Bioprinting 24 (2021) e00163

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Bioprinting
journal homepage: www.sciencedirect.com/journal/bioprinting

Alginate-based tissue-specific bioinks for multi-material 3D-bioprinting of

pancreatic islets and blood vessels: A step towards vascularized
pancreas grafts
Joanna Idaszek a, *, 1, Marina Volpi a, 1, Alessia Paradiso a, Martyna Nguyen Quoc a,
Żaneta Górecka a, Marta Klak b, Grzegorz Tymicki b, Andrzej Berman b, Mateusz Wierzbicki c,
Sławomir Jaworski c, Marco Costantini a, d, Agnieszka Kępczyńska e, Ewa Sawosz Chwalibóg c,
Michał Wszoła b, Wojciech Święszkowski a, **
a
Faculty of Materials Science and Engineering, Warsaw University of Technology, Warsaw, Poland
b
Foundation of Research and Science Development, Warsaw, Poland
c
Department of Nanobiotechnology and Experimental Ecology, Institute of Biology, Warsaw University of Life Sciences, Warsaw, Poland
d
Institute of Physical Chemistry, Polish Academy of Sciences, Warsaw, Poland
e
Laboratory of Electron Microscopy, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: Although allogeneic islet transplantation has been proposed as a therapy for type 1 diabetes, its success rate
Microfluidic-assisted multi-material 3D bio­ remains low. Disruption of both extracellular matrix (ECM) and dense vascular network during islets isolation are
printing referred to as some of the main causes of their poor engraftment. Therefore, the recapitulation of the native
Pancreatic islets
pancreatic microenvironment and its prompt revascularization should be beneficial for long-term islet survival.
Blood vessels
Bioink
In this study, we developed novel bioinks suitable for the microfluidic-assisted multi-material biofabrication of
ECM 3D porous pancreatic and vascular structures. The tissue-specific bioactivity was introduced by blending alginate
Fibrinogen either with pancreatic decellularized extracellular matrix powder (A_ECM) or fibrinogen (A_FBR). The formu­
Alginate lated bioinks, although they exhibited different rheological properties, were 3D-printed with high shape fidelity
using a multichannel microfluidic platform coupled with a co-axial needle system. The A_FBR scaffolds were
subjected to secondary crosslinking using thrombin, which significantly decreased their swelling and increased
mechanical properties. To investigate the bioactivity, the A_ECM and A_FBR bioinks were laden with, respec­
tively, porcine pancreatic islets and a mixture of vessel-forming cells (HUVEC and HMSC) and 3D-bioprinted
with high viability. Insulin secretion upon glucose stimulation and developed vessel-like structures composed
of CD31-positive cells validated the biomimetic potential of the two formulated bioinks. Moreover, we
demonstrated the feasibility of the secondary crosslinking to modulate cell behaviour and angiogenic potential
both in vitro and in vivo using a chick chorioallantoic membrane model. Finally, the successful 3D-printing of
heterogeneous 3D scaffolds with three different configurations demonstrated that our approach could be
considered as a potential step towards the biofabrication of a vascularized pancreas construct.

1. Introduction
Type 1 diabetes mellitus (T1DM) is a metabolic disorder character­
ized by an autoimmune response causing the destruction of insulin-
secreting β-cells [1]. Thus, T1DM results in the inability to control
blood glucose level and leads to disorders of glucose homeostasis, which
may, in turn, result in long-term, and potentially life-threatening,
co-morbidities such as retinopathy, cardiovascular diseases, and renal
failure [2]. The most widespread therapy for T1DM treatment includes
the delivery of exogenous synthetic insulin by subcutaneous injections.

* Corresponding author. Faculty of Materials Science and Engineering, Warsaw University of Technology, Warsaw, Poland.
** Corresponding author.
E-mail addresses: joanna.idaszek@pw.edu.pl (J. Idaszek), wojciech.swieszkowski@pw.edu.pl (W. Święszkowski).
1
These authors have equally contributed

https://doi.org/10.1016/j.bprint.2021.e00163
Received 23 February 2021; Received in revised form 23 July 2021; Accepted 26 July 2021
Available online 3 August 2021
2405-8866/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
J. Idaszek et al.
Even though it has been considered a life-saving procedure, such
treatment is associated with several limitations, mostly related to diffi­
culties in mimicking the physiological insulin release [3]. In this frame,
allogeneic pancreatic islet transplantation has emerged as a promising
therapy for T1DM [4,5]. Although the implantation procedure of islets
of Langerhans is less invasive than conventional pancreas trans­
plantation and allows the successful transplantation of lower quality
tissues (e.g., autologous islets transplantation) [6,7], the therapeutic
outcomes of such procedure are partially limited due to low efficiency of
islet engraftment [8].
Several factors are indicated as the underlying cause of high post-
transplantation islet loss [9]. Among them, deprivation of interactions
between the islets and extracellular matrix (ECM) molecules and
disruption of the dense vascular network intertwining the islets are a
direct consequence of the isolation from the donor pancreas [10,11].
Moreover, the destruction of the dense microvasculature not only im­
pairs efficient oxygenation, nutrient delivery, and metabolic waste
removal, but also disables the pancreatic islets from rapidly sensing
blood glucose fluctuations and responding accordingly by secreting in­
sulin [12]. Although transplanted islets can survive in avascular condi­
tions for up to several days, further delays in tissue vascularization leads
to necrosis and loss of endocrine function [10]. Therefore, recapitulation
of native pancreatic ECM-microenvironment and a dense vascular
network is considered crucial to increase the rate of positive therapeutic
outcomes related to islet transplantation procedures.
To this end, pancreatic tissue engineering (TE) explored bio­
fabrication strategies focused on combining islet encapsulation into
ECM-like hydrogels (e.g., fibrinogen [13], Matrigel [14], collagen [15])
with angiogenic factors (e.g., vascular endothelial growth factor (VEGF)
[14] and platelet-derived growth factor (PDGF) [13], among others
[16], or porous scaffolds able to stimulate vascular ingrowth upon im­
plantation [17]. In this frame, 3D-bioprinting (3D-BP) of pancreatic is­
lets has been proposed as a biofabrication method to engineer a suitable
biomimetic pancreatic microenvironment [18,19]. Indeed, 3D-BP offers
the advantage of precisely depositing islet-laden ECM-like hydrogel
strands in a layer-by-layer fashion in order to fabricate 3D porous
scaffolds with different geometries and pore sizes. For instance,
Marchioli et al. used an extrusion-based system to 3D bioprint human
islets in a wide range of alginate-based bioink formulations. They
demonstrated the positive effect of the introduction of porosity into
hydrogel bulk on insulin secretion [20]. Duin and co-workers success­
fully 3D bioprinted rat islets encapsulated in alginate blended with
methylcellulose [21]. Liu et al. further advanced the 3D-BP of islets by
employing a co-axial system to fabricate 3D porous scaffolds by
extruding murine islet-laden gelatin methacryoloyl (GelMA)/alginate
core-shell fibers, thus showing the control over islets spatial distribution
[22]. Kim and co-workers demonstrated enhanced effect of liquefied
pancreatic dECM bioink on insulin secretion by islets; however, the
powder dECM has not been applied in bioprinting of pancreatic islets yet
[18].
Although it has been already reported that 3D porous scaffolds can
stimulate and promote efficient vascular ingrowth once such engineered
tissues are implanted in vivo, this process requires up to 14 days to fully
develop [23,24]. To reduce the time required to achieve an appropriate
vascular supply in close proximity of transplanted islets, several strate­
gies for co-culture and co-transplantation of vascular supporting cells
have been recently developed [16,25,26]. However, the integration of
vascularized structures in islet-laden constructs by 3D-BP technologies
may be considered challenging, as different bioink formulations should
be designed to ensure a suitable tissue-specific microenvironment.
