Bio-ink properties and printability for extrusion printing living cells

TLDR: Alginate (Alg) was selected as the major component of the 'bio-ink' formulations for extrusion printing of cells and the viability of primary myoblasts delivered as a myoblast/Alg-Gel bio-ink was not affected by the printing process, indicating that the Alg-gel matrix provides a potential means to print 3D constructs that may find application in myoregenerative applications.

Abstract: Additive biofabrication (3D bioprinting) makes it possible to create scaffolds with precise geometries, control over pore interconnectivity and architectures that are not possible with conventional techniques. Inclusion of cells within the ink to form a “bio-ink” presents the potential to print 3D structures that can be implanted into damaged/diseased tissue to promote highly controlled cell-based regeneration and repair. The properties of an ‘ink’ are defined by its formulation and critically influence the delivery and integrity of structure formed. Importantly, the ink properties need to conform to biological requirements necessary for the cell system that they are intended to support and it is often challenging to find conditions for printing that facilitate this critical aspect of tissue bioengineering. In this study, alginate (Alg) was selected as the major component of the ‘bio-ink’ formulations for extrusion printing of cells. The rheological properties of alginate-gelatin (Alg-Gel) blends were compared with pre-crosslinked alginate and alginate solution to establish their printability whilst maintaining their ability to support optimal cell growth. Pre-crosslinked alginate on its own was liquid-like during printing. However, by controlling the temperature, Alg-Gel formulations had higher viscosity, storage modulus and consistency which facilitated higher print resolution/precision. Compression and indentation testing were used to examine the mechanical properties of alginate compared to Alg-Gel. Both types of gels yielded similar results with modulus increasing with alginate concentration. Decay in mechanical properties over time suggests that Alg-Gel slowly degrades in cell culture media with more than 60% decrease in initial modulus over 7 days. The viability of primary myoblasts delivered as a myoblast/Alg-Gel bio-ink was not affected by the printing process, indicating that the Alg-Gel matrix provides a potential means to print 3D constructs that may find application in myoregenerative applications.

Figures

Fig. 3. Previously, it has been reported that higher pressures 70 increase the shear stress at the nozzle which damages the cell membrane and thus lowers cell viability after extrusion 6. In contrast, cell viability across all the pressures used in this study was above 90% an hour following printing and was not significantly different even after 48 hours. This is in agreement 75 with findings in the literature where cells still demonstrated good viability across varying pressures of extrusion 50, 51. This suggested that the pressure selected to print optimally for this formulation did not induce adverse cellular response and were appropriate for the selected cell type. The fact that the cells were 80 surrounded within the gel may also shield them from the shear forces at the nozzle tip 51. Cell proliferation was not observed from the Alg-Gel scaffolds within the time period tested. This may suggest that the cells require longer times to overcome their surrounding structure and proliferate. A study conducted by 85 Gaetani et al. 14 reported an increase in the number of human cardiomyocyte progenitor cells (hCMPCs) within alginate matrices after 1 week of culture, whereas there were no signs of cell proliferation just after 24 hours in culture. Therefore, further studies into the proliferation and functions of these muscle cells 90 will need to be conducted at longer periods of time.

