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Bioink formulations to ameliorate bioprinting-induced loss of cellular

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Bioink formulations to ameliorate bioprinting-induced loss of cellular viability
Sudipto Datta, Ankita Das, Amit Roy Chowdhury, and Pallab Datta

Citation: Biointerphases 14, 051006 (2019); doi: 10.1116/1.5111392

View online: https://doi.org/10.1116/1.5111392
View Table of Contents: https://avs.scitation.org/toc/bip/14/5
Published by the American Vacuum Society
Bioink formulations to ameliorate bioprinting-induced loss of
cellular viability
Sudipto Datta,1 Ankita Das,1 Amit Roy Chowdhury,1,2 and Pallab Datta1,a)
1
Centre for Healthcare Science and Technology, Indian Institute of Engineering Science and Technology,
Shibpur, Howrah 711103, WB, India
2
Department of Aerospace Engineering and Applied Mechanics, Indian Institute of Engineering Science
and Technology, Shibpur, Howrah 711103, WB, India

(Received 25 May 2019; accepted 26 September 2019; published 14 October 2019)

Extrusion bioprinting, the most affordable and convenient bioprinting modality, is also associated
with high process-induced cell deaths. Mechanical stresses on the cells during pneumatic or piston
extrusion generate excessive reactive oxygen species and activate apoptosis, inflammatory path-
ways in the cells. In this study, a bioink formulation is augmented with an antioxidant, N-acetyl
cysteine (NAC) as a possible solution to abrogate the effect of bioprinting-associated cell survival
losses. The NAC addition to bioinks did not affect the bioprinting process, shape fidelity, or the
mechanical properties of the constructs to any large extent. However, the bioprinting process con-
ducted at 0.30 MPa pressure and 410 μm nozzle inner diameter with bioinks of 3% w/v alginate,
105 cells/ml resulted in survival losses of up to 25% for MC3T3 cells. In contrast, NAC bioinks
showed a significant ( p < 0.01) improvement in day 1 cell survival (91%), while the enhancement
in day 3 cell viability was still greater. It was further observed that the reactive oxygen species
(ROS) load of bioprinted constructs was approximately 1.4 times higher compared to control,
whereas NAC containing constructs reduced the ROS load at levels comparable to control
samples. The effect on apoptosis and inflammation markers showed that NAC had a greater role
in modulating apoptosis. It is concluded that the presented approach to preserve cell viability and
functionality would be advantageous over other contemporary methods (like alterations in extru-
sion pressure, nozzle diameter, polymer concentration, etc.) as viability can be preserved without
compromising the fabrication time or the resolution/mechanical properties of the constructs with
this bioink formulation approach. Published by the AVS. https://doi.org/10.1116/1.5111392

I. INTRODUCTION bioprinting (EB) processes, whereas there exists a critical

In the past few years, biofabrication techniques have
rapidly emerged as potential tools to generate tissue con-
structs conforming to the heterogeneity and architectural
complexity of native biological tissues. These techniques
raise expectations to reduce the clinical demand of organ
transplantations and address organ failure through a regen-
erative approach.1 Moreover, the tissue constructs can be
employed as biomimetic organ models for pathological and
pharmacological studies.2 Biofabrication techniques are
essentially distinguished from conventional manufacturing/
fabrication methods on the basis of their ability to process
cells in living conditions. However, the ability to process
cells in living conditions at best describes a relative expres-
sion as all biofabrication techniques are associated with
some extent of process-induced cell deaths, compromising
their eventual applications. For example, extrusion bioprint-
ing, one of the most facile, cost-effective, and widely used
biofabrication techniques known to produce mechanically
robust constructs, is reported to typically cause up to 60%
cell damage immediately due to the bioprinting process.3,4
Interestingly, most of the studies reported in the literature focus
on obtaining different tissue constructs with extrusion
a)
Electronic addresses: contactpallab@gmail.com and pd@chest.iiests.ac.in
need to improve cell viabilities associated with the process.
