Viscoll collagen solution as a novel bioink for direct 3D bioprinting

Abstract

Collagen is one of the most promising materials for 3D bioprinting because of its distinguished biocompatibility. Cell-laden constructs made of pure collagen with or without incorporated growth supplements support engineered constructs persistence in culture and are perfectly suitable for grafting. The limiting factor for direct 3D collagen printing was poor printability of collagen solutions, especially admixed with cells or tissue spheroids. In our study, we showed that concentrated solutions of native collagen branded Viscoll were effective as bioinks with high fidelity performance. Viscoll containing 20, 30, or 40 mg/ml collagen were used for direct extrusion 3D bioprinting to form scaffolds appropriate to support spatial arrangement of tissue spheroids into rigid patterns with resolution of 0.5 mm in details. Incorporated cells demonstrated sufficient viability. Associated rheological study showed that good printability of the collagen solutions correlates with their increased storage modulus value, notably exceeding the loss modulus value. The proper combination of these physical parameters could become technological criteria for manufacturing various collagen bioinks for 3D bioprinting.

Full text

Journal of Materials Science: Materials in Medicine (2019) 30:31
https://doi.org/10.1007/s10856-019-6233-y
S.I.: BIOFABRICATION AND BIOINKS FOR TISSUE ENGINEERING
Original Research
Viscoll collagen solution as a novel bioink for direct 3D bioprinting
Egor O. Osidak1,2 ●Pavel A. Karalkin3,4 ●Maria S. Osidak1●Vladislav A. Parfenov3●Dmitriy E. Sivogrivov1●
Frederico D. A. S. Pereira3●Anna A. Gryadunova3,5 ●Elizaveta V. Koudan3●Yusef D. Khesuani3●
Vladimir A. Кasyanov6●Sergei I. Belousov7●Sergey V. Krasheninnikov7●Timofei E. Grigoriev7●Sergey N. Chvalun7●
Elena A. Bulanova3●Vladimir A. Mironov3,5 ●Sergey P. Domogatsky1,8
Received: 14 April 2018 / Accepted: 4 February 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Collagen is one of the most promising materials for 3D bioprinting because of its distinguished biocompatibility. Cell-laden
constructs made of pure collagen with or without incorporated growth supplements support engineered constructs
persistence in culture and are perfectly suitable for grafting. The limiting factor for direct 3D collagen printing was poor
printability of collagen solutions, especially admixed with cells or tissue spheroids. In our study, we showed that
concentrated solutions of native collagen branded Viscoll were effective as bioinks with high fidelity performance. Viscoll
containing 20, 30, or 40 mg/ml collagen were used for direct extrusion 3D bioprinting to form scaffolds appropriate to
support spatial arrangement of tissue spheroids into rigid patterns with resolution of 0.5 mm in details. Incorporated cells
demonstrated sufficient viability. Associated rheological study showed that good printability of the collagen solutions
correlates with their increased storage modulus value, notably exceeding the loss modulus value. The proper combination of
these physical parameters could become technological criteria for manufacturing various collagen bioinks for 3D
bioprinting.
These authors contributed equally: Egor O. Osidak, Pavel A. Karalkin
*Egor O. Osidak
egorosidak@gmail.com
1Imtek Ltd., 3rd Cherepkovskaya 15A, Moscow, Russia
2Gamaleya Research Institute of Epidemiology and Microbiology
Federal State Budgetary Institution, Ministry of Health of the
Russian Federation, Gamalei 18, Moscow, Russia
3Laboratory of Biotechnological Research, 3D Bioprinting
Solutions, Kashirskoe Roadway, 68/2, Moscow, Russia
4P. A. Hertsen Moscow Oncology Research Center-branch of FSBI
NMRRC of the Ministry of Health of Russia, 3 2nd Botkinsky
drive, Moscow, Russia
5Institute for Regenerative Medicine, Ministry of Health of the
Russian Federation, Sechenov University, Trubetskaya 8/2,
Moscow, Russia
6Riga Stradins University and Riga Technical University,
Riga 1007, Latvia
7National Research Center «Kurchatov Institute», Akademika
Kurchatova pl.,1, Moscow, Russia
8Russian Cardiology Research and Production Center Federal State
Budgetary Institution, Ministry of Health of the Russian
Federation, 3 Cherepkovskaya 15A, Moscow, Russia
Supplementary information The online version of this article (https://
doi.org/10.1007/s10856-019-6233-y) contains supplementary
material, which is available to authorized users.