Full text

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Bio-ink properties and printability for extrusion printing living cells
Johnson H.Y Chung
a
, Sina Naficy
a
, Zhilian Yue
a
, Robert Kapsa
a,b
, Anita Quigley
a,b
, Simon E. Moulton*
a
and Gordon G. Wallace*
a
Additive biofabrication (3D bioprinting) makes it possible to create scaffolds with precise geometries,
5
control over pore interconnectivity and architectures that are not possible with conventional techniques.
Inclusion of cells within the ink to form a “bio-ink” presents the potential to print 3D structures that can
be implanted into damaged/diseased tissue to promote highly controlled cell-based regeneration and
repair. The properties of an ‘ink’ are defined by its formulation and critically influence the delivery and
integrity of structure formed. Importantly, the ink properties need to conform to biological requirements
10
necessary for the cell system that they are intended to support and it is often challenging to find
conditions for printing that facilitate this critical aspect of tissue bioengineering. In this study, alginate
(Alg) was selected as the major component of the ‘bio-ink’ formulations for extrusion printing of cells.
The rheological properties of alginate-gelatin (Alg-Gel) blends were compared with pre-crosslinked
alginate and alginate solution to establish their printability whilst maintaining their ability to support
15
optimal cell growth. Pre-crosslinked alginate on its own was liquid-like during printing. However, by
controlling the temperature, Alg-Gel formulations had higher viscosity, storage modulus and consistency
which facilitated higher print resolution/precision. Compression and indentation testing were used to
examine the mechanical properties of alginate compared to Alg-Gel. Both types of gels yielded similar
results with modulus increasing with alginate concentration. Decay in mechanical properties over time
20
suggests that Alg-Gel slowly degrades in cell culture media with more than 60% decrease in initial
modulus over 7 days. The viability of primary myoblasts delivered as a myoblast/Alg-Gel bio-ink was not
affected by the printing process, indicating that the Alg-Gel matrix provides a potential means to print 3D
constructs that may find application in myoregenerative applications.
1. Introduction 25
An emerging approach to create complex three dimensional
constructs containing biological cells is by a process referred to
as ‘biofabrication’ or ‘bioprinting’, using an appropriately
formulated bio-ink. Several biofabrication methods have been
used to create 3D scaffolds for tissue engineering applications
1, 2
.
30
3D bioplotting, first introduced by Landers et al.
3, 4
is an
extrusion based method that can continuously dispense materials
(i.e., ‘ink’) and biological cells from a movable dispensing head
or onto a moving stage to form patterns predesigned through
computer-aided design (CAD) tools
4
. This method has less
35
geometrical limits than most of the conventional methods and can
deposit material and cells within tens of minutes
5
.
Ink development can be considered as one of the most
challenging aspects in the bioprinting process. An ‘ideal’ ink
should satisfy biological needs from the cell compatibility point
40
of view, but also the physical and mechanical needs of the
printing process itself. Physically, the ink should exhibit gel-like
characteristics or be sufficiently viscous to be dispensed as a free
standing filament. However, if the gel is too strong, large shear
forces required to eject the ink can result in cell death and gel
45
fracture
6
. Mechanically, the individual printed filaments require
sufficient strength and stiffness to maintain structural integrity
after printing. Lastly, the formulation should not be cytotoxic,
allowing cell adhesion and proliferation. In some cases,
degradation of the scaffold in a controlled manner over time will
50
be appropriate. Hydrogels are polymeric materials commonly
used in tissue engineering due to their low cytotoxicity and
structural similarity to the extracellular matrix (ECM)
7
. The
highly hydrated network structure permits the exchange of gases
and nutrients and makes them an attractive option for the
55
formation of “inks” for bioprinting. Blending of hydrogels
provides an opportunity by which properties specific to each
respective hydrogel component can be combined to tailor the
overall hydrogel towards facilitating specific requirements
8, 9
.
Alginates (Alg) are naturally occurring polysaccharides
60
isolated from brown algae with linear blocks of (1, 4)-linked β-D-
mannuronate (M) acid and α-L-guluronic acid (G) residues
10, 11
.
Gel formation can be achieved through binding of divalent
cations with guluronic residues of the alginate chain,
subsequently forming junctions with adjacent chains creating an
65
egg-box structure
10, 12
. The viscosity of alginate solution depends
on the average molecular weight (MW), molecular weight
distribution, average chain segment ratio (G to M ratio),
concentration of the polymer and the pH of the solution
11, 12
. Due
to the structural similarity of alginate to ECM , these gels are
70
used in cell delivery vehicles
13
, matrices for tissue engineering
14
, drug delivery beads and ECM models for cell experiments
15
.
Gelatin, a denatured type of collagen, has been widely used in
wound dressing, as pro-angiogenic matrices and absorbents pads
for surgical applications
16-18
. At physiological temperature
75
(37ºC), gelatin is a solution, but can reversibly form a gel when
cooled (< 29 ºC). This is due to a conformational change from
coil to helix that leads to chain association and eventually the
formation of a three-dimensional network
2, 19-21
. The viscosity of
an alginate solution and thereby printability, can also be
80
controlled by incorporating gelatin and modulating the mixing