Common approaches employed for the 3D-BP of multi-material and
multi-cellular constructs involve the use of highly complex
multiple-printing heads apparatuses [27–29]. However, this results in
an overall time-consuming fabrication process. To address this limita­
tion, recent advances in biofabrication strategies integrated 3D printing
heads with a multi-inlet microfluidic platform, allowing a rapid
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Bioprinting 24 (2021) e00163
fabrication of complex heterogenous structures by simultaneously
extruding or rapidly switching between multiple bioinks [30–33].
Nevertheless, proper bioink formulations for microfluidic-assisted
3D-BP still remain challenging, as they have to meet technological and
biological criteria imposed by the bioprinting window.
In this study, we developed two biomimetic bioinks suitable for
microfluidics-assisted extrusion-based 3D-BP of heterogeneous scaffolds
consisting of pancreatic and vascular compartments. The two tissue-
specific bioinks were formulated by incorporating pancreatic decellu­
larized ECM (dECM) powder and fibrinogen into alginate to reproduce
pancreatic and vascular structure, respectively. The proposed bioinks
were successfully bioprinted into 3D high-resolution porous scaffolds
and investigated separately through morphological, physicochemical,
and mechanical characterization to validate their capacity for engi­
neering the tissue-specific function. Moreover, biological evaluation was
performed to further explore the biomimetic potential. In particular,
dECM- and fibrinogen-based bioinks were combined with, respectively,
porcine pancreatic islets and vessel-forming cells (i.e., a mixture of
human mesenchymal stem cells (HMSC) and human umbilical vein
endothelial cells (HUVEC)). As a result, an increased insulin secretion
upon glucose stimulation was detected, and the formation of in vitro
vessel-like structures was obtained. Moreover, potential pro-angiogenic
properties were evaluated in vivo in the chick chorioallantoic membrane
model. Finally, as a proof of concept, the two developed bioink formu­
lations were co-printed at different spatial configurations. Thus, a bio­
mimetic pancreatic-vascular 3D scaffold could be successfully
fabricated.
2. Results and discussion
2.1. Ink formulations and rheological characterization
In this work, a co-axial microfluidic chip was coupled with a com­
mercial 3D bioprinter for the fabrication of bio-mimetic multi-material
scaffolds to simultaneously engineer both pancreatic and vessel-like
structures (Fig. 1A). Co-axial 3D-BP is a TE strategy that combines the
microfluidic fabrication of cell-laden hydrogel-based microfibers with
layer-by-layer 3D deposition [34]. In the frame of microfluidic-assisted
applications, alginate-based hydrogel formulations have been widely
employed due to their instantaneous gelation ability via calcium ions
crosslinking [35]. Indeed, the simultaneous delivery of both alginate
solution and calcium chloride allows the fabrication of highly-defined
3D porous structures [36]. However, despite its high suitability for
microfluidic applications, alginate lacks cell-binding sites. Thus, the
maintenance of an appropriate micro-environment to support the
long-term viability and functionality of encapsulated cells is limited.
Therefore, alginate has been combined with more biomimetic bio­
polymers (i.e., chitosan, gelatin, GelMA) according to the specific tissue
engineering application [20,37,38]. In this work, alginate was used as a
templating agent, ensuring sufficient mechanical properties and high
shape fidelity, and tissue-specific bioactivity was introduced by blending
with pancreatic dECM and fibrinogen-based tissue glue for engineering
pancreatic and vessel-like structures, respectively (Fig. 1B).
DECM-powder was employed to closely mimic the complex molec­
ular composition of the pancreatic microenvironment. The introduction
of native-ECM components (e.g., collagen, laminin) into inert hydrogels
and the use of pristine solubilized pancreatic dECM improved the sur­
vival endocrine function of pancreatic islets and insulin-producing cells
[18,39,40]. However, liquid dECM is obtained by enzymatic solubili­
zation of the decellularized tissue, thus yielding degradation of its native
structure and mechanical strength [41,42]. Moreover, pepsin digestion
was reported to cause denaturation of some of the biochemical com­
ponents of dECM (e.g., proteins, growth factors) [43–45]. Therefore, in
this study, pancreatic dECM in the form of powder was selected over
conventional liquid dECM bioink. The median of the volumetric particle
size distribution of the dECM powder used in this study was equal 148 ±
J. Idaszek et al. Bioprinting 24 (2021) e00163

Fig. 1. A) Schematic of the experimental 3D-BP

setup. The microfluidic platform is adapted to fit
the bottom of the cartridge and connected with two
different digital pumps, loaded with pre-hydrogel
and crosslinking solution, respectively. The 3D-
printed fiber undergoes gelation as it is extruded
from the needle tip. B) Alginate-based tissue-spe­
cific pre-hydrogel formulations of cell-laden bio­
inks. C) Schematic model of a vascularized
pancreatic islet. D) Schematic of the microfluidic Y-
junction designed to simultaneously or alternatively
dispense the two formulated bioinks for the fabri­
cation of multi-layered heterogeneous structures.

10 μm. The DNA content was <50 ng per 1 mg of dECM powder, which
corresponds to 1.25 ± 0.11% DNA content of the native pancreas.
Fibrinogen has been widely employed in vascular tissue engineering
[46]. Indeed, in physiological conditions, fibrinogen is involved in the
repairment of tissues and blood vessels after injury by forming fibrin
clots upon thrombin-catalyzed enzymatic polymerization. Moreover,
fibrin possesses an effective potential to induce the growth of blood
vessels and the sprouting of newly formed capillaries (i.e., angiogenesis)
in the proximity of in vivo implanted scaffolds [47–49].
Alginate-dECM powder and alginate-fibrinogen bioinks were deno­
ted as A_ECM and A_FBR, respectively. To further tailor physiochemical
properties of the A_FBR hydrogel, we performed secondary crosslinking
with thrombin (A_FBR_T). Pristine alginate bioink was denoted as A_PRS
and used as a control. After material and biological characterization,
both bioink formulations were simultaneously 3D printed to reproduce a
vascularized pancreatic microenvironment using a custom-made Y-chip
(Fig. 1C and D).
Ink rheological characterization and 3D-printing of vascular and
pancreatic scaffolds.
Before 3D-printing, the rheological behaviour of the proposed bioink
formulations was tested by applying a shear rate ramp of 0.1–100 s− 1. As
shown in Fig. 2A, A_ECM showed a pronounced shear thinning behav­
iour, characterized by a decrease in viscosity over an increasing shear
rate. Both A_PRS and A_FBR exhibited a Newtonian plateau below shear
rates of approximately 0.5 s− 1, followed by a slight shear thinning
behaviour. Moreover, it was observed that all bioink formulations

Fig. 2. A) Dynamic viscosity of the formulated bioinks employed in 3D-BP experiments as a function of shear rate. Viscosity of alginate is depicted as a reference
bioink. Effect of 3D-printing parameters on deposited fibers and morphological characterization of 3D-printed scaffolds: B) Effect of deposition speed (v) and C)
bioink flow rate (Q) on fiber diameter (D). D-F) Optical and macroscopic images of 3D-printed scaffolds: D) A_PRS, E) A_ECM, and F) A_FBR. G) Average fiber
diameter and H) average pore size of 3D printed scaffolds upon optimized 3D-printing parameters (Q = 15 μl min− 1, v = 2.5 mm s− 1); * – p < 0.05.