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Graphical Abstract
1 Introduction
Modern methods of tissue engineering and regenerative
medicine enable the generation of human tissue in vitro by
using stem cells or differentiated ones [1,2]. To support cell
attachment, proliferation and differentiation the appropriate
hydrogel should be used to create bioengineered constructs.
The cells distribution throughout the scaffold is assumed to
be precise, supporting their spatial arrangement and pro-
viding their survival. Routinely, this is achieved by admix-
ing cells into a natural hydrogel made from extracellular
matrix components. The matrix provides appropriate
mechanical support, ensures sufficient permeability for
macromolecules, promoting the exchange of metabolites for
culturing cells. Various technologies [3–5] were developed
to create complex engineered constructs aimed for in vivo
implantation. 3D bioprinting displays the highest potential to
create heterogenic structures with complicated geometry and
specific properties [6–8]. Ultimate precision and fidelity of
3D bioprinting generally depend on printability of polymer
solutions used as bioinks. From a wide range of products
used for biofabrication, the most suitable one is collagen–the
major natural component of extracellular matrix (ECM).
Collagen hydrogels had shown their excellent biocompat-
ibility. Collagen-based structures retain full integrity during
the engraftment. Immunologic reactions to foreign collagen
are negligible on conditions of thorough depletion of
immunogenic admixtures. All this explains the broad
application of collagen for biofabrication of various struc-
tures [9–12]. However, pure collagen hydrogels showed
relatively weak mechanical properties compared to polymer-
based gels, stabilized by crosslinking. This drawback sub-
stantially hampers collagen application in pure form in 3D
bioprinting [12]. The majority of modern collagen-based
bioinks were developed from aqueous collagen solutions
with concentration below 5 mg/ml. They are not suitable for
direct bioprinting without additional stabilization by che-
mical crosslinking [10,13,14]. It should be noted that
chemical modification of native collagen molecules by
cross-linking agent could decrease biocompatibility and
increase antigenicity of the material [15–17].
Optimization of collagen solutions makes them suitable
for 3D bioprinting. Collagen hydrogel must be sufficiently
tough, supporting the sharp form after polymerization of the
solution extruded from thin needles or nozzles. This is
possible with concentrated collagen solutions [18]. More-
over, a high purity and sterility of the collagen material
must be also ensured. This is technically achievable with
completely soluble collagen, not for a suspension of
aggregated collagen molecules.
In our previous study, pure and sterile native collagen
has been prepared and used for bladder lesions substitution
[19] and for the improvement of biomechanical properties
of the artificial cornea [20]. Consequently, the homo-
geneous aqueous solutions of concentrated collagen type I
named Viscoll were developed. Due to their prominent
viscoelastic properties, Viscoll solutions serve as carriers
for growth factors. The controlled release of growth factors
from the hydrogel formed from injected Viscoll was
effectively shown in the model of cryptorchism con-
sequences treatment in rats [21]. Viscoll was also used as a
template for injection of stem cells into the damaged spinal
cord in the model of the spinal trauma [22].
Tissue spheroids (TS) are gaining extensively their place
in tissue engineering due to supreme cell organization
within these micro-aggregates and the intrinsic property for
self-assembly. Cells within TS are in a physiologically
relevant environment to mimic in vivo behavior with
appropriate cell–cell junctions, secreted and deposited
ECM, as well as with proper polarity exposure [23–25].
Over the last decades, it became obvious that the use of TS
as building blocks is a promising strategy for biofabrication
of human organ constructs [23]. TS represent living mate-
rial with certain measurable, evolving and controllable
composition, as well as mechanical and biological proper-
ties. Being closely placed together, TS undergo tissue
fusion, a process that represents a fundamental biological
and biophysical principle of developmental biology,
namely, the directed tissue self-assembly. The application
of TS for 3D bioprinting increases its speed and efficiency
31 Page 2 of 12 Journal of Materials Science: Materials in Medicine (2019) 30:31

due to easier and more rapid inclusion of bulk number of
cells into the hydrogel, while the standardized uniform TS
geometry opens the way to automation. On the top of that,
the maturation of the engineered tissue construct biofabri-
cated from TS with well-defined architecture and pre-
deposited ECM will be accelerated.
