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

Authors:
  • Imtek Ltd.
  • Laboratory for Biotechnological Research "3D Bioprinting Solutions"

Abstract and Figures

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.
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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. Osidak1Vladislav A. Parfenov3Dmitriy E. Sivogrivov1
Frederico D. A. S. Pereira3Anna A. Gryadunova3,5 Elizaveta V. Koudan3Yusef D. Khesuani3
Vladimir A. Кasyanov6Sergei I. Belousov7Sergey V. Krasheninnikov7Timofei E. Grigoriev7Sergey N. Chvalun7
Elena A. Bulanova3Vladimir 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 delity 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 sufcient 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 sufcient permeability for
macromolecules, promoting the exchange of metabolites for
culturing cells. Various technologies [35] 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
specic properties [68]. Ultimate precision and delity 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 collagenthe
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 [912]. 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 modication of native collagen molecules by
cross-linking agent could decrease biocompatibility and
increase antigenicity of the material [1517].
Optimization of collagen solutions makes them suitable
for 3D bioprinting. Collagen hydrogel must be sufciently
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 articial 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 cellcell junctions, secreted and deposited
ECM, as well as with proper polarity exposure [2325].
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 efciency
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-dened 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 delity. 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 ltration and obtained by acidic extrac-
tion from animal tendons, followed by purication 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
+4as 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 × 106ml1.
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 +4the 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 0.110
rad s1at +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 +5min
1The frequency and shear strain were maintained constant
at 10 rad s1and 1%, respectively. For investigation of the
gelation dynamics, all cooled samples were loaded onto the
cold stage followed by plate xture lowering into position
and quickly heated to +37 , 0.6 s1. The gelation time
of Viscoll was determined by measuring Gand G’’ as a
function of time (020 min) when Gand 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 × 103s1. 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 Youngs modulus (for 010% 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 delity of bioprinted constructs
Geometrical delity 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 Fabionextrusion type (3D Bio-
printing Sol., Russia) equipped with uid 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
orice 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 ow
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 microuidic 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 fulllment 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 uorescent 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 L1glutamine (Paneco, Russia).
The cells were cultured at 37 °C in humidied atmosphere
with 5% CO2 and were splitted at 8595% conuence.
2.4.2 Tissue Spheroid formation
TS were produced from NIH 3T3 broblasts using non-
adhesive scalable technology, as was described previously
[27]. Briey, 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 humidied
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 uorescent 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 uorescence) and
dead red (PI uorescence) 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 signicance 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 0.110 rad s1(Fig. 2a). The
frequency increase led to a signicant monotonous aug-
mentation of Gand 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 +5min1(Fig. 2d). The heating of
samples led to a slight decrease of Gand G’’. Nevertheless,
when the temperature was gradually increased to the gela-
tion temperature (2530 ), both Gand G’’ for all col-
lagen concentrations in Viscoll solutions demonstrated a
rapid growth due to the formation of permanent 3D network
with a xed modulus. For Gand 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 Gfrom 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
Gand G’’ increased drastically for all concentration and
became constant. It is worth to note, that the nal Gvalues
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
nal 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.cGand Gvalue as function of time at +37 .d
Gand Gvalue 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 delity
was conrmed (Fig. 3). For biofabricated constructs the
compression measurements illustrated that Youngs 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 delity 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. 4ac; Supl. Movies).