3
J. Idaszek et al.
exhibited high differences in terms of dynamic viscosity. Specifically,
A_ECM presented the highest dynamic viscosity, revealing values 240-
and 45-fold larger than A_PRS and A_FBR, respectively. A lower differ­
ence was detected comparing A_PRS to A_FBR, where A_FBR showed a
dynamic viscosity 3- to 6-fold larger than A_PRS. Despite those differ­
ences, all bioink formulations were easily extrudable by applying the
same flow rate. Hence, well-defined hydrogel filaments could be formed.
The effect of the deposition speed (v) and bioink flow rate (Q) on the
morphology of 3D-printed scaffolds was investigated for all bioinks
formulations (Fig. 2B and C). In particular, fiber diameter was modu­
lated by increasing v (i.e., 1–6 mm s− 1) and Q (i.e., 5, 10, 15, 20, 25 and
30 μl min− 1). A linear increase (R2 > 0.9) of fiber diameter following the
increase of Q was observed for all bioink formulations. Conversely, a
linear decrease (R2 > 0.9) of fiber diameter was measured as the v
increased. These findings are in accordance with the volumetric flow
rate equation, i.e., Q = Av, where A is the fiber cross-section area. The
diameter of the 3D printed fibers is directly dependent on the ink flow
rate and inversely dependent on the deposition speed. Continuous and
well-defined fibers were also obtained for most of the tested printing
parameters. The only exception was found for the lowest and the highest
deposition speeds (1 mm s− 1 and 6 mm s− 1, respectively). In the first
case, filaments appeared undulating rather than fully stretched, while
the highest speed reduced the chance of contact between 3D-printed
microfibers and the deposition plate. 3D scaffolds have been success­
fully printed using the same optimized printing parameters. However,
3D-printing of A_ECM presented more technical challenges when
compared to the other ink formulations. Specifically, nozzle-clogging
episodes occurred more frequently due to the relevant viscosity of
A_ECM and the presence of dispersed ECM particles. For all other for­
mulations, the 3D-printing process moved forward without any major
issues over the long printing sessions (i.e., 2–3 h).
3D-printed scaffolds were structurally stable and could be easily
manipulated by means of a spatula. Moreover, macroscopic images
(Fig. 2D–F) revealed overall high printing accuracy, as confirmed by a
distinct 0◦ –90◦ square grid pattern. In addition, 3D-printed scaffolds
presented a fully interconnected structure characterized by well-defined
square-shaped open pores. The investigation of fiber diameter (Fig. 2G)
showed a significantly higher fiber diameter for A_ECM scaffold (584.3
± 25.5 μm) compared to both A_PRS (445.2 μm ± 52.4) and A_FBR
(406.3 ± 28.9 μm). The increase in fiber dimension may be likely
attributed to lower crosslinking efficiency upon Ca2+ due to the high
concentration of ECM powder. Consequently, as shown in Fig. 2H,
thicker fibers resulted in a significant decrease in the pore size of A_ECM
scaffolds (360.8 ± 29.3 μm) compared to A_PRS (557.2 ± 52.4 μm) and
A_FBR (494.2 ± 29.9 μm). The obtained pore sizes and open porosity
may be beneficial in terms of in vivo vascularization since it has been
reported that pore sizes >200 μm increase vascular cell invasion and
ultimately lead to vessel ingrowth. Moreover, pore size >500 μm
Fig. 3. Physical and mechanical characterization of 3D-printed scaffolds (A_PRS, A_ECM, A_FBR_C, A_FBR_T). A) Averaged swelling ratio (ΔW%) of 3D-printed
scaffolds after 3 days of immersion in DI water determined by measuring the mass of both wet and freeze-dried samples. B) Average elastic modulus (E) ob­
tained from mechanical compression test. The stiffness of A_ECM and A_FBR_T scaffolds was significantly higher than A_PRS (p < 0.05).
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Bioprinting 24 (2021) e00163
facilitated nutrients supply and waste removal of products within the
scaffold [50–52]. After the 3D-printing process, A_FBR scaffolds were
subjected to secondary crosslinking by immersing in thrombin solution
(A_FBR_T). Not-crosslinked A_FBR scaffolds were used as a control
(A_FBR_C).
Taken together, the aforementioned data clearly shows that our
approach may be a good strategy for relatively fast and convenient
fabrication of high-resolution 3D porous structures using bioinks char­
acterized by inherently low printability and poor structural integrity (e.
g., ECM and fibrinogen), further advancing the 3D-BP of soft hydrogels
[53–56].
2.2. Swelling test
A swelling test was performed to investigate the water uptake ca­
pacity of the 3D printed scaffolds. The swelling ratio, expressed as a
difference in weight of wet and dry samples normalized to the weight of
dry samples (ΔW%), is reported in Fig. 3A. A significantly lower
swelling ratio was observed for A_ECM (546.2 ± 55.7 %) compared to
A_PRS (1180.2 ± 66.5 %). This finding is in contrast with results re­
ported in the literature, where liquid dECM increased the water ab­
sorption capacity when combined with pristine hydrogels [57]. We
hypothesize that the dECM powder incorporated in this study could
interfere with alginate polymer chains resulting in a packed network
with limited polymer chain mobility, leading to a decrease in the overall
water penetration. Another possible explanation is the effect of
nano-topography of dECM powder obtained during the freeze-drying
process, increasing its hydrophobicity despite its chemical composi­
tion [58]. This could be supported by the fact that we measured lower
water uptake by freeze-dried alginate and alginate-based samples upon
incubation in DI water, suggesting permanent structural changes
occurring during the lyophilization process (unpublished data). A
different water absorption effect was observed when fibrinogen was
incorporated into pristine alginate. Results showed a significantly higher
A_FBR_C swelling ratio (1493.5 ± 80.7 %) compared to A_PRS. This
could be attributed to the fibrinogen molecules disturbing the formation
of intramolecular bonds between alginate chains and Ca2+ ions and the
clearance of fibrinogen from the samples upon incubation in DI water
due to lack of thrombin crosslinking. This finding is confirmed by a
significantly higher swelling ratio of A_FBR_C compared to values ob­
tained for secondary crosslinked A_FBR_T (1013.1 ± 81.7 %), in which
fibrinogen was enzymatically converted into fibrin. Moreover, 40 mM
CaCl2 present in thrombin solution might induce a secondary cross­
linking effect also on the alginate network, thus further decreasing the
water uptake, as confirmed by significantly higher swelling ratio of
A_PRS compared to A_FRB_T [59].
J. Idaszek et al.
2.3. Mechanical characterization
Mechanical properties of all bioink formulations were investigated
by a static mechanical compression test on samples hydrated for 3 days
in DI water. Elastic modulus (E) values are reported in Fig. 3B. A_ECM
exhibited significantly higher stiffness (2.3 ± 0.1 kPa) compared to
A_PRS (1.0 ± 0.1 kPa). This increase could be induced by incorporating
high concentration dECM powder, which created a denser and more
compact structure than the A_PRS. Our finding is in accordance with the
results obtained by Kim et al., where E of the formulated gelatin-based
bioink was higher after dECM powder was incorporated [42]. On the
other hand, the incorporation of fibrinogen did not increase mechanical
properties. Indeed, A_FBR_C showed a similar E value (1.2 ± 0.4 kPa)
compared to A_PRS. This is probably due to the dual effect introduced by
the possible partial clearance of fibrinogen due to the absence of
thrombin secondary crosslinking and a lower crosslinking of alginate
chains resulting from lack of incubation with 40 mM CaCl2. This was
confirmed by A_FBR_T, which showed a significantly higher E value (1.6
± 0.3 kPa) compared to A_PRS. Indeed, secondary crosslinking intro­
duced by immersion in thrombin could keep fibrinogen inside the 3D
printed scaffold, thus creating a packed polymeric network which may
in turn increase E values under mechanical compression. Results are in
accordance with Duong et al., where higher E values were detected by
increasing thrombin concentration [60]. Moreover, the synergistic effect
of thrombin and 40 mM CaCl2 crosslinking was also reflected on algi­
nate, as confirmed by the significantly higher stiffness of A_FBR_T
compared to A_PRS. Finally, A_ECM, A_FBR_C, and A_FBR_T showed
mechanical properties similar to those of the native tissues they inten­
ded to mimic. In particular, elastic moduli of A_ECM, A_FBR_C, and
A_FBR_T are respectively in the range of the values found for pancreatic
(1–5 kPa) [61] and endothelium native tissue (1.2–2 kPa) [62].