In our study, systematic evaluation of collagen con-
centration impact at Viscoll properties was performed in
order to produce bioink supporting biofabrication of tissue
constructs with considerable printing fidelity. Additionally,
the novel bioinks were studied by oscillatory shear rheology
assay to determine their gelation kinetics. Structural repro-
ducibility of various 3D printed constructs and their
mechanical properties have been characterized as well.
Finally, the cell viability in Viscoll collagen bioprinted
scaffolds was estimated.
2 Materials and methods
2.1 Preparation of high concentrated collagen inks
for bioprinting
Collagen Viscoll represents a smoothly soluble collagen
fraction sterilized by filtration and obtained by acidic extrac-
tion from animal tendons, followed by purification with dif-
ferent salt precipitation and ion-exchange chromatography.
Lyophilized sterile collagen Viscoll from porcine tendons
(Viscoll, Imtek Ltd., Russia) was reconstituted in 20 mM
acetic acid to a stock concentrations 40, 60 and 80 mg/ml and
packed in individual syringes with a luer-lock connection. The
dilution and neutralization of collagen solutions were made at
+4℃as follows: (1) A syringe with the culture medium
(DMEM, 10% fetal bovine serum, 100 mM Tris-HCl) was
hermetically coupled with a syringe containing equal volume
of Viscoll collagen. (2) The collagen solution was carefully
mixed with the culture medium, resulting in neutralized
homogenous 20, 30, and 40 mg/ml collagen solution (Fig. 1,
Supl. 1). For bioprinting of cell-laden 3D constructs, the
neutralized Viscoll collagen solution was mixed just before
use with the culture medium containing the cells in 9:1 ratio
according to the method mentioned. The resulting cell density
in the collagen solutions was 0.5 × 106ml−1.
2.2 Viscoll collagen characterization
2.2.1 Rheological properties
Rheological measurements were performed under oscilla-
tory shear mode using a rotational Anton-Paar MCR 501
rheometer. Samples of neutralized collagen Viscoll solution
were placed between two parallel plates of 25 mm in dia-
meter and 0.53 mm gap. After cooling to +4℃the edges
were covered with a mineral oil to prevent dehydration. The
mechanical spectra and viscosity measurements were made
at a constant strain of 5% in the frequency range of 0.1–10
rad s−1at +4℃. The temperature dependence of storage
modulus (G’) and loss modulus (G’’) were measured using
a temperature sweep (oscillation) by increasing the tem-
perature from +4to+37 ℃at the heating rate of +5℃min
−1The frequency and shear strain were maintained constant
at 10 rad s−1and 1%, respectively. For investigation of the
gelation dynamics, all cooled samples were loaded onto the
cold stage followed by plate fixture lowering into position
and quickly heated to +37 ℃, 0.6 ℃s−1. The gelation time
of Viscoll was determined by measuring G’and G’’ as a
function of time (0–20 min) when G’and G’’ reached
steady state. 14 samples of each collagen concentration
were used for measurement.
2.2.2 Mechanical properties of bioprinted constructs
Mechanical testing was performed using Instron mechanical
universal tester (Model 5965, Instron, Norwood City, USA)
equipped with a 50 N load cell. Vertical compressive stress
force analysis of printed cubes (30 layers, 6 × 6 × 6 mm each)
made from various neutralized Viscoll collagen hydrogels,
was performed at initial shear rate of 8 × 10−3s−1. During the
Fig. 1 Preparation of Viscoll collagen solution as a bioink. a,ba syringe with the culture medium is hermetically coupled with a syringe
containing Viscoll. cThe collagen solution mixed with the culture medium results in neutralized homogenous collagen solution
Journal of Materials Science: Materials in Medicine (2019) 30:31 Page 3 of 12 31

test, the samples were deformed up to 60% of the starting
height. The measurements were performed at room tem-
perature. The Young’s modulus (for 0–10% deformation),
compressive strength, stress at 50% strain were obtained after
analysis of the stress-tension curve. Six cubes of each col-
lagen concentration were used for measurement.
2.2.3 Geometrical fidelity of bioprinted constructs
Geometrical fidelity of the bioprinted constructs was qua-
litatively characterized using a digital caliper (for cubes)
and high-quality image analysis using the imaging software
Image J (for meshes). Six cubes and 9 meshes of each
collagen concentration were used for measurement.