Typical EB processes require formulation of cells in a
polymeric hydrogel solution followed by deposition of the
bioink under pneumatic or piston pressure from a syringe res-
ervoir through a fine needle. The hydrogel polymer under-
goes cross-linking to a gel state after extrusion to yield the
construct. Though the chemical and physical nature of cross-
linking reactions plays a role in determining viability,5,6
pressure-induced mechanical stresses also act on the cells to
cause cell damage.7 In addition, it is often required to bioprint
with a high cell and/or polymer concentration to obtain
mechanically robust constructs—a factor that attenuates the
fluid shear stresses on the cells. Mechanical stresses cause
cell damage by several mechanisms like stretching of cell
membranes8–11 as well as generation of excessive reactive
oxygen species,12 which eventually lead to damaging the
nucleic acids.13 In the case of cell damage mediated by
damage to cell membrane, membrane integrity can be pre-
served only up to a certain point of deformation, beyond
which rupture happens.14 On the other hand, ROS can have
temporal effects on the activation of several signaling pathways
leading to cell damage over a period of time.15 Interestingly,
shear-induced biomechanical damages are also associated with
several pathological processes indicative of inherent shear
sensitivity of mammalian cells.16

051006-1 Biointerphases 14(5), Sep/Oct 2019 1934-8630/2019/14(5)/051006/9/$30.00 Published by the AVS. 051006-1
051006-2 Datta et al.: Bioink formulations to ameliorate bioprinting-induced loss of cellular viability
Several studies have illustrated the detrimental effects of
shear stress during bioprinting processes. For example,
using gelatin-alginate bioinks, Ouyang et al. demonstrated
an exponential relationship between shear stress and cell
viability. They found that to maintain 90% viability, applied
stresses should be lower than 100 Pa, constraining rapid fab-
rication of the constructs.17 In a seminal study on micro-
valve bioprinting, Blaeser et al. optimized the bioprinting
process with respect to shear stress and cell viability.7 In order
to better understand the cell death, several computational
models based on fluid flows and fluid dynamics have also
been proposed considering different parameters that are the
cause of shear stresses at the nozzle. In such studies, strain
energy density and immersed boundary method methods are
employed to model the cell movement-deformation and cell
damage during the fluid-cell interactions.10 Investigations by
Tabriz et al. have shown that cell proliferation (at 88% over a
11 day period) is affected to a greater extent compared to
short-term cell viability (93%), indicating the temporal aspects
of process-induced damage to cells.18 This observation also
suggests that bioprinting process optimization studies being
performed with cell viability may be of limited impact on the
long-term functionality of the constructs. However, most of
these studies have aimed to reduce shear-induced damages
only by process optimization, i.e., change of extrusion pres-
sure. Moreover, the computational models tend to account
for shear damages occurring mostly at the nozzle point while
neglecting the shears experienced by the cells during move-
ment through the whole syringe. In order to overcome these
limitations, we thus hypothesized that a certain amount of
cell damage can be minimized by bioink optimization, thus
allowing bioprinting to be carried out with a larger number
of process parameters without affecting construct functional-
ity. Though concentration optimization of cell and polymer in
bioink suspensions results in improving cell viability,18 we
propose that using an antioxidant as a bioink formulation
component along with polymer and cells can be an effective
strategy to minimize stress-induced cell damages and improve
long-term functionality of constructs. Among antioxidants,
N-acetyl cysteine (NAC) has a documented role in preventing
ROS-induced cell damages19–21 and was, therefore, selected
for this investigation. NAC-incorporated bioinks were first
bioprinted to ensure that structural fidelity can be main-
tained at the desired compositions followed by evaluation
of intracellular ROS, viability, and apoptosis activity of the
bioprinted constructs. This was followed by evaluation of
osteogenic activity of cells in the constructs.
II. EXPERIMENT
A. Bioink formulation
Alginic acid sodium (viscosity ≥ 2000 cP, 2%) and NAC
(MW ≥ 163.19 g/mol) were procured from Sigma Aldrich,
USA. Dulbecco’s modified Eagle’s medium (DMEM) was
obtained from Gibco, Thermofisher, USA and reconsti-
tuted in ASTM Type I Grade water (Wasserlab, Spain)
for alginate solution preparation. A 3% w/v solution of
Biointerphases, Vol. 14, No. 5, Sep/Oct 2019
051006-2
alginate was prepared and 1 mM solution of NAC was
added to get the BP-NAC designated samples. To prepare
cell-laden bioinks, cells at a concentration of 1 × 105 cells/
ml were suspended in the mixture to obtain the bioinks.7
B. Extrusion-based bioprinting
An extrusion-based 3D printer was obtained from M/s
Alfatek Systems, India for bioprinting of scaffolds. CURA
(v15.04.5, Ultimaker, the Netherlands), an open source soft-
ware was used to slice the printed structures designed in
SKETCHUP 2017 software and the sliced .stl file was converted to
.GCODE format by using PRONTERFACE (version 2014.03.10,
Printrun), as described earlier. For the cell-laden bioprinting,
the bioprinter was housed inside a hood cabinet.
C. Scaffold design
A solid square 3D shape of size 34 × 34 mm2 was designed
by SKETCHUP 2017 software and saved in the .stl format.
Further slicing of the 3D square structure was done by CURA.
The file was saved in the G-Code format and uploaded to
the printer server for printing. The adjustment of the mesh
structure was done by changing the fill density from 100%
to 18%. The input parameters were taken as follows at a
layer-upon-layer orientation: Z-layer stacking—3.961 mm,
strand-to-strand distance—4.4 mm, strand thickness—2.0 mm,
layer height—0.1 mm, filament diameter—1.75 mm, bottom/
top thickness—0.6 mm, nozzle size—0.410 mm, printing
speed—50 mm/s (0.30 MPa), and shell thickness—0.8 mm.
D. Printing of alginate ink
The bioinks were charged in a 5 ml syringe attached
with a nozzle of diameter 0.4 mm. At a fixed printing speed
of the head of the printed 50 mm/s, the extrusion of the
bioinks was performed on a glass coverslip at a temperature
of 25 °C. As per the desired dimension, the printer printed
the desired scaffold dot by dot on a 1M calcium chloride
bath contained in a glass Petri dish and the cross-linking
was allowed to proceed for 10 min.22
E. Characterization of the constructs
The images of the constructs were captured by a Sony
Cybershot (DSC-WX220/B) digital camera. Mechanical
testing under uniaxial tensile loading was performed by a
universal testing machine with 125 N load cell (Tinius
Olsen 5KT, Tinius Olsen, UK). Sample dimensions were
used and gripped with a clip-type sample holder. Samples
were loaded at a crosshead speed of 0.1 mm/min. Five
samples for each bioink formulation scaffold were tested.