temperature during printing to form a gel that retains biological
aspects of the original alginate solution while satisfying physical
extrusion criteria.
Alginate-gelatin (Alg-Gel) blends have been reported as
potential drug delivery carriers
8, 9
, enzyme immobilisation beads 5
22
, wound dressing fibres
23
, and sponges for tissue matrices
24
.
Among the studies related to bioprinting, Yan and co-workers
25-
27
have attempted to print 3D scaffolds from alginate-gelatin
blends. Here, we elaborate this approach with particular attention
paid to the ink properties required for effective printing with
10
respect to both the delivery and integrity of structure formed.
Interestingly, there have been limited studies aimed at
understanding the specific ink properties suitable for extrusion
printing. This study establishes a systematic approach to
characterise the specific requirements needed to print a 3D
15
scaffold successfully for TE. The printability of ink formulations
was assessed by comparing a viscous solution, semi-crosslinked
gel and hybrid hydrogels. Alginate was selected as the major
component of the ink formulations used in this work due to its
potential in biomedical applications, and its versatility in
20
generating a range of possible inks by ionically crosslinking it or
blending with another component. The techniques examined here
provide the criteria and tools by which the printability of a
hydrogel-based ink can be evaluated. In addition, the mechanical
properties and cell compatibility of the optimum ink formation
25
will be investigated.
2. Materials and methods
2.1 Materials
Alginic acid sodium salt (MW ~ 50,000 Da, M/G ratio of 1.67, 30
viscosity of 100-300 cP for 2 wt% solution, 25
o
C) and gelatin
(MW ~ 50,000 - 100,000 Da, type A from bovine skin) were
obtained from Sigma-Aldrich Pty Ltd. Other reagents were all
analytical grade and used as received.
35
2.2 ‘Ink’ preparation
To prepare the ink solution, three different concentrations of
sodium alginate solution (1, 2 and 4% w/v) were prepared in
phosphate buffered saline (PBS, pH 7.4) and blended with 10%
w/v gelatin solution (4 parts alginate solution: 1 part gelatin
40
solution, kept constant for all blended samples). The ink solution
was mixed using vortex and centrifuged for 1 min (1000 rpm) to
remove air bubbles. The ink solution was then transferred into
syringe barrels or appropriate moulds for characterisation and
cooled on ice for 15 mins. Ink solutions comprising alginate at 1,
45
2 or 4% blended with gelatin were labelled as 1% Alg-Gel, 2%
Alg-Gel and 4% Alg-Gel respectively, while alginate solutions
without added gelatin were labelled as 1% Alg, 2% Alg and 4%
Alg respectively. For comparison, an alginate solution (4% w/v)
pre-mixed with calcium chloride (0.2% v/v) was also prepared
50
and labelled 4% Alg +Ca
2+
.
2.3 Fabrication of scaffolds
Samples were extrusion printed using a custom modified 55
computer numerical control (CNC) milling machine (Sherline
Products, CA). The system was equipped with a three-axis
positioning platform and designed using EMC2 software
(LinuxCNC). An attachment for syringe deposition was built and
connected to a controllable gas flow regulator (1-100 psi). The
60
regulator was controlled using a Pololu SciLabs USB-to-serial
microcontroller and with an in-house software interface. The ink
solution was loaded into a disposable syringe, kept at 5ºC and
(Nordson EFD) fitted with a 200 µm diameter nozzle. Three
layers of the ink solution were extruded onto a glass slide at a
65
feed rate of 100 mm min
-1
, with strands spacing of 1 mm, to a
final size of 10 mm × 10 mm. The gas pressures used for
extruding the various ink solutions were selected to produce the
most reproducible and defined structure and were 4, 8 and 9.5 Psi
for 1%, 2% and 4% Alg-Gel respectively. 4% Alg+Ca
2+
was 70
printed at RT (25ºC, 2 Psi). Samples required for further
characterization were ionically crosslinked in 2% w/v CaCl
2
for
10 mins. The macroscopic structure of extruded scaffolds was
imaged using Leica M205A optical microscope (Leica
Microsystems, Germany).
75
2.4 Ink consistency measurement
The consistency of the ink solutions were measured using the
method described by Cohen et al.
31
. The method was based on
80
measuring the variations in extrusion force during deposition of
ink in real time. Ink solutions were loaded into a syringe with the
plunger connected to the upper clamp of an EZ-S mechanical
tester (Shimadzu, Japan). The measurements were performed in
compression mode while the nozzle end of the syringe (200µm in
85
diameter) was held perpendicularly in position by a plastic rack.
A 10 N load cell was used and the testing was carried out by
applying a constant strain at 0.2 mm s
-1
and recording the force
over time (see Supporting Information). Distilled water was used
as a control for the experiment and regions where the force
90
showed consistent fluctuations over 300 s was used.
2.5 Rheology
The rheological behaviour of ink solutions was analysed using an
95
AR-G2 rheometer (TA Instruments, DE) equipped with a Peltier
plate thermal controller. A 2 º/40 mm cone and plate geometry
was used in all measurements (see Supporting Information). The
solutions were allowed to reach the equilibrium temperature for 1
min prior to performing the experiments. Storage modulus (G')
100
and loss modulus (G'') were measured as a function of
temperature and frequency by varying, respectively, temperature
(at a constant frequency) and frequency (at a constant
temperature). Temperature sweep experiments were conducted at
a rate of 6 ºC min
-1
from 50 ºC to 5 ºC, at a fixed strain and
105
frequency of 1% and 1 Hz respectively. Frequency sweep
experiments (5 ºC for Alg-Gel, 25 ºC for all Alg) were conducted
at a fixed strain of 1% from 0.01 to 10 Hz. A temperature of 5ºC
was selected for conducting experiments on Alg-Gel to ensure the
ink maintains a gel-like structure.
110