2.3 Bioprinting
Multifunctional bioprinter “Fabion”extrusion type (3D Bio-
printing Sol., Russia) equipped with fluid dispenser LDAV-
HT-BA (Fishman Corporation, USA) was used to prepare
collagen constructs. Digital 3D models for bioprinting were
created with «SolidWorks 2015» software suite (Dassault
Systemes, France) and then converted to CAM format by
SprutCAM 11 (SPRUT-Technology, Russia), which was also
used as a power software for 3D bioprinter. Pure or cell-laden
Viscoll hydrogels were extruded into sterile plastic 60 mm
Petri dishes (Corning, USA) through straight plastic needle
Fisnar 5601280 (Fisnar Inc., Germantown, USA) with inner
orifice diameter 0.25 mm. The temperature of Viscoll collagen
in syringes was maintained 15 °Сduring the bioprinting pro-
cess, while Petri dishes bottom was heated up to 37 °Сto
allow the immediate collagen polymerization. The bioink flow
rate in experiments was 5 mm/min. TS with 500 μm diameter
were patterned into the pores of collagen meshes by robotic
deposition using custom-designed printing head, integrated in
hardware components of Fabion system. This device com-
prises precision “turnstile”-valve microfluidic device, provid-
ing the alternate delivery of single spheroids, suspended in
culture medium through the micro channels and the registra-
tion of each printed spheroid by the sensor [26] (Fig. Supl. 2).
Immediately after the fulfillment of 3D printing, the constructs
were covered with a pre-heated culture medium to support the
collagen polymerization process.
2.4 In vitro cell studies
2.4.1 Cell culture
NIH 3T3 cell line expressing green fluorescent protein (GFP)
was obtained from Evrogen (Russia). Cells were grown in
DMEM (Gibco, USA) containing 10% fetal bovine serum
(Gibco, USA) and supplemented with antibiotic/antimycotic
mix (Gibco, USA), 1 mM L−1glutamine (Paneco, Russia).
The cells were cultured at 37 °C in humidified atmosphere
with 5% CO2 and were splitted at 85–95% confluence.
2.4.2 Tissue Spheroid formation
TS were produced from NIH 3T3 fibroblasts using non-
adhesive scalable technology, as was described previously
[27]. Briefly, NIH 3T3 cells were detached from the substrate
using 0.25% trypsin/0.53 mM EDTA (Gibco, USA) and sus-
pended in the complete growth medium to the concentration
2×10
4cells per milliliter. 100 μl of cell suspension were
dispensed into each well of Corning spheroid microplate (cat.#
4520) according to the manufacturing protocol. The spheroid
microplates were then incubated at 37 °C in a humidified
atmosphere with 5% CO2for 3 days. Biofabricated TS with
~500 µm diameter consisted of approximately 2 × 103cells.
2.5 3D cell viability assay
NIH 3T3 cell viability was estimated within one-layer 5 × 5
mm square constructs, printed with cell-laden Viscoll
bioinks 20, 30, and 40 mg/ml. After 1, 3 and 7 days in
culture, cells encapsulated within collagen were stained by
5μg/ml propidium Iodide (PI) solution (Sigma-Aldrich,
Saint Louis, USA) for 15 min at RT. Then, after washing
with PBS, cells were imaged using the fluorescent micro-
scope Eclipse Ti-E (Nikon, Japan) and their amount was
automatically counted using ImageJ 1.48v software (NIH,
Bethesda, MD, USA). The percent of cell viability was
calculated as a ratio of live green (GFP fluorescence) and
dead red (PI fluorescence) cells. The content of living cells
counted immediately after printing and polymerization of
cell-laden collagen constructs represented 100% viability.
The viability in constructs printed applying each testing
concentration of Viscoll bioink was estimated in triplicates.
2.6 Statistical analysis
Unpaired t-tests were applied when two groups were
compared, while ANOVA followed by a post hoc test was
performed when more than two groups were compared.
The statistical data was analyzed using GraphPad Prism
software (GraphPad Software, Inc., La Jolla, CA) and
represented as mean ± S.E.M. Statistical significance was
determined at p<0.05.