Bioprinted samples that varied by more than 10% in geo-
metrical dimensions from the average parameters were dis-
carded, and the actual dimension for each scaffold was
used for the determination of cross-sectional area. Before
testing each sample, samples were preconditioned up to 10%
strain for ten cycles to observe for structural integrity. The
stress at break of the samples failed was recorded as the
051006-3 Datta et al.: Bioink formulations to ameliorate bioprinting-induced loss of cellular viability
tensile strength. To measure the swellability of these con-
structs, equilibrium water content was measured by immers-
ing the dried bioprinted constructs in DMEM at 37 °C for
24 h. Swelling extent was calculated as per the equation
S = [(Wt − Wi)/Wi)], where Wi is the initial weight and Wt is
the weight of the samples after 24 h.
F. Cell culture study
Cells (MC3T3) were cultured at 37 °C and 5% CO2 incu-
bator (Esco, Singapore) in DMEM (Gibco, USA) containing
10% fetal bovine serum and 1% antibiotic ( penicillin-
streptomycin). Cells at 105 cells ml−1 were taken for bioink
formulations. Cell viability was measured on day 1 by
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide
(MTT) assay (EZcountTM MTT Assay Kit, Himedia,
India) in a 96 well plate. The MTT reagent was added and
incubated with the constructs for 4 h. Formazan crystals
appearing after incubation were dissolved in solubilization
buffer, absorbance at 570 and 670 nm was recorded using
Multi-Scan Go (Thermo Fisher, Finland), and cell viability
was determined according to the manufacturer’s protocol.
To evaluate cell proliferation, the same assay was repeated
after incubating a set of bioprinted constructs for 3 days. All
cell viability was expressed in terms of percentage of cell via-
bility obtained on control samples (constructs obtained by
involvement of steps of only pipetting of the cell-laden bioink
suspensions at the same concentrations on glass coverslips) on
day 1. The bioprinted constructs are designated as BP (only
cell-laden constructs) and BP-NAC (cell-laden constructs with
the antioxidant NAC).
G. Live/dead assay
The cell culture was done on the two alginate and
alginate-NAC scaffolds for 3 days, and staining was done
by 100 μl staining solution and 15 min incubation was done at
37 °C according to the protocol given by the manufacturer
(QIA76 Live/Dead Double Staining kit, Merck, India).
H. Antioxidant/free radical scavenging capability
study
To measure the release of free radical from the BP and
BP-NAC scaffolds (1.5 × 1.5 cm2), the constructs were
immersed in sterile phosphate buffered saline (PBS, 10 ml)
and a supernatant was obtained after 6 h. The control group
alginate constructs deposited by a pipette on glass coverslips
and without containing NAC were taken as a blank. After incu-
bation for the designated time points, to determine the func-
tionality of scaffolds with time, 2,2-diphenyl-1-picrylhydrazyl
(DPPH) activity was measured at each time point following
the procedure.23 All the samples were incubated in dark for
30 min, and the measurement was done by a Multi-Scan
Go (Thermo Fisher, Finland) spectrometer at a wavelength
of 520 nm. Higher radical scavenging power is related to
Biointerphases, Vol. 14, No. 5, Sep/Oct 2019
051006-3
decreasing purple color intensity, which is calculated by
 
Asam
Scavenging power ¼ 1   100: (1)
Ablk
Here, Asam is the absorbance of the sample and Ablk is the
absorbance of the blank at 30 min reaction time.24
I. ROS assay
200 μl of cell suspension was mixed with 3% alginate and
3% alginate with 1 mM NAC-alginate. The solutions were
then 3D printed, and the bioprinted scaffolds were incubated
in complete high-glucose DMEM for 24 h. The bioprinted
scaffolds were washed thoroughly with 1× PBS after removal
of media. Incomplete high-glucose DMEM and DCF-DA
(20 ,70 -dichlorodihydrofluorescein diacetate) (Cellular Reactive
Oxygen Species Detection Assay Kit, Abcam, AB113851,
USA) at a concentration of 20 μM at 37 °C was further incubated
for 30 min, as per manufacturer’s instructions. The scaffolds
counterstained with 40 ,6-Diamidine-20 -phenylindole dihydro-
chloride (DAPI) for 5 min after thorough washing with 1× PBS.
J. Immunocytochemistry
Cell constructs (control, BP, and BP-NAC) were placed in
a 48-well plate and treated with 4% paraformaldehyde for fix-
ation for 15 min and then with 0.1% Triton X-100 for permea-
bilization at room temperature. Constructs were incubated with
an anti-caspase3 primary antibody (Merck Millipore, India)
overnight at 4 °C after blocking with goat serum.25 After thor-
ough washing, goat antimouse IgG conjugated-Alexa Fluor
488 (1:500) (secondary antibody) (Santa Cruz Technology,
Shanghai) was treated with the constructs for 2 h at room tem-
perature. Counterstaining with DAPI (Sigma Aldrich, Saint
Louis, MO, USA) was performed subsequently. Mounting
media (Sigma Aldrich, USA) was used to mount the cells on
the slide after thorough washing of the cells with 1× PBS.
