrospraying apparatus may affect
cell viability and function. So far, neuronal cells (Eddaoudi et al.,
2010; Jayasinghe \& Townsend-Nicholson, 2006; Townsend-Nicholson \&
Jayasinghe, 2006), smooth muscle cells (Jayasinghe et al., 2007;
Odenwälder et al., 2007; Patel et al., 2008), lymphocytes (Kempski et
al., 2008), mononuclear cells (Hall et al., 2008), primary cardiac
myocytes and endothelial cells (Barry et al., 2008; Ng et al., 2011),
kidney cells (Kwok et al., 2008), embryonic stem cells (Abeyewickreme et
al., 2009), mesenchymal stem cells (Mongkoldhumrongkul et al., 2009) to
hematopoietic stem cells (Bartolovic et al., 2010), and even for
multicellular organisms (Clarke \& Jayasinghe, 2008) have been
electrosprayed and survived with no significant influence on a genetic,
genomic and physiological level. Yet, so far, no study has reported the
bio-electrospray of chondrocyte suspensions. So, the aim of the present
study is to understand the impact of the electrospraying process and the
respective parameters on the viability and proliferative behavior of
chondrocytes, so that this technology might be implemented for the
fabrication of chondrocyte-laden scaffolds for cartilage TE.

\section*{MATERIALS AND METHODS}

{\label{materials-and-methods}}

\subsection*{Electrospraying apparatus}

{\label{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).

\subsection*{Chondrocyte culture}

{\label{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\% CO\textsubscript{2} 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.

\subsection*{Chondrocyte
electrospraying}

{\label{chondrocyte-electrospraying}}

5 mL of Phosphate-Buffered Saline (PBS; Sigma Aldrich) supplemented with
2.5 \selectlanguage{greek}µ\selectlanguage{english}g/mL Amphotericin B was passed through the electrospraying
apparatus. C28/I2 chondrocyte were split into three groups, each with
1\selectlanguage{ngerman}×10\textsuperscript{6} chondrocytes suspended in 300 \selectlanguage{greek}µ\selectlanguage{english}L of culture
medium with 0.25 \selectlanguage{greek}µ\selectlanguage{english}g/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 \emph{n} = 5 was considered for each group
and for each electrospraying parameter test.

\subsubsection*{Chondrocyte viability and
morphology}

{\label{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 \selectlanguage{ngerman}ºC for 4 h in the
dark, after which 100 \selectlanguage{greek}µ\selectlanguage{english}l 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).

\subsubsection*{Chondrocyte proliferative
behavior}

{\label{chondrocyte-proliferative-behavior}}

The proliferative ability of the electrosprayed C28/I2 chondrocytes
subjected to different NG and NCD parameters was assessed. Briefly,
2x10\textsuperscript{4} 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.

\subsection*{Statistical analysis}

{\label{statistical-analysis}}

All the quantitative data are expressed as mean \selectlanguage{ngerman}± 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 \emph{p} -values inferior to 0.001,
0.01 and 0.05.

\section*{RESULTS}

{\label{results}}

\subsection*{Establishment of electrospray
stability}

{\label{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
(\emph{p} \textless{} 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 (\emph{p} \textless{} 0.001 at 5cm and\emph{p}
\textless{} 0.01 at 10 cm). Also, wider stability voltage ranges were
obtained with decreasing NG.

\subsection*{Impact of electrospraying parameters on chondrocytes
viability}

{\label{impact-of-electrospraying-parameters-on-chondrocytes-viability}}

\subsubsection*{Electrospraying process and needle
gauge}

{\label{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
\%;\emph{p} \textless{} 0.05) and for 30G (35 ± 22 \%; \emph{p}
\textless{} 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 \%; \emph{p} \textless{} 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).

\subsubsection*{Applied voltage}

{\label{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 \%; \emph{p} \textless{}
0.001) and 30G (from 53 ± 15 to 17 ± 12 \%, \emph{p} \textless{} 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 \%,
\emph{p}\textless{} 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).

\subsubsection*{Needle to collector
distance}

{\label{needle-to-collector-distance}}

A higher NCD (10 cm) substantially lowered the number of viable
chondrocytes when 25G (from 76 ± 18 to 44 ± 3 \%; \emph{p} \textless{}
0.05) and 27G (from 85 ± 14 to 44 ± 11 \%; \emph{p} \textless{} 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,
\emph{p}\textless{} 0.001; and 21 ± 7 \% at 10 cm, \emph{p} \textless{}
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).

\subsubsection*{Flow rate}

{\label{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 \%, \emph{p} \textless{} 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 \%; \emph{p} \textless{}
0.01).

\subsection*{Influence of the electrospraying parameters on chondrocyte
long-term proliferative
behavior}

{\label{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
(\emph{p}\textless{} 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
(\emph{p}\textless{} 0.05). At day 7, statistically significant
differences were found on viable chondrocyte percentage between 25 and
27 NG for both NCD tested (\emph{p} \textless{} 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).

\section*{DISCUSSION}

{\label{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 \emph{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 (\selectlanguage{english}~25-27 oC) and
CO\textsubscript{2} concentration (\selectlanguage{english}~0.04\%) may have contributed to
chondrocyte death (Braghirolli et al., 2013; Paletta et al., 2011).
Actually, Braghirolli \emph{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.

\section*{CONCLUSIONS}

{\label{conclusions}}

Here, an alternative seeding methodology has been proposed and evaluated
to assess its possible use for a direct incorporation of chondrocytes
onto electrospun scaffolds. Bio-electrospraying proved to be
non-detrimental for chondrocytes under certain operational conditions.
These include an intermediate NG (27G), not only to prevent cell
shearing from a smaller NG, but also to avert the solicitation of higher
voltages to establish a stable cone-jet mode from the higher NG; lower
applied voltage (9 kV), since higher voltages can induce electrical and
thermal damages to the cells; smaller NCD (5 cm), to prevent cell
desiccation and increase the number of recovered cells; and an
intermediate FR (2 to 5 mL/h) to prevent not only the shear stress on
the cells of higher FR, but also to reduce the electrospraying duration
and, the consequent, prolonged exposure to the electric field and lower
temperatures and CO\textsubscript{2} concentrations. The long-term
proliferation studies revealed that chondrocyte proliferative ability
was not affected, regardless of the employed operational conditions.
These results suggest that in fact this technology might be a promising
alternative to the existent cell seeding techniques, promoting not only
cells safe delivery to the scaffold, but also the development of highly
cellularized and uniform tissue constructs for cartilage repair.

\section*{ACKNOLEDGEMENTS}

{\label{acknoledgements}}

This work was supported by the Portuguese funding of Program
COMPETE-FEDER, Programa Operacional Competitividade e
Internacionalizacao through the projects POCI-01-0145-FEDER-028424 and
CENTRO-01-0145-FEDER-022083. Also by Fundacao para a Ciencia e
Tecnologia I.P. (FCT, IP) through the projects PTDC/EME-SIS/28424/2017,
UIDB/00481/2020 and UIDP/00481/2020. The authors thank to FCT for the
PhD grant SFRH/BD/133129/2017. The authors would also like to thank the
valuable input of Andre F. Girao, Dr. Susana Pinto and Dr. Nuno Almeida.

\textbf{Conflict of Interests Statement}

The authors hereby certify that there are any conflicts of interests of
any kind to declare.

\textbf{REFERENCES}

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