3 Results
3.1 Rheological and mechanical properties
In order to determine the biomechanical suitability of the
Viscoll collagen solution as a bioink for 3D bioprinting, the
31 Page 4 of 12 Journal of Materials Science: Materials in Medicine (2019) 30:31

systematic evaluation of the collagen concentration, tem-
perature and gelation time was performed.
The mechanical spectra analyses were performed at +4
℃in the frequency range of 0.1–10 rad s−1(Fig. 2a). The
frequency increase led to a significant monotonous aug-
mentation of G’and G’’ for all variants of Viscoll bioinks
(Fig. 2b). Next temperature sweep (oscillation) was con-
ducted by Viscoll collagen solution heating from 4 ℃to 37
℃at heating rate of +5℃min−1(Fig. 2d). The heating of
samples led to a slight decrease of G’and G’’. Nevertheless,
when the temperature was gradually increased to the gela-
tion temperature (25–30 ℃), both G’and G’’ for all col-
lagen concentrations in Viscoll solutions demonstrated a
rapid growth due to the formation of permanent 3D network
with a fixed modulus. For G’and G’’ studies, precooled
samples were put on the plate with temperature 4℃, then
the plate was rapidly heated to 37 ℃. It was shown that the
heating of samples from 4 ℃to the gelation temperature led
to reduction of G’from 1790 ± 235 Pa to 1270 ± 138 Pa,
from 1120 ± 95 Pa to 827 ± 41 Pa, from 724 ± 36 Pa to 497
± 13 Pa and G’’ from 502 ± 50 Pa to 416 ± 29 Pa, from 289
± 32 Pa to 255 ± 20 Pa, from 176 ± 9 Pa to 162 ± 8 Pa for
4%, 3 and 2% Viscoll solution respectively. After that point
G’and G’’ increased drastically for all concentration and
became constant. It is worth to note, that the final G’values
reached 4720±280 Pa, 4210 ± 295 Pa and 3890±156 Pa for
Viscoll solution at concentrations of 4%, 3 and 2%,
respectively (Fig. 2c). At the same time, the corresponding
final G’’ values changed to 1280 ± 102 Pa, 1080 ± 43 Pa and
955 ± 48 Pa, respectively. Thus, the results showed the
ability of Viscoll solution to maintain the predesigned shape
once being extruded from the needle, avoiding immediate
deformation and allowing subsequent polymerization at
+37 ℃.
The mechanical properties of the bioprinted 6 × 6 × 6 mm
cubes consisting of 30 layers were analyzed by comparing
Fig. 2 Characterization of Viscoll collagen solution. aMechanical
spectra of Viscoll bioinks at +4℃.bViscosity as function of angular
frequency at +4℃.cG’and G”value as function of time at +37 ℃.d
G’and G”value as function of temperature. Date are represented by
mean for n=14 samples
Journal of Materials Science: Materials in Medicine (2019) 30:31 Page 5 of 12 31

the compressive modulus of the constructs made applying
neutralized Viscoll collagen hydrogels of different con-
centration, 40, 30, 20 and 15 mg/ml. The structural fidelity
was confirmed (Fig. 3). For biofabricated constructs the
compression measurements illustrated that Young’s mod-
ulus improved from 7.2 ± 0.6 kPa to 8.2 ± 0.9 kPa, 9.5 ± 0.4
kPa and 21.5 ± 1.4 kPa when concentration of the collagen
increased from 15 mg/ml to 20, 30, and 40 mg/ml, respec-
tively. Table 1summarizes some mechanical characteristics
of bioprinted 3D constructs.
3.2 Geometrical fidelity of 3D constructs
The capability of the Viscoll bioinks to produce 3D con-
structs is essential to achieve its functionality. First, 10 layer
30 × 30 × 2 mm meshes using 2%, 3 and 4% Viscoll bioinks
were successfully bioprinted (Fig. 4a–c; Supl. Movies).
Analysis of the wall thickness and pore size confirmed that
the 3D model (wall thickness 0.5 mm and pore size 1.0 mm)
was comparable to 3D printed 10 layers meshes, however it
should be noted that wall thickness increased and as a
consequence the pore size decreased. Analysis comparing
the 3D model in planexy (Fig. 4d–f) with the 3D printed
meshes showed good fidelity. Data obtained showed that
mean percent difference in surface area was 19%, 16 and
12% for 20, 30 and 40 mg/ml Viscoll bioink respectively.