Cyclooxygenase-2 (Cox-2) study was done by culturing
cells in a 48 well plate treated with control, BP, and
BP-NAC scaffolds and were first fixed-permeabilized with
paraformaldehyde-Triton X as mentioned for caspase 3. The
protocol was adapted from a previous report.26 Cells were
incubated overnight at 4 °C with anti-Cox2 primary antibody
(Cell Signaling Technology) after blocking with goat serum.
After thorough washing, cells were incubated with secondary
antibody for 2 h at room temperature [goat anti-rabbit IgG
conjugated with Texas Red (1:200)] (Santa Cruz Technology,
Shanghai). Immunostained cells were counterstained with
DAPI (Sigma Aldrich, Saint Louis, MO, USA). Mounting
media (Sigma Aldrich, USA) was used to mount the cells on
the slide after thorough washing of the cells with 1× PBS.
Intensity (green intensity for caspase 3 and red for Cox) was
calculated for three constructs, and the mean is reported.
K. Alizarin red assay
Constructs were cultured for 3 days in serum-free media,
stained with 40 mM Alizarin Red S (Himedia, India) for
051006-4 Datta et al.: Bioink formulations to ameliorate bioprinting-induced loss of cellular viability
15 min at room temperature, and solubilized in 10% acetic
acid for 1 h, and the optical density (OD) was taken at
405 nm. Results are expressed relative to the OD obtained
for manually pipetted pure alginate constructs.
L. Image acquisition
For fluorescence microscopy, images were acquired
with a Nikon eclipse Tί U, Japan microscope equipped
with 20× objective. Three different fluorescence filters
(λex340–380 nm and λem435–485 nm, λex465–495 nm and
λem515–555 nm, and λex512–552 nm and λem565–615 nm
for blue, green, and red emission, respectively) were
used for imaging. Fluorescence intensity of the images
was measured using IMAGEJ software (version 1.44p).
The mean intensity was normalized per cell and repre-
sented graphically. In the case of alizarin red assay,
cell imaging was performed through bright-field mode
using 20× objective lens. Dimensions of fluorescence
and bright-field images are 1280 × 1024 and 2560 × 1920
pixels, respectively.
M. Statistical analysis
All measurements were performed in triplicate, and the
results are reported as mean ± SD. A one-way analysis of the
variance coupled to Bonferroni post hoc test was employed
to assess the significance of the differences between the mea-
surements. Results with a p value less than 0.01 were consid-
ered different from each other.
FIG. 1. Characterization of constructs prepared by extrusion of alginate and alginate with cells showing (a) higher spreading in cell-alginate bioinks in morphol-
ogy and (b) the extent of effect on mechanical properties of the constructs.
Biointerphases, Vol. 14, No. 5, Sep/Oct 2019
051006-4
III. RESULTS AND DISCUSSION
A. Characterization of the constructs
Figure 1(a) depicts that the constructs including cell-laden
alginate bioinks were bioprinted into well-demarcated struc-
tures but with slightly different shape fidelity. Inclusion of
cells into alginate bioink solutions caused distortion of bio-
printed constructions, which is the characteristic of under-
gelation compared to pure alginate extrusion. However, the
addition of NAC did not cause any significant difference in
the geometrical attributes of the bioprinted constructs. On the
other hand, the addition of cells decreased the tensile strength
of the constructs by 36% (0.3 MPa) compared to pure alginate
while the presence of NAC resulted in 0.25 MPa strength for
the cell-alginate constructs, as represented in Fig. 1(b). The
swelling experiments showed that both BP and BP-NAC
showed a swelling ratio of 15–20%, a range which is compa-
rable to the literature reported values.22,27 The constructs
were then advanced for viability analysis.
B. Effect of NAC on cell viability of bioprinted
constructs
Cell viabilities on days 1 and 3 of post-bioprinting were
measured using the MTT assay and are represented in Fig. 2.
The % viabilities on day 1 were 75 and 91% on BP and
BP-NAC samples. There was a significant difference between
BP and control samples BP-NAC (p < 0.01). This showed that
the adopted bioprinting process can result in cell death ∼25%
on day 1 compared to a control sample extruded through
manual pipetting under the same conditions. However, the
051006-5 Datta et al.: Bioink formulations to ameliorate bioprinting-induced loss of cellular viability
FIG. 2. Effect of antioxidant N-acetyl cysteine on bioprinting process cell
viability investigated by MTT assay.
addition of NAC to bioinks used for bioprinting showed a sig-
nificantly reduced cell viability on day 1 up to ∼90% of the
control samples. Day 3 viability results further showed that
cells proliferated in both the samples. In the control samples,
day 3 cell viability was 144%, which was significantly differ-
ent from day 1 control value taken as 100%. In contrast, BP
group had a viability of 104% and BP-NAC showed 138%.
Cell viability in BP was significantly less (p < 0.01) in com-
parison to BP-NAC, whereas no significant difference was
found between BP-NAC and control value on day 3. The
effect of NAC in restoring cell viability was thus clearly
visible in BP-NAC, wherein the viability was found to be
comparable to control. The increase in viability from day 1
and day 3 for BP-NAC was also higher compared to BP, indi-
cating that NAC had a cytoprotective effect on cell damage
induced due to the bioprinting process. The observations of
MTT assay were corroborated with Live-dead assay imaging
(Fig. 3) wherein a larger fraction of live cells compared to
dead cells were evident in BP-NAC compared to BP on day 3.