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Bioprinting 24 (2021) e00163
2.4. 3D-bioprinting of the pancreatic islets
After thorough characterization of the developed ink in terms of
printability and the investigation of physicochemical properties of the
3D scaffolds, 3D-BP experiments with pancreatic islet-laden bioink were
performed. One of the major concerns related to the 3D-BP technique
was the effect of shear stress on islet viability during extrusion of the
A_ECM bioink. In Fig. 4A, results of live/dead (L/D) staining are pre­
sented. Despite the overall difficulties in distinguishing islets from flake-
like dECM particles due to their green autofluorescence, the absence of
red fluorescence led us to conclude that all islets survived the 3D-BP
process. This result was further confirmed by the evaluation of the
response of the islets to glucose fluctuations in a culture medium, as
shown in Fig. 4C. The GSIS assay was performed 24 h and 48 h after the
3D-BP. The difference in secretion of insulin by the islets encapsulated in
A_PRS and A_ECM bioinks and the corresponding controls (i.e., the free-
floating islets designated as C_A_PRS and C_A_ECM) could be due to
biological diversity, as the islets were isolated from two different ani­
mals in two independent isolation procedures. Interestingly, both types
of islets encapsulated in the bioinks produced in the low-glucose me­
dium (LG) more insulin than the free-floating controls. Similar results
were obtained by Marchioli and co-workers [9]. Although the islets
encapsulated in the A_ECM bioink secreted in the LG over 3 times less
insulin than those bioprinted in A_PRS, both groups secreted a similar
concentration of insulin upon stimulation with high-glucose medium
(HG). Therefore, the stimulation index (SI, i.e., the ratio of insulin
secreted in a high glucose condition to the concentration secreted in a
low glucose condition) was approximately 2-fold higher for the former
(SI equal 2,8 ± 1,4 vs. 1,3 ± 0,2 respectively; see Fig. 4D). Those results
suggest loss of functionality by the islets bioprinted in the pristine
alginate. Unfortunately, after additional 24 h of culture, the
Fig. 4. Biological evaluation of the A_PRS and
A_ECM bioinks. A, B) Viability of islets after 3D-BP
in A_PRS (A) and A_ECM (B) bioinks determined by
means of L/D staining. The islets encapsulated in
both bioinks were viable (green) after the 3D-BP
process; no dead islets were detected (lack of red
fluorescence). Scale bar 200 μm. C) Secretion of
insulin upon stimulation with high-glucose medium
(HG, 16.7 mM of glucose) 24 h after the 3D-BP.
Both 3D bioprinted constructs and controls (free-
floating islets, C_A_PRS, C_A_ECM) contained
approximately 3000 IEQ isolated from two different
pancreases in two independent isolation proced­
ures. LG – low-glucose medium (2.5 mM of
glucose). D) Stimulation index (SI) measured for
islets encapsulated in controls, A_PRS and A_ECM
24 h after 3D-BP. E) Diffusion of glucose through
A_PRS and A_ECM disc; the diffusion followed
Fick’s law of diffusion. F) Diffusion of insulin
through A_PRS and A_ECM membranes; after 1 h,
the diffusion was significantly higher in the case of
A_ECM membrane.
J. Idaszek et al.
glucose-stimulated insulin secretion was significantly reduced for both
the 3D bioprinted and the free-floating islets, indicating loss of their
functionality (data not shown). Loss of functionality after 24 h of in vitro
culture, i.e., a decrease in SI below 2, has been reported for the
free-floating islets by Wang and co-workers [63].
To further investigate the enhanced endocrine function of the 3D
bioprinted porcine islets, we measured diffusion of glucose and insulin
through A_PRS and A_ECM membranes. The working hypothesis of this
experiment was that, although dECM particles increase the overall
density of the hydrogel, they could disrupt the alginate network, thus
increase its diffusivity. However, obtained results of glucose diffusion
led us to a different conclusion (see Fig. 4E). The lag time, i.e., the time
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Bioprinting 24 (2021) e00163
needed for the diffusion of glucose molecules through the whole thick­
ness of the hydrogel membrane (l), could not be determined from the
diffusion curve, as it was below 15 min. Based on extrapolation of the
linear region of the curve representing constant glucose diffusion flux,
the lag time was estimated to be approximately 5 min and 11 min for the
A_PRS and A_ECM disc, respectively. Those values correspond to diffu­
sion coefficients D equal around 6.6 × 10− 6 cm2 s− 1 for A_PRS (3 % w/v)
and 3.2 × 10− 6 cm2 s− 1 for A_ECM hydrogels. Both values are in the
range of D determined for a blend of alginate (4 % w/v) with gelatin (5
% w/v) equal 1.13 × 10− 6 cm2 s− 1 and water equal 6.7 × 10− 6 cm2 s− 1
[20,64]. Moreover, the D of the A_PRS membrane, i.e., containing 3 %
(w/v) of alginate, was higher than the coefficient reported for
Fig. 5. Biological evaluation of A_FBR bio­
ink. After bioprinting, viability of cells was
visualized by means of L/D staining (A – all
cells, B – dead cells) and quantified using
MTS assay over 1 week of culture (C). The
MTS was performed for samples bioprinted
at two cell densities, i.e., 12 × 106 and 24 ×
106 cells per ml, which were either submit­
ted to secondary crosslinking with thrombin
(A_FBR_T) or left unmodified (A_FBR_C).
Morphology (D–F) and expression of CD31
marker by HUVEC (G–I) after 1 (D, E, G, H)
and 2 weeks (F, I) of in vitro culture. D and G
– A_FBR_C; E, F, H, I – A_FBR_T. Sprouting of
vessels (blue) in vitro from the 3D bioprinted
constructs after 2 weeks of culture in fibrin
hydrogel: J) A_FBR_C, K) A_FBR_T. Scale bar
100 μm.
J. Idaszek et al.
membrane made of 4 % (w/v; D = 5 × 10− 6 cm2 s− 1) [65]. On the other
hand, the increased insulin diffusion through A_ECM discs (depicted in
Fig. 4F) cannot be explained by Fick’s law of diffusion, as it resembles
the super-diffusion variant of anomalous diffusion. Increased insulin
uptake was measured by Duin and coworkers in the case of alginate and
methylcellulose discs, although no difference was observed for the
glucose [21]. However, having taken into account the higher molecular
weight and bigger hydrodynamic radius of an insulin molecule, the
result obtained in the present study is surprising, and the underlying
mechanism of this phenomenon should be better understood.
Because the presence of dECM powder in the alginate-based hydrogel
significantly reduced the diffusion of glucose, the enhanced function­
ality of the 3D-printed islets could likely be attributed to the recapitu­
lation of native niches and matrix-islet interactions provided by dECM
powder. Similar results were obtained by Weber et al., who demon­
strated that incorporating biomolecules derived from pancreatic ECM (e.
g., collagen type IV and laminin) significantly improved secretion of
insulin compared to the bioinert hydrogel [39]. A similar trend was
reported by Kim and colleagues, who showed improved functionality of
insulin-producing cells for increasing degrees of tissue biomimicry [18].