Dimension of 3D printed cubes (Fig. 4i) were measured
with digital calipers and used to calculate the bulk volume.
For cubes printed applying 15 mg/ml Viscoll bioink the
mean (M) and standard deviation (SD) in sidexwas 7.10 ±
0.20 mm, in sideywas 6.90 ± 0.12 mm, in sidezwas 6.32 ±
0.20 mm. For cubes printed with 20, 30 and 40 mg/ml
Viscoll bioink M and SD in sidexwas 6.30 ± 0.30 mm, 6.30
± 0.18 and 6.29 ± 0.09 mm, in sideywas 6.50 ± 0.18 mm,
6.42 ± 0.18 and 6.41 ± 0.06 mm, in sidezwas 6.59 ± 0.40
mm, 6.43 ± 0.90 and 6.30 ± 0.60 mm respectively. A com-
parison with digital calculations of bulk volume revealed
that cubes applying 15 mg/ml Viscoll bioink were generated
with about 70% structural fidelity, 20, 30 and 40 mg/ml
Viscoll bioink–with 80%, 83 and 85% fidelity respectively.
Fig. 3 Mechanical properties of
bioprinted constructs. a,b
Deformation of 3D bioprinted
cubes by vertical compressive
stress. cCharacteristic stress-
tension curves of various 3D
bioprinted cubes
31 Page 6 of 12 Journal of Materials Science: Materials in Medicine (2019) 30:31

Table 1 Mechanical properties
of the 3D constructs printed
using various Viscoll collagen
bioinks
Collagen
concentration
Young’s
modulus
(kPa)
Maximum
tension force
(kPa)
Deformation at
brake (%)
Stress at 50%
deformation
(kPa)
Residual
deformation (%)
1,5 % 7,2 ± 0,6 41,6 ± 3,1 54,9 ± 2,2 34,3 ± 0,9 –
2 % 8,2 ± 0,9 68,7 ± 3,4 57,3 ± 0,6 45,6 ± 1,0 –
3 % 9,5 ± 0,4 ––33,8 ± 3,6 22 ± 4
4 % 21,5 ± 1,4 ––75,2 ± 9,4 17 ± 4
Fig. 4 Direct bioprinting of 3D constructs using Viscoll collagen
solution. Ten layer meshes bioprinted with a,dViscoll, 20 mg/ml; b,e
Viscoll, 30 mg/ml, c,fViscoll, 40 mg/ml. Dashed lines indicates
dimensions and locations of the wall of meshes in CAD. CAD model
of mesh (g) and cube (h). icube printed with Viscoll, 3%
Journal of Materials Science: Materials in Medicine (2019) 30:31 Page 7 of 12 31

3.3 Cell culture
3.3.1 Cell Viability inside the bioprinted constructs
NIH 3T3 cells were mixed within the 2%, 3 and 4% Viscoll
collagen bioinks and printed using the 27G cone-shaped
nozzle thus producing one-layer 5 × 5 mm constructs. Cell
growth within the hydrogel was analyzed on days 1, 3 and
7. Figures 5a–6c show the cell-laden constructs after 3 days
in culture, live cells expressing GFP (green,) and dead cells
stained with PI (red). The cells survived after extrusion
through the nozzle during the bioprinting, as it was shown
in Fig. 5d and maintained their viability within the collagen
constructs for at least 1 week in vitro. The viability of NIH
3T3 cells inside the bioprinted constructs on 7th day in
culture was 97.2% ± 1.2%, 95.2% ± 1.3% and 87.2% ±
2.1% for Viscoll bioinks at 2%, 3 and 4% collagen con-
centrations respectively. A noticeable increase of spread
Fig. 5 The effect of bioprinting process on cell viability. a,b,cCell
viability within printed constructs at 7th day of culture for 2%, 3%, 4%
collagen hydrogels, respectively; dQuantification of viability; e,fcell
spreading within 3% collagen hydrogel mesh at 3rd day in culture.
Scale bars 500 µm e, 200 µm f.g,h,i3D bioprinting withTS.