However, in control samples too, some red cells were
observed showing that even low shear pressure on cells in
high viscosity bioinks can also be a cause of cell deaths.
C. Antioxidant, apoptotic, and anti-inflammatory
levels of cells
To understand if the shear pressure causes cell death with
the involvement of oxidative mechanisms, ROS assay was
FIG. 3. Viability of cells after bioprinting with and without NAC observed by (a) representative Live/Dead stain imaging. Images acquired in 20× magnification.
Biointerphases, Vol. 14, No. 5, Sep/Oct 2019
051006-5
performed on the constructs. Figures 4(a) and 4(b) qualita-
tively and quantitatively demonstrate that the ROS load of BP
constructs is higher than BP-NAC constructs. In BP con-
structs, MC3T3 cells showed 1.4 times higher ROS load than
the control (p < 0.01) constructs, indicating that after bioprint-
ing, extrusion pressures cause extensive intracellular accumu-
lation of ROS. This is because the control samples have been
fabricated by following exactly the same procedure as BP and
the only difference being the extrusion pressures associated
with the bioprinting process, and the ROS load can be
assumed to be originated from the shear stresses. As seen in
Fig. 4(c), % free radical scavenging was significantly higher
(p < 0.01) in BP-NAC compared to the BP group. These
results show that the ROS load is around 1.4 times higher
compared to control constructs whereas the difference in cell
viability was around 20%. This indicates that cells also proba-
bly possess some intrinsic mechanism to reduce viability
losses due to oxidative stress; however, such mechanisms are
not adequately robust to provide 100% short-term or long-
term viability. The addition of NAC, on the other hand,
resulted in a free radical scavenging activity of 78%, which
was closer to the control group and significantly higher
(p < 0.01) than the BP group, while no significant difference
in the ROS load between control and BP-NAC was observed.
Correlating with viability assay, it can be seen that NAC nev-
ertheless allows significant enhancement of cell viability and
mitigates the stress-induced effects.
The mechanism of NAC action of cells was further inves-
tigated through caspase and Cox expressions. As shown in
Fig. 5(a), high amount of cleaved caspase 3 proteins could
be observed in BP whereas a lesser number of caspase 3 was
present in BP constructs on day 3. Quantitative interpretation
of the immunoflorescence micrographs further confirmed the
visual observation wherein significantly higher ( p < 0.01)
apoptotic cells were observed in BP compared to the control
group [Fig. 5(b)]. Therefore, shear stress was shown to
reduce cell deaths by inducing ROS-mediated apoptosis of
the cells. However, NAC had a positive role in preventing
apoptosis as BP-NAC showed a significantly lower apoptotic
cell compared to BP ( p < 0.01). Examination of Cox-2
expression in the constructs further revealed that Cox-2 was
significantly higher in BP [Figs. 6(a) and 6(b)] compared to
051006-6 Datta et al.: Bioink formulations to ameliorate bioprinting-induced loss of cellular viability 051006-6

FIG. 4. Reactive oxygen species accumulated in cells inside the bioprinted constructs with and without NAC estimated by (a) representative imaging and (b)
quantification; (c) % free radical scavenging activity by DPPH assay. Images acquired in 20× magnification.

FIG. 5. Apoptosis of cells inside the bioprinted constructs containing NAC observed by (a) caspase 3 imaging and (b) quantification of caspase 3. Images
acquired in 20× magnification.

Biointerphases, Vol. 14, No. 5, Sep/Oct 2019

051006-7 Datta et al.: Bioink formulations to ameliorate bioprinting-induced loss of cellular viability
BP-NAC (p < 0.05). However, the difference in Cox-2 expres-
sion in between BP and BP-NAC was considerably less than
the difference in caspase 3 activity between the two groups.
This indicates that NAC has probably a greater role in reduc-
ing apoptosis and cell viability compared to inflammatory pro-
cesses in the cells. The differences between control and BP
samples indicate that bioprinting process stresses may be more
susceptible to cause apoptosis-mediated cell deaths compared
to inflammation-mediated damages. However, an NAC will
have beneficial effects in both cases.
D. Functional characterization of constructs
To investigate the functionality of the constructs, alizarin
red staining was performed on day 5 as a marker for bone
tissue engineering application and the results are shown in
Fig. 6(c). Compared to the control group, BP constructs
showed no significant difference in alizarin red S (ARS)
activity. However, BP-NAC showed significantly higher
ARS stain compared to BP (p < 0.01). Interestingly, the
number of viable cells on day 1 in BP-NAC was lower than
control whereas ARS activity on control samples was lower
than BP-NAC on day 5. However, the results also indicate
that the bioprinting process induces not only lower cell viabil-
ity during fabrication but also diminishes their long-term
functionality, thus justifying the development of bioink for-
mulations to mitigate such process-induced effects. Results
confirm that NAC has a positive role in improving the func-
tionality of constructs. The observation that ARS activity of
FIG. 6. (a) Expression of inflammatory marker Cox by MC3T3 cells after bioprinting with and without NAC and (b) its quantification; (c) expression of
Alizarin red by the cells as a functional marker. Images acquired in 20× magnification.
Biointerphases, Vol. 14, No. 5, Sep/Oct 2019
051006-7
BP-NAC was higher than the control samples, suggesting that
NAC has direct involvement in improving cell functionalities.