Moreover, the results suggest that the small fiber diameter obtained in
this study (i.e., ~580 μm), combined with high open porosity, was suf­
ficient to overcome the mass transfer limitation observed by others in 3D
plotted scaffolds (fiber diameter of 1.55 ± 0.13 mm) [20]. Improved
functionality of rat islets encapsulated in a small-diameter (<250 μm)
alginate-collagen fibers was demonstrated both in vitro and in vivo by
Jun and coworkers [66].
2.5. 3D-bioprinting of vessel-like structures
Cell viability: LD staining of the HMSC/HUVEC-laden A_FBR scaffolds
was performed after the 3D-BP process. Due to the high cell density, it
was difficult to precisely number the overall amount of cells (green
stain, Fig. 5A). However, the low number of dead cells (depicted in
Fig. 5B) suggested relatively high cell viability, which we estimated to
be around 80 % (based on the ratio of live cells (green stain - red stain
(G-R)) and all cells (i.e., live and dead cells, green stain (G)). To quan­
titatively investigate cell viability, an MTS assay was performed on day 1
and day 7 of culture. Cell metabolic activity increased with the initial
cell density. However, it did not change significantly over the culture
period (see Fig. 5C). The thrombin-catalyzed fibrin polymerization did
not affect the MTS conversion except for the 3D scaffolds bioprinted at a
density of 24 × 106 cells⋅ml− 1 at day 7 of culture. This result could be
explained by significantly lower swelling of the samples crosslinked
with thrombin in the presence of Ca2+ ions due to a higher crosslinking
degree, which led to reduced diffusivity and lower cell metabolic ac­
tivity [67,68]. Another reason for the reduced diffusion could be a layer
of cells that covered the surface of A_FBR_T fibers and thus reduced
diffusion of nutrients into the fibers (see Fig. 5E). Of note, the cell sheet
could also limit the accessibility of the MTS substrate to cells residing
inside the fibers; some of the MTS molecules could not diffuse through
the cell layer because they were irreversibly reduced to formazan upon
interacting with the cells. To overcome this issue in future, we will use
an assay based on detection of ATP to measure cell viability.
2.6. CD31 expression
The early endothelialization of scaffolds containing pro-angiogenic
cells was evaluated by means of immunocytochemical staining against
CD31, a specific marker for endothelial cells (ECs). Moreover, staining
against F-actin was performed to visualize the cytoskeleton. To inves­
tigate the angiogenesis of the constructs, long-culture experiments (up
to 14 days) were performed. Confocal images of the cell-laden constructs
after 7 and 14 days of culture are depicted in Fig. 5. At week 1 (Fig. 5D,
E, G, H), there was evidence of cell migration towards the fiber surface.
Angiogenic activity on spreading cells was visible, as well as the
7
Bioprinting 24 (2021) e00163
formation of EC intracellular junctions (indicated by an arrow on the
insert in Fig. 5H) [69]. This result could be a consequence of adhesion
motives presented by both fibrinogen and fibrin layer. Moreover, in case
of scaffolds crosslinked with thrombin, a significant presence of
HUVEC-bridging points between adjacent fibers was detected (see
Fig. 5H). This is likely due to the mature intercellular junction stabili­
zation occurring between adjacent cells and to the pro-angiogenic signal
transduction resulting from the secreted ECM. Indeed, ECs acquired a tip
cell’s phenotype and form filopodia, leading to preliminary motility
through the fibers [70,71]. 3D bioprinted scaffolds crosslinked with
CaCl2 did not show HUVEC-bridging points, suggesting a
thrombin-mediated angiogenic feedback at the cell-surface interface.
At week 2, much more CD31-positive cells were observed on the
surface of A_FBR_T fibers (Fig. 5G). Moreover, such cells revealed typical
tight junctions-patterns at the intercellular level, which plays a pivotal
role in the adhesion of endothelial cells [72]. In this frame, confluent
HUVEC cells formed a monolayer on the hydrogel surface and guided
characteristic endothelial morphogenesis. In addition, established filo­
podia contacts started tip cell anastomosis by fusion of endothelial
sprouts, and a microvascular network was formed (Fig. 5I). However,
CD31-positive cells did not form a homogenous monolayer on the entire
surface of the constructs. This may be attributed to the different cell
attachment occurred over the whole cell-laden scaffolds [41,73,74]. In
case of A_FBR_C 3D bioprinted scaffolds, the loss of mechanical integrity
occurred after 2 weeks of culture, making the confocal imaging of the
constructs virtually impossible.
In vitro sprouting: After implanting in vivo, the scaffolds would be
covered by fibrin clots due to bleeding induced by the surgical proced­
ure. Therefore, we wanted to investigate the sprouting of blood vessels
in such environment. To investigate this phenomenon, A_FBR_C and
A_FBR_T 3D bioprinted constructs were embedded in fibrin hydrogel and
incubated in co-culture medium for 2 weeks. Representative images of
cells with fluorescently-labeled cell membranes are presented in Fig. 5J
and K, where consistent differences in cell density can be appreciated.
Secondary crosslinking significantly reduced the blood vessel sprouting
from 3D constructs. This could be explained by the higher crosslinking
density of the A_FBR_T hydrogels due to incubation in thrombin/Ca2+
solution. The secondary crosslinking induced the formation of fibrin
shells around the fibers and increased the crosslinking of alginate mol­
ecules, both likely contributing to hindering the cell escape. Similar
correlation between crosslinking density and cell release from alginate
fibers was observed in another study (unpublished data). Our findings
suggest the potential of the secondary crosslinking to modulate cell
release kinetics from 3D bioprinted constructs.
2.7. In ovo CAM assay
In ovo CAM assay on A_FBR scaffolds was performed to investigate
the potential in vivo angiogenic response induced by the transplanted
heterogeneous scaffolds. The chick embryo chorioallantoic membrane
(CAM) is known to possess many advantages [75]. Compared to in vivo
experiments, CAM studies are cost-effective and allow immediate visu­
alization. In addition, it can be considered an intermediate stage be­
tween the in vitro and in vivo models, which enables effective screening
of novel biomaterial configurations. In this frame, CAM assay also pro­
vides reliable outcomes in terms of material-tissue biocompatibility at
the material interface, as it is commonly considered a potential target for
the angiogenesis response [76].
In this study, the CAM assay was used to test the effect of the
hydrogel-based formulations on vascular sprouting and angiogenesis in
a physiological environment. Herein, cell-free and cell-laden scaffolds
were investigated. Results showed the effect of secondary crosslinking
and incorporation of cells on A_FBR scaffolds stability, as well as their
interaction with the membrane (Fig. 6). Evidence-based differences in
scaffold integrity were observed. This was likely due to the different
crosslinking conditions. Indeed, secondary crosslinking with thrombin
J. Idaszek et al.
Fig. 6. In vivo evaluation of the samples in CAM assay. Representative images of A_FBR_C (A, B) and A_FBR_T (C, D) samples without cells (A, C) and with
encapsulated cells (B, D). Histological evaluation of the explanted 3D bioprinted constructs. H&E (E, G) and Sirius red (F, H) stainings were performed to visualize the
overall structure of the explanted samples, CAM, and the presence of blood vessels. Scale bar 200 μm (E–G) and 100 μm (H). SC – acellular A_FBR_T hydrogel scaffold;
SCC – cell-laden A_FBR_T hydrogel scaffolds; black arrows indicate vessels and grey arrow the interface between cell-laden hydrogel fiber and CAM.
reduced the swelling behaviour and increased the stability of the 3D
structures. This was confirmed by the dissolution of some A_FBR_C
samples during the incubation time in CAM (almost 20% of samples),
furtherly enhanced by the incorporation of cells (almost 40 % of dis­
solved scaffolds). It could also be speculated that the higher presence of
Ca2+ - alginate ionic bonds formed during the secondary crosslinking
caused the overall increased stability of A_FBR_T scaffolds, as well as the
presence of fibrin shell surrounding the alginate-fibrinogen core (see
swelling test, Fig. 3A). Moreover, secondary crosslinking with thrombin
likely improved the overall interactions between samples and CAM, as
highlighted in Fig. 6H. On the other hand, acellular scaffolds showed
poor integration with the chorioallantoic membrane (Fig. 6E and F).