Analysis of the wall thickness and pore size conrmed 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. 4df) with the 3D printed
meshes showed good delity. 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 delity, 20, 30 and 40 mg/ml
Viscoll bioinkwith 80%, 83 and 85% delity 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
Youngs
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 5a6c 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; dQuantication 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
conrmed 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. 5gi) 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 [2830]. 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 delity [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 signicantly stronger
[18]. Constructs with such a stiffness (820 kPa) could be
applied for engineering of the liver tissue (26kPa) [36],
the arterial wall (215 kPa) [37], the heart or muscle tissue
(820 kPa) [3840] and the chondron (2030 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 2225 °C
if samples were gradually heated from 4 °C to 37 °C. Rapid
increase in storage modulus Gand loss modulus G’’
accompanied temperature raise before gelation. This
observation was valid for 2%, 3 and 4% collagen con-
centrations. Our nding 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 delity (data no shown). Value of storage mod-
ulus Gsignicantly 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 Glower 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 Gand Gat approximately
equal values. They have found a shear-thinning behavior for
all collagen solutions studied. To all appearances only
collagen solutions with Gmuch 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 riboavin cross-
linking etc. This suggestion is supported by the previous
nding correlating improved printability of collagen bioink
with increased Gbefore extrusion [14]. Viscoll collagen
solution possesses characteristics of solid hydrogels before
polymerization though retaining the uidity 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 delity 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 delity 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 8085% printing accuracy was
achieved using Viscoll collagen concentrations from 20 to
40 mg/ml. Reducing collagen concentration to 15 mg/ml
impaired nal geometric delity to 70%. These results
correspond well to the delity evaluation method using a
3D scanner [18].
Cell viability and performance differs signicantly 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 Williams 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-dened
patterns onto hydrogels applying an automated drop-on-
demand dispenser setup and further preserved within bri-
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 denitely suitable for
grafting. Moreover, the biocompatibility of the Viscoll
bioinks could be further improved by incorporating addi-
tional ECM proteins, such as brin, bronectin and growth
factors.
Compliance with ethical standards
Conict of interest The authors declare that they have no conict of
interest.
Publishers note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
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... Models. Collagen, 111,112,118 fibrin, 113,114,117 and gelatin 112,114 are the common ECM-based bioinks for bioprinted organotypic skin models. Numerous studies have employed collagenbased bioinks in generating bioprinted skin models using EBB 102 and LBB. ...
... 119 Although collagen-based bioinks are highly biocompatible, they are often limited by their long gelation time and poor mechanical strength. 111,119 To solve this, collagen concentration can be increased to improve its rheological property. Osidak et al. have used a highly concentrated Col-I solution (Viscoll) and demonstrated that the shape of the extruded polymer was well-retained while achieving gelation at 37°C. 111 In situ cross-linking is another method to accelerate the gelation time of bioinks. ...
... Osidak et al. have used a highly concentrated Col-I solution (Viscoll) and demonstrated that the shape of the extruded polymer was well-retained while achieving gelation at 37°C. 111 In situ cross-linking is another method to accelerate the gelation time of bioinks. Min et al. coated the collector surface with sodium bicarbonate, which allows the extruded collagen to be cross-linked via neutralization upon contact with the coated surface. ...
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... The storage modulus has been correlated with an improved shape fidelity (Diamantides et al., 2017;Gao et al., 2018;Lee et al., 2020). Storage modulus also correlates with increased polymer concentration (Osidak et al., 2019). A study on gelatin-alginate blends suggested that bioinks with a G′/G″ between 0.25-0.45 ...
... To improve its application for extrusion-based bioprinting, higher concentrations of COL 1 have been used to formulate a bioink (Rhee et al., 2016). The use of higher concentrations collagen to formulate bioink increases difficulty in cell mixing and bioionk loading into the bioprinter New methods have been developed to produce more concentrated COL 1 solutions with more favorable properties (Shim et al., 2016;Osidak et al., 2019;Lee et al., 2020). This COL 1, branded Viscoll collagen, is a fractionated collagen solution extracted from animal tendons, and purified using salt precipitation and ion-exchange chromatography (Osidak et al., 2019). ...
... The use of higher concentrations collagen to formulate bioink increases difficulty in cell mixing and bioionk loading into the bioprinter New methods have been developed to produce more concentrated COL 1 solutions with more favorable properties (Shim et al., 2016;Osidak et al., 2019;Lee et al., 2020). This COL 1, branded Viscoll collagen, is a fractionated collagen solution extracted from animal tendons, and purified using salt precipitation and ion-exchange chromatography (Osidak et al., 2019). ...
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... The corneal stroma is rich in collagen, proteoglycan, and matrix metalloproteinase, which play an important role in the mechanical strength and transparency of the cornea. Due to the good biocompatibility and low immunogenicity, collagen is widely used as a bioink in 3D bioprinting [38] . However, low mechanical property of the pure collagen is the main limitation of using it as the bioink to form a stable structure. ...