IV. DISCUSSION
Though extrusion bioprinting is most convenient and
affordable among the various bioprinting techniques, it is asso-
ciated with the highest viability losses. Several authors have
attempted to understand the mechanism of viability losses in
extrusion bioprinting. Though some hypothesized surface
imperfections in nozzles and initial alginate solution tonicity
are some of the reasons, the general consensus has been attri-
bute to cell damages to pneumatic force-generated stresses on
the cells.11,28 These stresses are a combination of hydrostatic
pressure, shear forces, and extensional stresses.29 It is thus
expected that attempts to reduce shear-induced cell viability
will have significance for the bioprinting research. Moreover,
process modifications should also focus on long-term cell func-
tionality apart from the immediate post-bioprinting viability, as
shear stresses beyond a threshold can not only affect viability
but also the functionality of the viable cells.30
The extrusion bioprinted constructs fabricated in this study
using MC3T3 cells and alginate bioinks showed similar mor-
phological characteristics as reported in our previous studies
as well as the lattice structures generally bioprinted to assess
bioprintability of new bioinks.31,32 From Fig. 1, it was
evident that embedding cells into the bioinks caused construct
morphologies to be more characteristic of under-gelation and
hence over-flowing of structures due to delayed gelation.17
051006-8 Datta et al.: Bioink formulations to ameliorate bioprinting-induced loss of cellular viability
The same reason could be attributed to the decrease in
mechanical properties of the cell-laden constructs as the cells
interfere with the network formation and effectively act as
pores to reduce the construct strength. Indeed, Zhao et al. have
shown that including cells in alginate-based bioink suspensions
can result in a decrease of gel modulus and affect alginate rhe-
ology.33 Similarly, results of Duan et al. also illustrate how
cell-laden hydrogels usually have a lesser mechanical strength
compared to cell-free bioinks.34 However, as they have also
shown, cell-laden hydrogels usually increase in strength and
stiffness over time due to the deposition of extracellular matrix.
The range of mechanical properties reported in their study
closely approximates the properties obtained in our study, as
also other tough hydrogels based on alginate fabricated by
extrusion bioprinting techniques reported in the literature.35
The presence of the antioxidant, NAC, at the concentrations
studied had no remarkable effect on these properties.
Examination of cell viability after day 1 and day 3
revealed the positive role of antioxidant supplementation in
the bioink formulation. Both short-term and long-term via-
bility had improved, with a more pronounced effect on
long-term viability, which attained near-to control sample
viability. The bioprinting process-associated cell viability
loss in our study was around 25%, which is near to the viability
reported in the literature for alginate (2%–3%), extrusion pres-
sure (0.2–0.4 MPa), and nozzle diameters (100–300 μm),9,11
close to conditions investigated in this work. There have been
several efforts on improving cell viability in such bioprinting
applications mostly by reducing the extrusion pressures or
alginate concentrations or increasing nozzle size. However,
any such changes invariably result in a compromise on prop-
erties of the bioprinted constructs. For example, reducing
extrusion pressure results in an increase in residence time of
cells during the bioprinting process, which will offset the via-
bility gains. Decreasing the alginate concentration reduces the
mechanical properties of the constructs, whereas increasing
nozzle dimensions would result in affecting resolution of the
bioprinted constructs.5,36–38 A limitation of the present study
is that a higher concentration of CaCl2 has been used for
cross-linking (1M), which can compromise cell membrane
integrity due to high osmotic effects. However, the advantage
of NAC addition at an extreme CaCl2 concentration also indi-
cates that better outcomes may be expected if bioprinting is
conducted at lower cross-linker concentrations. An optimiza-
tion may be required between the gelator concentration and
cell shocks, and in some cases an osmotic regulator may be
employed.39 In contrast, the approach presented by us shows
that cell viability can be increased during the bioprinting
process by not changing the bioprinting process parameters
and without any significant compromise in the physico-
chemical properties of the constructs. Therefore, the present
solution of adding an antioxidant as bioink formulations may
provide a new paradigm in bioprinting process research. The
antioxidant NAC has been previously shown to abrogate
stress-induced cell deaths and also known as a bioactive mol-
ecule to enhance bone regeneration.40,41 It is also known that
hydrostatic pressure, shear stresses due to syringe flow, and
Biointerphases, Vol. 14, No. 5, Sep/Oct 2019
051006-8
the presence of a viscous medium can induce excessive oxi-
dative stress in the cells, causing cell death apart from
causing membrane damage.42–44 ROS generated in cells is
further associated with activation of apoptosis pathways by
several mechanisms.45 Alternatively, inflammatory markers
such as Cox-2 are also induced in response to mechanical
stresses on the cells.46 The interlinking between ROS, apo-
ptosis, and inflammatory pathways have been observed by
many researchers.47 Therefore, the role of NAC was compre-
hensively analyzed with respect to these cellular markers and
it was evident that NAC can have multiple protective effects
on cell viability. Apart from long-term viability, a positive
role of NAC was also evidenced on the functionality of the
cells. Our results are in close agreement with the recent find-
ings of Watanabe et al.,48 who have demonstrated that pre-
conditioning of mesenchymal stem cells with NAC improves
the long-term functionality and bone differentiation of stem
cells by reducing oxidative stress and building apoptosis
resistance. Indeed, NAC has been shown to suppress
mechanical stretch-induced cell hypertrophy in cardiomyo-
cytes.49 In another study, Shaik et al. have demonstrated that
an antioxidant silymarin-containing scaffold aids in the
revival of cells after exposure to UV radiation-induced oxida-
tive stresses50—a phenomenon that has a resemblance to the
results observed by us with shear stress-induced oxidative
stresses. The study by Lin et al. showed that conditioning
with NAC counteracted the negative influence of initiator
and light sources used during cellular photoencapsulation in
a gelatin methacrylate hydrogel system and resulted in
improved cell proliferation. In conjunction with these results,
our study further proves that the bioactive role of NAC can
be efficiently employed to improve process viability in bio-
printing for different hydrogel systems.51
V. SUMMARY AND CONCLUSIONS
In this work, we reported that the addition of the antiox-
idant N-acetyl cysteine to bioink formulations of MC3T3
cells-alginate can reduce the extrusion process-associated
cell deaths. The NAC addition results in improvement of
both short-term (day 1) and long-term (day 3) cell viabil-
ity. This observation was correlated with reduction in the
ROS load of the cells. Additionally, it was also seen that
expression of apoptotic marker (caspase 3) and inflamma-
tory marker (Cox-2) was also lowered due to the addition
of NAC during the bioprinting process. It was plausibly
due to the uptake of NAC by the cells during the extrusion.