Interestingly, it was not possible to obtain histological sections of CAM
incubated with cell-laden A_FBR_C scaffolds due to the disintegration of
the membrane. This finding suggested that increased dissolution of
A_FBR_C samples led to the weakening of the CAM structure. Hence, the
underlying cause should be investigated in future studies. Cell-laden
A_FBR_T scaffolds revealed a dense capillary network at the CAM
interface (depicted in Fig. 6D), suggesting increased angiogenesis. The
macroscopic observation was confirmed by histological analysis (Fig. 6G
and H). The improved angiogenesis could be a result of the paracrine
action of encapsulated cells. Indeed, HMSC secrete pro-angiogenic fac­
tors (i.e., FGF-2, VEGF, and IL-6) [77,78]. Thus, they could promote
tissue vascularization upon incorporation in engineered constructs.
8
Bioprinting 24 (2021) e00163
Interestingly, the study showed no effective angiogenesis on neither
A_FBR_C nor A_FBR_T acellular scaffolds, suggesting the poor influence
of crosslinking methods at this stage of the investigation.
The obtained results highlight the potential of the proposed bioinks
and hydrogel-based scaffolds for engineering heterogeneous tissues.
However, further investigation on the angiogenic potential and the
ability of the encapsulated cells to anastomose with host vasculature
should be undertaken.
2.8. Multi-material 3D-printing: a proof-of-concept
After A_ECM and A_FBR were thoroughly investigated to ultimately
validate the respective TE applications, the two developed bioinks were
co-printed. As a proof-of-concept, this approach was addressed to
determine the potentiality of our 3D-printing method to recreate a
bioartificial vascularized pancreatic microenvironment (Fig. 7A). In this
frame, a microfluidic device bearing a Y-junction was employed to
simultaneously or alternatively deliver A_ECM and A_FBR bioinks
(Fig. 7B and C). Three different scaffold configurations were 3D printed:
i) Janus-scaffold, obtained by the simultaneous delivery of A_ECM and
A_FBR; ii) alternating-layer scaffold, obtained by the alternative delivery
of either A_ECM or A_FBR bioinks, and iii) hybrid scaffold, which is a
hybrid of Janus- and alternating-layer geometries.
Although all three scaffold configurations were successfully 3D
Fig. 7. Proof of concept of the multi-
material 3D printed scaffolds by using the
two developed hydrogel-based inks (A_FBR
and A_ECM). Geometries and representative
optical images of multi-material 3D printed
scaffolds, where material “A” indicates
A_FBR bioink and material “B” refers to
A_ECM formulation. Herein, AB/AB (A), A/B
(B), and AB/B (C) 2-layer scaffolds are dis­
played. (A) In the Janus scaffold geometry,
two filaments of both bioinks are simulta­
neously compartmentalized in the same 3D-
printed fiber, while in the alternating-layer
scaffold (B) the geometry was obtained by
alternating a layer of A_FBR with a layer of
A_ECM (A/B). The hybrid scaffold (C) was
obtained by alternating a Janus-layer with a
layer of A_ECM fibers.
J. Idaszek et al.
printed, the fabrication of alternating-layer scaffolds was more chal­
lenging due to the inertial forces present in the A_ECM bioink, which did
not allow the precise and controlled switching of A_ECM and A_FBR
bioinks. This might lead to the fabrication of multiple layers not entirely
composed of a single specific ink formulation. To overcome this issue,
higher flow rates up to 50 μl min− 1 were applied for a very short time (i.
e., 10 s) prior to the 3D printing of the layer. However, an increase of
bioink flow rate induces higher shear stress, especially for high-viscous
A_ECM, which may hamper cell viability and functionality. Therefore,
simultaneous delivery of A_ECM and A_FBR for 3D-printing of Janus-
microfiber scaffolds has been selected as a potential method to fabri­
cate vascularized-pancreatic bio-hybrid structures. Moreover, the Janus-
fiber configuration may provide a biological advantage by increasing the
paracrine communication between pancreatic islets and HMSC/HUVEC
due to higher physical proximity than the other scaffold configuration.
3. Conclusion
In this study, we have presented a novel approach for microfluidics-
assisted multi-material 3D bioprinting of vascularized pancreatic sub­
stitutes. The incorporation of pancreatic dECM and fibrinogen into
alginate enabled the development of two distinct tissue-specific bioinks
and 3D bioprinting of hydrogel scaffolds characterized with high shape
fidelity and open porosity. The bioinks were suitable for the 3D bio­
printing of islets- and HMSC/HUVEC-laden constructs and supported
endocrine function of porcine pancreatic islets (secretion of insulin upon
glucose stimulation) and vascularization (formation of intercellular
junctions and vessel-like structures by CD31-positive cells). Secondary
crosslinking with thrombin in the presence of Ca2+ increased the sta­
bility of the A_FBR hydrogels and enabled the modulation of in vitro
vessel sprouting, which was higher in the absence of secondary cross­
linking. On the other hand, the higher angiogenic potential in vivo was
demonstrated by more stable hydrogel scaffolds in CAM assay.
To better understand the effect of secondary crosslinking on angio­
genesis, further work will include evaluation of the A_FBR scaffolds by
means of prolonged CAM assay. Moreover, we will investigate the
impact of paracrine activity of HMSC/HUVEC on islets functionality by
co-printing the islets and cells within one scaffold. Another aspect to be
pursued in the future study is the effect of different sizes of dECM par­
ticles on the 3D-BP and behaviour of pancreatic islets.
In conclusion, the presented results can be considered a significant
advancement both in the field of biofabrication of vascularized
pancreatic substitutes and microfluidic-assisted multi-material/multi-
cellular 3D bioprinting, as they expand the biofabrication window by
application of alginate-based biomimetic bioinks.
4. Experimental section
4.1. Materials
Sodium alginate with high molecular weight (75–200 kDa, PRO­
NOVA UP LVG) and low molecular weight (<75 kDa, PRONOVA UP
VLVG) were purchased from Novamatrix (Norway). Fibrinogen and
thrombin were purchased from Baxter (Austria) as a part of tissue glue
kit Tisseel Lyo. Calcium chloride (CaCl2) was purchased from Eurochem
BGD (Poland).
4.2. Decellularization of porcine pancreas
Pig pancreases (total weight of 5 kg) were collected at a local
slaughterhouse. After harvesting, all organs were frozen at − 20 ◦ C. Prior
the decellularization process, the pancreases were thawed, manually
cleaned of fat and rinsed with streptomycin solution (0.1 mg ml− 1). The
decellularization process was carried out by immersing the pancreases
in a 1 % (v/v) solution of Triton X-100 in PBS and placing it in an
incubator shaker (Eppendorf Innova) maintained at 4 ◦ C and mixed at
9
Bioprinting 24 (2021) e00163
150 rpm. The solution of the detergent was changed every 4 h. The ef­
ficiency of the decellularization was evaluated by measuring the total
DNA content with a DNA quantification assay (Quant-iT PicoGreen
dsDNA Assay kit, Invitrogen). The obtained dECM was frozen in liquid
nitrogen, ground into fragments of about 0.5 cm, and subjected to the
lyophilization at − 32 ◦ C. The obtained lyophilizate was subsequently
ground in a cryogenic mill. The volumetric particle size distribution was
determined by means of diffraction aerosol spectrometer Spraytec
(Malvern Instruments, UK) operating at a flow rate of 100 dm3 min− 1.