Spheroids fabricated from NIH 3T3 GFP+cells were patterned using
robotic distribution into the pores of 3D constructs printed with 3%
collagen-based hydrogel. Scale bars 500 µm h,i
31 Page 8 of 12 Journal of Materials Science: Materials in Medicine (2019) 30:31

cells was observed on 3rd day for all variants of Viscoll
bioinks applied (Fig. 5e, f). Additionally, observed
cell adhesion and proliferation in printed Viscoll meshes
confirmed good biocompatibility of the collagen bioink
(Fig. Supl. 3A, B). Cell migration was more intense within
constructs printed applying less concentrated Viscoll (Fig.
Supl. 3C).
3.3.2 Tissue Spheroids bioprinting
In our study, TS were patterned by extrusion 3D bioprinter
Fabion equipped with a special printing head adopted for
delivery of single TS at a time into the collagen meshes
according digital CAM model. Fluorescence microscopy
revealed the precise distribution of TS (Fig. 5g–i) in the
mesh pores. TS viability was not affected during the
deposition procedure. All TS were attached to the walls of
printed meshes and were fully visible after 7 days of
observation. A small portion of the cells migrated into the
collagen matrix (data not shown).
4 Discussion
Collagen is a promising material for application in tissue
engineering [28–30]. The main obstacle in collagen solution
application as a bioink is rather long gelation time and low
mechanical strength of the formed gels [31]. Collagen
should be crosslinked rapidly after deposition to achieve
favorable shape fidelity [14,32]. But the crosslinking is not
approvable for tissue engineering since it might cause graft
rejection over time [33]. Additionally, crosslinking of the
collagen matrix impairs survival of embedded cells [34,35].
Another strategy to overcome these limitations is increasing
collagen concentration. The proof of this concept was made
by Rhee et al. [18], but they failed to print constructs
applying 20 mg/ml collagen. They found that at this con-
certation range the collagen gel polymerized in the
deposition tool and only the liquid from the gel was
extruded. The goal of this work was to develop a 3D bio-
printing method using collagen solution with concentration
more than 20 mg/ml. Our study showed that maintaining
temperature from 4 °C to 15 °C for Viscoll collagen solution
with concertation more than 20 mg/ml during the printing
process could overcome problems mentioned by Rhee et al.
[18]. The resulting compressive moduli of the bioprinted
constructs was 7.2 ± 0.6 kPa for 1.5% collagen gel and 21.5
± 1.4 kPa for 4% collagen gel, respectively. This result
correlates with the published data where increased collagen
concentration in the hydrogel made it significantly stronger
[18]. Constructs with such a stiffness (8–20 kPa) could be
applied for engineering of the liver tissue (2–6kPa) [36],
the arterial wall (2–15 kPa) [37], the heart or muscle tissue
(8–20 kPa) [38–40] and the chondron (20–30 kPa) [41].
Our rheological studies revealed that Viscoll collagen
solution exhibits shear-thinning properties. Viscosity
diminished for all the samples at higher shear rates. Though,
viscosity increased concomitantly with elevated collagen
concentration. Collagen concentration had direct impact
both on storage modulus (G’) and on loss modulus (G’’).
The data were consistent with previously published results
on collagen solutions behavior with concentration less than
15 mg/ml [42]. The temperature of the collagen bioink
during printing is critical for collagen bioprinting. Poly-
merization of Viscoll collagen solution started at 22–25 °C
if samples were gradually heated from 4 °C to 37 °C. Rapid
increase in storage modulus G’and loss modulus G’’
accompanied temperature raise before gelation. This
observation was valid for 2%, 3 and 4% collagen con-
centrations. Our finding was applied for optimization of
printing process. Increasing the bionk initial temperature
from 4 °C to 15 °C before printing led to faster gelation and
improved fidelity (data no shown). Value of storage mod-
ulus G’significantly prevailed over G’’ loss modulus before
and after polymerization for Viscoll solution with collagen
concentration exceeding 20 mg/ml. In this way, con-
centrated collagen solutions differ from the previously
described collagen solutions having G’lower than G’’ [42,
43]. Also, Lai et al. [42] investigated dilute collagen solu-
tions (below 15 mg/ml) and found frequency and tempera-
ture dependent variations of G’and G”at approximately
equal values. They have found a shear-thinning behavior for
all collagen solutions studied. To all appearances only
collagen solutions with G’much more than G’’ are suitable
Fig. 6 Scheme of three possible variants of using Viscoll collagen solution as a bioink in 3D bioprinting technology. acollagen hydrogel loaded
with cells. bcollagen hydrogel with TS inserted into the pores. ccollagen hydrogel loaded with cells and TS inserted into pores
Journal of Materials Science: Materials in Medicine (2019) 30:31 Page 9 of 12 31

for direct extrusion bioprinting without applying any other
addition methods such as UV-activated riboflavin cross-
linking etc. This suggestion is supported by the previous
finding correlating improved printability of collagen bioink
with increased G’before extrusion [14]. Viscoll collagen
solution possesses characteristics of solid hydrogels before
polymerization though retaining the fluidity at the same
time. That is the rationale why all the preparations of Vis-
coll collagen solution tested were extruded smoothly
through the bioprinting nozzle.