... Crosslinking using different methods (e.g., chemical, physical, or biological) or a mixture with other components (e.g., fibrin, agarose, and alginate) can be performed to improve the properties of collagen bioinks [39][40][41] . Depending on the types of strategies selected, the bioinks can be tuned to prepare suitable low-viscosity solutions for jetting-based printing [42,43] or hydrogels with increased storage modules for extrusion-based printing [38] . In addition, synthesized peptide-based collagen is considered a good option to reduce the batch-to-batch variation effect and improve the mechanical properties in bioprinting [41] . ...
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The use of 3D printing to produce a bioengineered cornea is emerging as an approach to help alleviate the global shortage of donor corneas. Collagen Type 1 (Col-1) is the most abundant collagen in the human cornea. However, Col-I presents challenges as a bioink. It can self-assemble at neutral pH, making phase transitions as required for 3D printing difficult to control. Furthermore, low concentration solutions required for the transparency of printed Col-I lead to weak mechanical properties in its printed structures. In this study, Col-I at high concentrations, was tested with 15 different solutions to identify the composition preventing Col-I self-assembly. A stable Col-I bioink was then developed using riboflavin as a photoinitiator and UV irradiation-induced crosslinking. The mechanical properties and transparency, of the structures produced, were evaluated. The optimised Col-I bioink with corneal stromal cells was tested using a spiral printing method. The printed structure was transparent, and the encapsulated corneal stromal cells had over 90% viability after three weeks of culturing.
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3D Printing technology offers a vast range of applications for tissue engineering applications. Over the past decade a vast range of new equipment has been developed; while, 3D printable biomaterials, especially hydrogels, are investigated to fit the printability requirements. The current candidates for bioprinting often requires post-printing cross-linking to maintain their shape. On the other hand, dynamic hydrogels are considered as the most promising candidate for this application with their extrudability and self-healing properties. However, it proves to be very difficult to match the required rheological in a simple material. Here, we present for the first time the simplest formulation of a dynamic hydrogel based on thiol-functionalized hyaluronic acid formulated with gold ions that fulfill all the requirements to be printed without the use of external stimuli, as judged by the rheological studies. The printability was also demonstrated with a 3D printer allowing to print the dynamic hydrogel as it is, achieving 3D construct with a relatively good precision and up to 24 layers, corresponding to 10 mm high. This material is the simplest 3D printable hydrogel and its mixture with cells and biological compounds is expected to open a new era in 3D bioprinting. This article is protected by copyright. All rights reserved.
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3D bioprinting has received great attention in recent years because of its remarkable advantage in the fabrication of personalized biomaterial designs. In order to meet the full potential of bioprinting, it is necessary to develop new or improved bioinks with appropriate physicochemical properties supporting biological function. One potential bioink is sodium alginate (SA), an anionic polymer which can form an “egg-box” three-dimensional network complex with the introduction of cations. The rapid network formation allows for stabilization of extrudable alginate, making it suitable for use as a bioink in bioprinting applications. In this study, the complexation of positively charged chitosan (CS) and laponite (Lap) with negatively charged SA was investigated. 1-Layer, 2-layer, and multi-layer models were established to evaluate and compare the printability of SA, SA-CS, and SA-Lap bioinks, including metrics of extrudability, filament stability, pore circularity, and structure fidelity. In addition, material properties of each bioink, including viscosity, compressive modulus, swelling rate, and biocompatibility, were assessed. The results showed the printability of SA was improved with the addition of CS or Lap. Moreover, the swelling rate of composite bioinks was reduced to half of that of sodium alginate counterparts. In addition, cell compatibility studies demonstrated that all bioinks were capable of maintaining cell proliferation over five days. As a whole, this work highlights the importance of electrostatic interactions of alginate-based bioinks for bioprinting.