4.3. Ink preparation
A_PRS was prepared by dissolving 3 % (w/v) alginate (2 % (w/v)
high molecular weight, 1 % (w/v) low molecular weight) in HEPES
buffer (25 mM) containing 10 % (v/v) of fetal bovine serum (FBS) and 1
% of antibiotic-antimycotic solution (AA; 10 000 units⋅ml− 1 penicillin,
10 mg ml− 1 streptomycin and 25 μg ml− 1 amphotericin B).
A_ECM was prepared by dissolving 15 % (w/v) dECM porcine pow­
der in A_PRS. A_FBR was prepared by dissolving 9 % (w/v) fibrinogen in
a solution of 3000 KIU aprotinin at 35 ◦ C. After complete dissolution, 1
ml of fibrinogen solution was added under stirring condition to 1 ml of a
2-times concentrated solution of A_PRS to obtain a final concentration of
A_PRS equal 3 % (w/v) and 4.5 % (w/v) of fibrinogen. The thrombin
solution used for the post-process crosslinking of A_FBR was prepared by
dissolving 500 U/ml of thrombin in a solution of 40 mM CaCl2.
4.4. Rheology
The rheological properties of A_PRS, A_FB, and A_ECM were inves­
tigated by using an ARES rheometer (TA Instruments, Delaware, USA)
with cone and plate geometry, setting the gap at 51 μm. Shear viscosity
behaviour was evaluated by applying shear rates ranging from 0.1 to
100 s− 1. All measurements were performed at a fixed temperature of
25 ◦ C, controlled by a Peltier system on the plate.
4.5. 3D-bioprinting set up
Design of the microfluidic printing head: 3D-bioprinting was performed
by coupling a microfluidic platform with a 3D-printer (3D-Bioplotter™
System, EnvisionTEC, Germany). The microfluidic platform was
designed with a Y-junction (2 inlets, 1 outlet) to be purposely compat­
ible with simultaneous or alternative multi-material deposition. The
microfluidic device was fabricated as previously reported [34]. The
outlet of the microfluidic platform was fluidically connected to a
co-axial nozzle system formed by inner needle (21G) and outer needle
(16G), as already described in previous studies [32,36,79]. Finally, the
dispensing co-axial system was screwed at the bottom of a
low-temperature cartridge of 3D-Bioplotter.
Dispensation of the bioink: In multi-material 3D-printing, both bioinks
were loaded into 3 ml syringes subsequently placed into programmable
microfluidic syringe pumps (neMESYS low pressure, CETONI GmbH),
and the CaCl2 solution (0.2 M) was accommodated on a single-channel
microfluidic pump (New Era Pump Systems, Inc). Inks and CaCl2 were
connected, respectively, to the inlets of the microfluidic system and to
the outer of the co-axial nozzle system by polyethylene (PE) microfluidic
tubings. In case of single-bioink 3D-printing, only one inlet of the Y-
junction was used and connected to the system. Once either A_ECM or
A_FBR came in contact with CaCl2 at the tip of the co-axial system, the
instantaneous gelation process occurred.
4.6. 3D-bioprinting
The 3D-printed fiber diameter was investigated by increasing the
deposition speed (i.e., 1–6 mm s− 1) and ink flow rate (i.e., 5, 10, 15, 20,
25, and 30 μl min− 1). Microfibers were printed on cover glasses, and
microfiber diameter was measured using a confocal microscope (Leica
J. Idaszek et al.
TCS SP8). Five microfibers were measured for each combination of pa­
rameters and each ink formulation.
3D printed scaffolds were designed to have dimensions correspond­
ing to 1 × 1 × 0.3 cm and 0◦ –90◦ orientation. Inks and CaCl2 were
dispensed at flow rates of 15 and 8.5 μl min− 1, respectively. The printing
speed was set to 2.5 mm s− 1. Hydrogel microfibers were printed as
continuous filaments with a fiber distance of 900 μm and 190 μm layer
thickness. Immediately after 3D-printing, A_FBR_T was subjected to
secondary crosslinked by incubation in thrombin solution at 37 ◦ C for
15 min. Scaffolds without secondary crosslinking (A_FBR_C) were used
as a control. Fiber diameter and pore size of the 3D printed scaffolds
were measured using a confocal microscope. Five different samples (n =
5) were analyzed for each ink formulation.
Multi-material 3D printing: As a proof of concept, the fabrication of a
multi-material 3D printed scaffold was also performed. To obtain Janus-
fiber constructs, A_FBR and A_ECM were simultaneously delivered at
flow rates of 10 and 5 μl min− 1, respectively. To obtain 3D scaffolds
formed by alternating layers of A_ECM and A_FBR, inks were alterna­
tively delivered at 15 μl min− 1.
4.7. Swelling test
To investigate water absorption capacity, 3D printed scaffolds were
weighed after 72 h of immersion in DI water at 37 ◦ C to obtain swollen
weight (Ws). Samples were then frozen at − 80 ◦ C overnight, lyophilized
for 1 day, and weighed again to obtain dry weight (WD). Water ab­
sorption capacity (ΔW%) was obtained as: ((Ws- WD)/WD) × 100.
4.8. Mechanical characterization
The compressive mechanical test was performed on 3D-printed
scaffolds (n = 4 per ink formulation) after 72 h-incubation in DI water
using a Dynamic Mechanical Analyzer (DMA Q800, TA instruments).
Mechanical tests were conducted at 37 ◦ C with a pre-load of 0.001 N, by
applying a compressive strain ramp at 2.5 % min− 1 until 30 % strain.
From stress-strain (σ–ε) curves, elastic modulus (E) was calculated as the
slope in the 0–5% strain range, R2 > 0.9).
4.9. Diffusion of glucose and insulin
Solid hydrogel discs with a diameter of 15 mm were fabricated using
ion eluting molds. Briefly, 200 μl of either A_PRS or A_ECM bioinks was
cast into molds made of agarose (1 % (w/v) in deionized (DI) water) and
immersed in 0.2 M CaCl2 for 25 min. The surface of the mold was
covered with MCE membrane (MF-Millipore). The samples were washed
with DI water and placed between 2 rubber seals in an in-house made
diffusion chamber. Porcine insulin (Sigma) was dissolved in a RPMI
1640 (Gibco, Thermo Fisher) medium at a concentration of 2.5 μg ml− 1
and pipetted into one of the chambers. RPMI 1640 medium without
glucose (Gibco, Thermo Fisher) was added to the other chamber; to
measure the diffusion of glucose and insulin, 0.5 ml of the medium was
removed at 15 min intervals for up to 1 h. According to the manufac­
turer’s protocols, glucose concentration was determined using glucose
colorimetric detection assay (Invitrogen) and insulin by means of
porcine insulin ELISA assay (Invitrogen).