Printing fidelity of non-rigid constructs made of various
hydrogels by means of 3D bioprinting could be estimated
using different criteria, sometimes rather sophisticated [44–
46]. In our study, percent of fidelity was calculated by
relating the average height of the printed 30 layers cubes
measured directly to the dimension preset in the printing
program. It was found that 80–85% printing accuracy was
achieved using Viscoll collagen concentrations from 20 to
40 mg/ml. Reducing collagen concentration to 15 mg/ml
impaired final geometric fidelity to 70%. These results
correspond well to the fidelity evaluation method using a
3D scanner [18].
Cell viability and performance differs significantly in
dense collagen matrices. In our work, the viability of NIH
3T3 cells, printed into the Viscoll collagen constructs with
protein concentrations 20, 30 and 40 mg/ml, were evaluated
at 1, 3 and 7 days after 3D bioprinting. The cell survival rate
was more than 90% for 2 and 3% collagen and did not drop
down during the observation period. The small decrease of
viability to 87 ± 2.1% was found for 4% collagen only.
The application of prefabricated TS instead or together
with single cells printing is a promising rapidly advancing
strategy in tissue engineering. The main advantage of the
cell aggregates utilization is the delivery of thousands of
cells in one stroke during the bioprinting. TS display a
complex cell and matrix composition which enables phy-
siological environment for cells within the structures and
facilitates further maturation of produced tissue upon self-
assembly. Natural scaffolds provide a vigorous temporal
support to enable TS maturation and further effective
fusion. Stuart William’s group developed a method to
produce stromal-vascular fraction cell-laden spheroids with
collagen type I as a gel forming biomatrix [47]. Collagen I
promoted cells survival and functionality thus producing
pre-vascularized cell aggregates suitable for immediate
transplantation and therapeutic application. Combining TS
with scaffolds, enabling the fusion of neighboring spheroids
can be found in the literature throughout the recent years
[48,49]. The precise positioning of TS within the hydrogels
remains tricky because of TS intrinsic capacity to fuse
rapidly then closely placed to each other which causes
needle clogging in course of extrusion printing. In Gutz-
weiler et al, TS were deposited individually in user-defined
patterns onto hydrogels applying an automated drop-on-
demand dispenser setup and further preserved within fibri-
nogen gel enabling initial angiogenic steps, sprouting for-
mation [50]. In our study, we have used the turnstile system
to print single TS a time which was applied already for
biofabrication of vascularized and functional mouse thyroid
gland [26]. In our previous work collagen type I hydrogel
was also used as a temporal construct support.
In our study, the technology for precise tissue spheroids
positioning in the pores of printed collagen mesh was
developed ensuring accurate of aggregates location. The
additive technology applying the combination of bio-
compatible collagen-based hydrogels in a wide concentra-
tion range with patterned distribution of multicellular tissue
spheroids was accomplished by means of highly sophisti-
cated 3D bioprinter (Fig. 6). It gives the possibility for
biofabrication of complex constructs with different mor-
phological and biomechanical parameters. The proposed
biofabrication method using cell-laden collagen hydrogels
and uniformly sized TS seems to be promising for
future applications in regenerative medicine and drug
discovery.
5 Conclusion
In our work we reported a new application of Viscoll col-
lagen as a novel bioink for biofabrication. Improved
mechanics and homogeneity of Viscoll bioink allow to
create constructs with complex geometry, maintaining the
predesigned shape without any molding or chemical/photo
crosslinking. Such structures are definitely suitable for
grafting. Moreover, the biocompatibility of the Viscoll
bioinks could be further improved by incorporating addi-
tional ECM proteins, such as fibrin, fibronectin and growth
factors.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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