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Collagens from a wide array of animals have been explored for use in tissue engineering in an effort to replicate the native extracellular environment of the body. Marine-derived biomaterials offer promise over their conventional mammalian counterparts due to lower risk of disease transfer as well as being compatible with more religious and ethical groups within society. Here, collagen type I derived from a marine source (Macruronus novaezelandiae, Blue Grenadier) is compared with the more established porcine collagen type I and its potential in tissue engineering examined. Both collagens were methacrylated, to allow for UV crosslinking during extrusion 3D printing. The materials were shown to be highly cytocompatible with L929 fibroblasts. The mechanical properties of the marine-derived collagen were generally lower than those of the porcine-derived collagen; however, the Young’s modulus for both collagens was shown to be tunable over a wide range. The marine-derived collagen was seen to be a potential biomaterial in tissue engineering; however, this may be limited due to its lower thermal stability at which point it degrades to gelatin.
Chapter
The skeletal system comprises multiple tissues which converge in the formation of complex biological structures such as hierarchical bone, organized cartilage, joints, and tissue interfaces. However, different human conditions derived from aging, lifestyle, illness, or trauma can damage the components of the human skeletal system and lead to loss of function and reduced life quality. In the context of skeletal tissue engineering, physical and biological demands play a key role in the successful construction and implantation in bone, cartilage, and blood vessel tissue formation. After a brief summary of the biological properties of supportive connective tissues, namely bone and cartilage, the chapter is organized in three different technological sections: (i). grafting-based tissue engineering techniques, (ii). 3D bioprinting technology, and (iii). in vitro organ modeling based on organ-on-a-chip. With scientific progress and emergence of different in vitro models, knowledge of the entire organism behavior is growing, but the possibility to mimic the complexity of human skeletal tissues and their functionality is still a significant ongoing challenge.
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Three-dimensional (3D) bioprinting enables the creation of tissue constructs with heterogeneous compositions and complex architectures. It was initially used for preparing scaffolds for bone tissue engineering. It has recently been adopted to create living tissues, such as cartilage, skin, and heart valve. To facilitate vascularization, hollow channels have been created in the hydrogels by 3D bioprinting. This review discusses the state of the art of the technology, along with a broad range of biomaterials used for 3D bioprinting. It provides an update on recent developments in bioprinting and its applications. 3D bioprinting has profound impacts on biomedical research and industry. It offers a new way to industrialize tissue biofabrication. It has great potential for regenerating tissues and organs to overcome the shortage of organ transplantation.
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For three-dimensional bio-printed cell-laden hydrogel tissue constructs, the well-designed internal porous geometry is tailored to obtain the desired structural and cellular properties. However, significant differences often exist between the designed and as-printed scaffolds because of the inherent characteristics of hydrogels and cells. In this study, an iterative feedback bio-printing (IFBP) approach based on optical coherence tomography (OCT) for the fabrication of cell-laden hydrogel scaffolds with optimal geometrical fidelity and cellular controllability was proposed. A custom-made swept-source OCT (SS-OCT) system was applied to characterize the printed scaffolds quantitatively. Based on the obtained empirical linear formula from the first experimental feedback loop, we defined the most appropriate design constraints and optimized the printing process to improve the geometrical fidelity. The effectiveness of IFBP was verified from the second run using gelatin/alginate hydrogel scaffolds laden with C3A cells. The mismatch of the morphological parameters greatly decreased from 40% to within 7%, which significantly optimized the cell viability, proliferation, and morphology, as well as the representative expression of hepatocyte markers, including CYP3A4 and albumin, of the printed cell-laden hydrogel scaffolds. The demonstrated protocol paves the way for the mass fabrication of cell-laden hydrogel scaffolds, engineered tissues, and scaled-up applications of the 3D bio-printing technique.
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This comparative study aims to identify a biocompatible and effective crosslinker for preparing gelatin sponges. Glutaraldehyde (GTA), genipin (GP), 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC), and microbial transglutaminase (mTG) were used as crosslinking agents. The physical properties of the prepared samples were characterized, and material degradation was studied in vitro with various proteases and in vivo through subcutaneous implantation of the sponges in rats. Adipose-derived stromal stem cells (ADSCs) were cultured and inoculated onto the scaffolds to compare the cellular biocompatibility of the sponges. Cellular seeding efficiency and digestion time of the sponges were also evaluated. Cellular viability and proliferation in scaffolds were analyzed by fluorescence staining and MTT assay. All the samples exhibited high porosity, good swelling ratio, and hydrolysis properties; however, material strength, hydrolysis, and enzymolytic properties varied among the samples. GTA-sponge and GP-sponge possessed high compressive moduli, and EDC-sponge exhibited fast degradation performance. GTA and GP sponge implants exerted strong in vivo rejections, and the former showed poor cell growth. mTG-sponge exhibited the optimal comprehensive performance, with good porosity, compressive modulus, anti-degradation ability, and good biocompatibility. Hence, mTG-sponge can be used as a scaffold material for tissue engineering applications.