It was further revealed that the extent of reduction in apo-
ptosis was higher compared to the inflammatory marker.
The presented process assumes significance in light of the
fact that other methods to reduce cell deaths during extru-
sion bioprinting are associated with a compromise over the
resolution or mechanical integrity of the constructs. However,
by the addition of an antioxidant to the cell-laden bioink
suspension, cell survival can be enhanced by a simple
process without interfering with other process parameters
or construct properties.
 051006-9 Datta et al.: Bioink formulations to ameliorate bioprinting-induced loss of cellular viability
ACKNOWLEDGMENTS
The authors acknowledge the DST-Inspire Faculty grant
to P.D. (No. IFA-12-LSBM-48) and the IIEST institute fel-
lowship to S.D. and A.D. The authors also acknowledge
TEQIP-II for the Nikon Microscopy facility.
1 30
D. J. Ravnic, A. N. Leberfinger, S. V. Koduru, M. Hospodiuk, K. K. Moncal,
P. Datta, M. Dey, E. Rizk, and I. T. Ozbolat, Ann. Surg. 266, 48 (2017).
2
F. Pati, J. Gantelius, and H. A. Svahn, Angew. Chem. Int. Ed. 55, 4650
(2016).
3
R. Chang, J. Nam, and W. Sun, Tissue Eng. Part A 14, 41 (2008).
4
K. Nair, M. Gandhi, S. Khalil, K. C. Yan, M. Marcolongo, K. Barbee, and
W. Sun, Biotechnol. J. 4, 1168 (2009).
5 34
P. Datta, A. Barui, Y. Wu, V. Ozbolat, K. K. Moncal, and I. T. Ozbolat,
Biotechnol. Adv. 36, 1481–1504 (2018).
6 35
A. K. Nguyen, P. L. Goering, V. Reipa, and R. J. Narayan, Biointerphases
14, 21007 (2019).
7 36
A. Blaeser, D. F. Duarte Campos, U. Puster, W. Richtering,
M. M. Stevens, and H. Fischer, Adv. Healthc. Mater. 5, 326 (2016).
8 37
X. Y. Tian, M. G. Li, and X. B. Chen, J. Biomimetics Biomater. Tissue
Eng. 17, 1 (2013).
9 38
L. Ning, N. Betancourt, D. J. Schreyer, and X. Chen, ACS Biomater. Sci.
Eng. 4, 3906 (2018).
10
M. Li, X. Tian, J. A. Kozinski, X. Chen, and D. A. E. K. U. N. Hwang,
J. Mech. Med. Biol. 15, 1550073 (2015).
11
B. A. Aguado, W. Mulyasasmita, J. Su, K. J. Lampe, and S. C. Heilshorn,
Tissue Eng. Part A 18, 806 (2011).
12
K.-Y. Lo, Y. Zhu, H.-F. Tsai, and Y.-S. Sun, Biomicrofluidics 7, 64108
(2013).
13
M. Mayr, Y. Hu, P. Hainaut, and Q. Xu, FASEB J. 16, 1423 (2002).
14
K. A. Barbee, Ann. N. Y. Acad. Sci. 1066, 67 (2006).
15
S. Tan, Y. Sagara, Y. Liu, P. Maher, and D. Schubert, J. Cell Biol. 141,
1423 (1998).
16
L. Valon and R. Levayer, Biol. Cell 111, 51 (2019).
17
L. Ouyang, R. Yao, Y. Zhao, and W. Sun, Biofabrication 8, 35020 (2016).
18
A. G. Tabriz, M. A. Hermida, N. R. Leslie, and W. Shu, Biofabrication
7, 45012 (2015).
19
L. Sun, L. Gu, S. Wang, J. Yuan, H. Yang, J. Zhu, and H. Zhang, PLoS
One 7, e32503 (2012).