2.6 Mechanical properties
The modulus of alginate and alginate-gelatin samples was
determined using both compression and indentation tests. Ink
5
solutions were casted in custom-made moulds (compression:
cylindrical, 10 mm ID, 4 mm in thickness; indentation: square, 10
mm × 10 mm × 2mm). The samples were crosslinked in 2% w/v
CaCl
2
for 10 mins, washed and equilibrated in Dulbecco’s
modified eagle medium (DMEM, Sigma Aldrich) supplemented
10
with 10% foetal bovine serum and 1% penicillin/streptomycin
(P/S) for 30 mins to remove the excess calcium ions. For
compression testing, the diameter and thickness of each sample
was measured using a digital micrometer. Each sample was tested
at a strain rate of 2 mm min
-1
using the EZ-S mechanical tester 15
fitted with a 10 N load cell (see Supporting Information). The
initial linear slope of stress-strain curve was used to calculate the
compression modulus (E
comp
). At least three different samples
were used for each composition and the average values are
reported.
20
A flat stainless steel indenter (1 mm in diameter) was used
along with a 2 N load cell to perform the indentation test at a rate
of 0.1 mm min
-1
. At least three different samples were used for
indentation testing and the test was carried out on a minimum of
4 different points for each sample. The indentation modulus (E
ind
)
25
was calculated from the recorded force and the indenter
displacement. The applied force () can be related to the
indentation depth () by taking in account the reduced modulus
(

) and the indenter geometry (radius ):
30
 = 2

 (1)
where the reduced modulus in Equation 1 can be expressed as a
function of the indenter modulus (

) and the substrate modulus
(

)
32
:
35
(

)

=
(
1  


)



+
(
1  


)



(2)
here, 

and 

are the Poisson’s ratios of the indenter and the
substrate, respectively.
(
1  


)



of Equation 2 becomes
negligible when indenter is much stiffer than the substrate. For
40
swollen hydrogels 

is taken as 0.5, and Equations 1 and 2
become:
~
(
8 3
 )