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Three dimensional (3D) printing is a hot topic in today’s scientific research and commercial areas. It is recognized as the third revolution in industrial as well as biomedical fields. Recently, human organ 3D bioprinting has been put forward into equity market as a concept stock and attracted a lot of attention. A large number of outstanding scientists have flung themselves into this area and made some remarkable headways. Nevertheless, organ 3D bioprinting is a sophisticated procedure which needs profound scientific/technological backgrounds/knowledges to accomplish Especially, large organ 3D bioprinting encounters enormous difficulties and challenges. One of them is to build implantable branched vascular networks in a predefined 3D construct. At present, organ 3D bioprinting still in its infancy and a great deal of work needs to be done. Here we briefly overview some of the achievements of 3D bioprinting in three large organs, such as the bone, liver and heart.
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Replacement of the removal site of the spinal cord on a collagen implant restores the motor function of the hind limbs in rats to the level of movements in the two joints for 8 weeks. After intravenous administration of mononuclear cells of human umbilical blood, recovery accelerated, significantly improved to the level of motion in the three joints, and there is a tendency to improve further recovery of movements.
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Fibrillar type I collagen-based hydrogels are commonly used in tissue engineering and as matrices for biophysical studies. Mechanical and structural properties of these gels are known to be governed by the conditions under which fibrillogenesis occurs, exhibiting variation as a function of protein concentration, temperature, pH, and ionic strength. Deeper understanding of how macroscopic structure affects viscoelastic properties of collagen gels over the course of fibrillogenesis provides fundamental insight into biopolymer gel properties and promises enhanced control over the properties of such gels. Here, we investigate type I collagen fibrillogenesis using confocal rheology—simultaneous confocal reflectance microscopy, confocal fluorescence microscopy, and rheology. The multimodal approach allows direct comparison of how viscoelastic properties track the structural evolution of the gel on fiber and network length scales. Quantitative assessment and comparison of each imaging modality and the simultaneously collected rheological measurements show that the presence of a system-spanning structure occurs at a time similar to rheological determinants of gelation. Although this and some rheological measures are consistent with critical gelation through percolation, additional rheological and structural properties of the gel are found to be inconsistent with this theory. This study clarifies how structure sets viscoelasticity during collagen fibrillogenesis and more broadly highlights the utility of multimodal measurements as critical test-beds for theoretical descriptions of complex systems.
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During extrusion-based bioprinting, the deposited bioink filaments are subjected to deformations, such as collapse of overhanging filaments, which compromises the ability to stack several layers of bioink, and fusion between adjacent filaments, which compromises the resolution and maintenance of a desired pore structure. When developing new bioinks, approaches to assess their shape fidelity after printing would be beneficial to evaluate the degree of deformation of the deposited filament and to estimate how similar the final printed construct would be to the design. However, shape fidelity has been prevalently assessed qualitatively through visual inspection after printing, hampering the direct comparison of the printability of different bioinks. In this technical note, we propose a quantitative evaluation for shape fidelity of bioinks based on testing the filament collapse on overhanging structures and the filament fusion of parallel printed strands. Both tests were applied on a hydrogel platform based on poloxamer 407 and poly(ethylene glycol) (PEG) blends, providing a library of hydrogels with different yield stresses. The presented approach is an easy way to assess bioink shape fidelity, applicable to any filament-based bioprinting system and able to quantitatively evaluate this aspect of printability , based on the degree of deformation of the printed filament. In addition, we built a simple theoretical model that relates filament collapse with bioink yield stress. The results of both shape fidelity tests underline the role of yield stress as one of the parameters influencing the printability of a bioink. The presented quantitative evaluation will allow for reproducible comparisons between different bioink platforms.