20
G. Spagnuolo, V. D’Antò, C. Cosentino, G. Schmalz, H. Schweikl, and
S. Rengo, Biomaterials 27, 1803 (2006).
21
G. Aldini, A. Altomare, G. Baron, G. Vistoli, M. Carini, L. Borsani, and
F. Sergio, Free Radic. Res. 52, 751 (2018).
22
Z. Li, S. Huang, Y. Liu, B. Yao, T. Hu, H. Shi, J. Xie, and X. Fu, Sci.
Rep. 8, 1 (2018).
23
S. Datta, R. Sarkar, V. Vyas, S. Bhutoria, A. Barui, A. Roy Chowdhury,
and P. Datta, J. Mater. Res. 33, 2029 (2018).
24
B. Ates, L. Abraham, and N. Ercal, Free Radic. Res. 42, 372 (2008).
25
Q. Zhang, P. D. Nguyen, S. Shi, J. C. Burrell, D. K. Cullen, and A. D. Le,
Sci. Rep. 8, 6634 (2018).
Biointerphases, Vol. 14, No. 5, Sep/Oct 2019
View publication stats
051006-9
26
R. Sarkar, A. Ghosh, A. Barui, and P. Datta, J. Mater. Sci. Mater. Med.
29, 31 (2018).
27
S. Deepthi and R. Jayakumar, Bioact. Mater. 3, 194 (2018).
28
D. E. Godar, in Tissue Regeneration, edited by Hussein Abdelhay and
Essayed Kaoud (IntechOpen, 2018).
29
Extrusion Bioprinting of Scaffolds for Tissue Engineering
Applications, edited by D. X. B. Chen (Springer, Cham, 2019),
pp. 117–145.
W. Y. Sim, S. W. Park, S. H. Park, B. H. Min, S. R. Park, and S. S. Yang,
Lab Chip 7, 1775 (2007).
31
S. Datta, A. Das, P. Sasmal, S. Bhutoria, A. Roy Chowdhury, and
P. Datta, Int. J. Polym. Mater. Polym. Biomater. 1 (2018).
32
C. Li et al., Biomed. Mater. 14, 25006 (2019).
33
Y. Zhao, Y. Li, S. Mao, W. Sun, and R. Yao, Biofabrication 7, 45002
(2015).
B. Duan, L. A. Hockaday, K. H. Kang, and J. T. Butcher, J. Biomed.
Mater. Res. Part A 101A, 1255 (2013).
J. Wei, J. Wang, S. Su, S. Wang, J. Qiu, Z. Zhang, G. Christopher,
F. Ning, and W. Cong, RSC Adv. 5, 81324 (2015).
N. Paxton, W. Smolan, T. Böck, F. Melchels, J. Groll, and T. Jungst,
Biofabrication 9, 044107 (2017).
P. S. Gungor-Ozkerim, I. Inci, Y. S. Zhang, A. Khademhosseini, and
M. R. Dokmeci, Biomater. Sci. 6, 915 (2018).
J. Snyder, A. Rin Son, Q. Hamid, C. Wang, Y. Lui, and W. Sun,
Biofabrication 7, 44106 (2015).
39
A. F. Carvalho, L. Gasperini, R. S. Ribeiro, A. P. Marques, and R. l. Reis,
J. Tissue Eng. Regen. Med. 12, e1063 (2018).
40
M. Yamada et al., Biomaterials 34, 6147 (2013).
41
K. Niwa, J. Sakai, T. Karino, H. Aonuma, T. Watanabe, T. Ohyama,
O. Inanami, and M. Kuwabara, Free Radic. Res. 40, 167 (2006).
42
D. A. Chistiakov, A. N. Orekhov, and Y. V. Bobryshev, Acta Physiol.
219, 382 (2017).
43
M. Rouhanizadeh, W. Takabe, L. Ai, H. Yu, and T. Hsiai, in Nitric
Oxide, Part G Oxidative Nitrosative Stress in Redox Regulation on Cell
Signalling, edited by E. Cadenas and L. Packer (Academic, 2008),
pp. 111–150.
44
Q. Liu, W.-K. Ju, J. G. Crowston, F. Xie, G. Perry, M. A. Smith,
J. D. Lindsey, and R. N. Weinreb, Investig. Ophthalmol. Vis. Sci. 48, 4580
(2007).
45
M. Redza-Dutordoir and D. A. Averill-Bates, Biochim. Biophys. Acta
Mol. Cell Res. 1863, 2977 (2016).
46
N. Shimizu, Y. Ozawa, M. Yamaguchi, T. Goseki, K. Ohzeki, and
Y. Abiko, J. Periodontol. 69, 670 (1998).
47
Y. Chao, P. Ye, L. Zhu, X. Kong, X. Qu, J. Zhang, J. Luo, H. Yang, and
S. Chen, J. Cell. Physiol. 233, 1384 (2018).
48
J. Watanabe, M. Yamada, K. Niibe, M. Zhang, T. Kondo, M. Ishibashi,
and H. Egusa, Biomaterials 185, 25 (2018).
49
R. Aikawa et al., Biochem. Biophys. Res. Commun. 289, 901 (2001).
50
M. M. Shaik, A. Dapkekar, J. M. Rajwade, S. H. Jadhav, and
M. Kowshik, J. Mater. Sci. Mater. Med. 30, 13 (2019).
51
C.-H. Lin, K.-F. Lin, K. Mar, S.-Y. Lee, and Y.-M. Lin, Tissue Eng. Part
C Methods 22, 792 (2016).