 (3)
Comparisons between Alg and Alg-Gel were made using a two-
45
way analysis of variance (ANOVA). A p-value <0.05 was used to
indicate a significant difference.
2.7 Degradation analysis
50
Degradation study was undertaken by monitoring the loss in
material strength of the samples in cell culture medium at 37ºC
(Dulbecco’s Modified Eagle Medium supplement foetal bovine
serum (10%) and penicillin/streptomycin (1%)) using
compression and indentation tests. Materials for degradation
55
studies were cast and the experiment conducted as previously
described in Section 2.6. Measurements were taken after 1, 4, 7
and 14 days incubated in cell culture media with fresh media
being replenished at every second day. At every time point for
measurement, samples were tested by indentation first followed
60
by compression testing.
2.8 Cell compatibility
In order to facilitate efficient cell attachment and proliferation
within the alginate-based scaffolds, the peptide sequence GRGDS 65
(Auspep) was covalently linked to the alginate using aqueous
carbodiimide chemistry under sterile conditions
30-32
. Briefly,
sodium alginate was dissolved in MES buffer (0.1 M, 0.3 M
NaCl, pH=6.5). 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC, Sigma-Aldrich) and N-hydroxysulfosuccinimide (sulfo-
70
NHS, Sigma-Aldrich) were added to activate 5% of the
carboxylic acid groups of alginate. The solution was stirred for 15
mins followed by addition of peptide where the reaction was
allowed to proceed for 24 hrs. The product was then dialysed for
4 days, lyophilised and stored at -80ºC. The grafting procedure
75
was conducted accordingly to the study by Rowley et al
28
, where
they have optimized the chemistry with reaction efficiency
reaching up to 80%. Based on values reported by Rowley et al.
28
and the alginate molecular weight (given by the manufacturer),
the average number of peptide grafted per alginate chain was
80
calculated to be 8 grafts/chains.
Primary cells used to conduct biological assays in this study
were derived from two week-old C57BL10/J back-crossed
C57BL6-(GTRosa) mice. After euthanasia by cervical
dislocation, the muscles were removed from the mice and
85
macerated with sharp scissors in Hams F10 media devoid of
serum. Primary myoblasts were then cultured in Hams F10 media
supplemented with foetal bovine serum (20%), bFGF (2.5ng ml
-
1
), ʟ -glutamine (2mM) and penicillin/streptomycin (1%, P/S) as
described elsewhere
33
. A 2% Alg-Gel ink solution was prepared
90
as described previously (Section 2.2) under sterile conditions.
Briefly, alginate-GRGDS was mixed with appropriate amounts of
gelatin and BL6 primary myoblast at a cell density of 5 × 10
5
cells ml
-1
. The ink solutions were printed onto glass slides at 3
different pressures denoted as P1 (8 psi), P2 (16 psi) and P3 (24
95
psi) and crosslinked in 2% w/v CaCl
2
for 10 mins. To determine
the viability of printed cells, samples were stained with calcein
AM and propidium iodide (PI). In brief, samples were incubated
with calcein AM for 10 mins in dark, washed with cell culture
media and stained with PI for 2 mins. Samples were imaged using
100
a Leica DM IL fluorescent microscope (Leica Microsystems,
Germany) and analysed using Image J software. The viability of
printed samples was tested 1 hr and 48 hr after printing. Results
are presented as mean ± standard deviation. Differences between
groups were analysed using Tukey's method. A p-value <0.05
105
was used to indicate a significant difference.
3. Results

Frequently asked questions

Q1. What are the contributions mentioned in the paper "Bio-ink properties and printability for extrusion printing living cells" ?

In this study, alginate ( Alg ) was selected as the major component of the ‘ bio-ink ’ formulations for extrusion printing of cells. The viability of primary myoblasts delivered as a myoblast/Alg-Gel bio-ink was not affected by the printing process, indicating that the Alg-Gel matrix provides a potential means to print 3D constructs that may find application in myoregenerative applications. Decay in mechanical properties over time suggests that Alg-Gel slowly degrades in cell culture media with more than 60 % decrease in initial modulus over 7 days.