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Bioprinting can be defined as additive biofabrication of 3D tissues and organ constructs using tissue spheroids, capable of self-assembly, as building blocks. Thyroid gland, a relatively simple endocrine organ, is suitable for testing the proposed bioprinting technology. Here we report the bioprinting of functional vascularized mouse thyroid gland construct from embryonic tissue spheroids as a proof of concept. Based on the self-assembly principle, we generated thyroid tissue starting from thyroid spheroids (TS) and allantoic spheroids (AS), as a source of thyrocytes and endothelial cells (EC), respectively. Inspired by mathematical modelling of spheroid fusion, we used an original 3D bioprinter to print TS in close association with AS within collagen hydrogel. During the culture, closely placed embryonic tissue spheroids fused into a single integral construct, endothelial cells from AS invaded and vascularized TS, and epithelial cells from the TS progressively formed follicles. In this experimental setting, we observed formation of capillary network around follicular cells, as observed during in utero thyroid development when thyroid epithelium controls the recruitment, invasion and expansion of EC around follicles. To prove that EC from AS are responsible for vascularization of thyroid gland construct, we depleted endogenous EC from thyroid spheroids before bioprinting. EC from allantoic spheroids completely revascularized depleted thyroid tissue. Cultured bioprinted construct was functional as it could normalize blood thyroxin levels and body temperature after grafting under the kidney capsule of hypothyroid mice. Bioprinting of functional vascularized mouse thyroid gland construct represents further advance in bioprinting technology exploring self-assembling properties of tissue spheroids.
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Collagen has shown promise as a bioink for extrusion-based bioprinting, but further development of new collagen bioink formulations is necessary to improve their printability. Screening these formulations by measuring print accuracy is a costly and time consuming process. We hypothesized that rheological properties of the bioink before, during, and/or after gelation can be used to predict printability. In this study, we investigated the effects of riboflavin photocrosslinking and pH on type I collagen bioink rheology before, during, and after gelation and directly correlated these findings to the printability of each bioink formulation. From the riboflavin crosslinking study, results showed that riboflavin crosslinking increased the storage moduli of collagen bioinks, but the degree of improvement was less pronounced at higher collagen concentrations. Dots printed with collagen bioinks with riboflavin crosslinking exhibited smaller dot footprint areas than those printed with collagen bioinks without riboflavin crosslinking. From the pH study, results showed that gelation kinetics and final gel moduli were highly pH dependent and both exhibited maxima around pH 8. The shape fidelity of printed lines was highest at pH 8–9.5. The effect of riboflavin crosslinking and pH on cell viability was assessed using bovine chondrocytes. Cell viability in collagen gels was found to decrease after blue light activated riboflavin crosslinking but was not affected by pH. Correlations between rheological parameters and printability showed that the modulus associated with the bioink immediately after extrusion and before deposition was the best predictor of bioink printability. These findings will allow for the more rapid screening of collagen bioink formulations.
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The therapeutic infusion of adipose-derived stromal vascular fraction cells (SVF) for the treatment of multiple diseases, has progressed to numerous human clinical trials; however, the often poor retention of the cells following implantation remains a common drawback of direct cell injection. One solution to cellular retention at the injection site has been the use of biogels to encapsulate cells within a microenvironment prior to and upon implantation. The current study utilized 3D bioprinting technology to evaluate the ability to form SVF laden spheroids with collagen I as a gel forming biomatrix. A superhydrophobic surface was created to maintain the bioprinted structures in a spheroid shape. A hydrophilic disc was printed onto the hydrophobic surface to immobilize the spheroids during the gelation process. Conditions for the automated, rapid formation of SVF laden spheroids were explored including time/pressure relationships for spheroid extrusion during bioprinting. The formed spheroids maintain SVF viability in both static culture and dynamic spinner culture. Spheroids also undergo a time dependent contraction with the retention of angiogenic sprout phenotype over the 14 day culture period. The use of a biphilic surface exhibiting both superhydrophobicity to maintain spheroid shape and a hydrophilicity to immobilize the spheroid during gel formation produces SVF laden spheroids that can be immediately transplanted for therapeutic applications.