4.10. Isolation of pancreatic islets
Pancreatic islets were isolated from the pancreases of breeding pigs,
which died as a result of bleeding out within 2 h of the organs being
harvested. The entire process of preparing the organs for isolation was
carried out under hypothermic conditions. After cleansing of the fatty
tissue, the pancreas was injected with a solution of NB8 collagenase at a
concentration of 2.73 mg of collagenase per 1 g of pancreas and neutral
protease (100 DMC-U). The solution was administered directly into the
Wirsung’s duct. Then, the organ was mechanically fragmented. The
10
Bioprinting 24 (2021) e00163
entire isolation procedure was done using Ricordi’s chamber. The iso­
lated pancreatic islets were pooled and stored at 4 ◦ C until the start of
the 3D-BP process.
4.11. Cell culture
Human bone marrow-derived mesenchymal stem cells (HMSC) were
obtained from Dr. Jan E. Brinchman of Oslo University Hospital. The
cells were isolated and validated as HMSC according to protocols
developed in the past [80–82] and expanded in a medium consisting of
α-MEM (Gibco, UK) supplemented with 10 % fetal bovine serum (FBS,
Biowest, South America origin), 1 % of antibiotics (10′ 000 U ml− 1
penicillin, 10 μg ml− 1 streptomycin; Gibco, USA) and 1 ng ml− 1 human
basic fibroblast growth factor 2 (both Sigma-Aldrich). Human umbilical
vein endothelial cells (HUVEC) were purchased from Lonza and cultured
in a dedicated medium (EGM-2 Endothelial Medium Bullet Kit, Lonza).
Cells used in the 3D-BP experiments were from passages 4 and 5. For the
co-culture experiments, a mixture of EGM-2 and supplemented α-MEM
at ratio 4:1 was used.
4.12. 3D-bioprinting process of islets- and cell-laden scaffolds
For the bioinks preparation, A_PRS and CaCl2 (0.2 M) were sterile-
filtered (pore Ø = 0.22 μm). The other bioink components (i.e., 2×
A_PRS, fibrinogen, and dECM powder) were provided already sterile.
Islets-laden A_PRS and A_ECM bioinks were prepared by mixing A_PRS
and A_ECM with the islets at density, ensuring approximately 3000 IEQ
per construct. Cell-laden A_FBR bioink was prepared by mixing A_FBR
with HUVECs and HMSC at 1:2 ratio [83,84], respectively, to achieve a
final density of 2.4 × 107 cells mL− 1. The as-prepared bioinks were
loaded into syringes and then 3D-bioprinted with the same printing
parameters used for acellular inks.
4.13. Evaluation of viability
Live/dead (L/D) staining: Islets and cell viability was evaluated post-
printing using live and dead staining. Briefly, the printed islets and cell-
laden constructs were incubated in a solution of acridine orange (Sigma,
3 μg mL− 1, green fluorescence, all cells (G)) and propidium iodide
(Sigma, 10 μg mL− 1, red fluorescence, dead cells (R)) dissolved in 25 mM
HEPES for approximately 5 min and analyzed with a fluorescent mi­
croscope (Leica TCS SP8) at wavelengths corresponding to fluorophores
of interest. The cells visible in green and red were counted, and the
viability was calculated according to the formula: % viability = 100 %⋅
(G-R)/G.
MTS assay: The HMSC/HUVEC-laden bioprinted constructs were
washed with α-MEM w/o FBS and moved to new wells containing 1 ml of
pre-incubated α-MEM w/o FBS. Subsequently, 200 μL of MTS (Cell Titer
96 Aqueous One Solution Cell Proliferation Assay, Promega, USA) was
added, and the samples were incubated at 37 ◦ C and 5 % CO2 for 1 h,
followed by the addition of 250 μL of 10 % SDS and shaking for 30 min
100 μL, aliquots of the supernatant were transferred quadruplicates into
a 96-well plate, and the absorbance was measured at λ = 490 nm.
4.14. Evaluation of islets functionality
Glucose-stimulated insulin secretion (GSIS): The solution of 2.5- and
16.7 mM glucose was prepared by diluting glucose in sterile Creb’s
buffer. Before use, the solutions were heated to 37 ◦ C, and the time zero
samples were taken from both solutions. Islets-laden scaffolds were
placed on the inserts and submerged in 2.5 mM glucose solution. After
30 min, inserts were dried on tissue paper and placed in 16.7 mM
glucose solution for 60 min. This was followed by drying inserts on
tissue paper and placing them in 2.5 mM glucose solution for another 60
min. During the entire assay, the plates were incubated in the cell cul­
ture incubator at 37 ◦ C, and 100 μL of supernatant was taken every 15
J. Idaszek et al.
min and frozen at − 80 ◦ C for further analysis by means of ELISA assay
(Demeditec Diagnostics GmbH (Insulin ELISA ref: DE2935) according to
manufacturer’s protocol.
4.15. Evaluation of cell morphology and formation of endothelial cell
intercellular junctions
The cytoskeleton was visualized by staining of F-Actin (Alexa Fluor
Phalloidin). Scaffolds were fixed with 4 % paraformaldehyde for 30 min
and then washed with HEPES thrice, followed by permeabilization with
0.2 % Triton X-100 (v/v) in HEPES for 15 min. In order to block un­
specific binding, the constructs were incubated in 10 % (v/v) goat serum
(GS) for 30 min at RT. Subsequently, the samples were incubated with
rabbit polyclonal anti-CD31 primary antibody (1:20, Abcam ab28364)
and Alexa Fluor 546 phalloidin (1:40, Invitrogen) overnight at 4 ◦ C.
After washing thrice with HEPES, scaffolds were furtherly incubated
with Alexa Fluor 488 anti-rabbit secondary antibody produced in goat
(1:300) for 3 h in the darkness at RT. Finally, cell nuclei were stained
with Draq5 solution (1:1000, Thermo Scientific) and incubated for 15
min in the dark and washed thrice with HEPES before imaging by
confocal microscope (Nikon A1R).
4.16. In vitro vessel sprouting
The 3D-bioprinted scaffolds were embedded in fibrin matrices
(TISSEEL, Baxter) with a final concentration of 2.5 mg/ml fibrinogen
and crosslinked with 0.2 IU/ml thrombin for 30 min. The cell-laden
scaffolds were cultured in co-culture medium and fixed with 4 % para­
formaldehyde buffered with HEPES after 2 weeks. Prior to the obser­
vations, the cells were labeled with DiD dye (Molecular Probes) and
washed extensively with HEPES. Due to the high opacity of the samples,
glycerine was applied to improve the visibility of the embedded cells
[85]. The constructs were analyzed with a confocal microscope (Leica
TCS SP8) at excitation wavelengths of 633 nm.
4.17. Evaluation of angiogenic properties of the bioinks by means of in
ovo CAM assay
Fertilized eggs from Ross line 308 hens were obtained from a certi­
fied hatchery. The eggs were cleaned, sterilized with UVC light and
divided into four groups (4 × 15 eggs). Embryos were incubated at
standard conditions (temperature 37 ◦ C, humidity 60 %, and turned
once per hour). At day 9 of embryonic development, scaffolds were
placed on the chorioallantoic membrane (CAM). A hole of approxi­
mately 1.5 cm was made in the shell and, subsequently, the inner
membrane was gently stripped off, and a scaffold was placed on CAM.
Chicken embryos were incubated to day 11 of embryonic development
when scaffolds with CAM were prefixed with 1 ml of 4 % para­
formaldehyde. After 5 min of incubation, CAM with implants were cut
out and fixed at 4 ◦ C in 4 % paraformaldehyde for 60 min. Scaffolds with
CAM were imaged using stereomicroscope under a 6.3-fold magnifica­
tion (SZ×10, CellD software version 3.1; Olympus Corporation, Tokyo,
Japan). Subsequently, the samples were incubated in a gradient of su­
crose (10, 20, and 30 w/v%), embedded in Shandon Cryomatrix
(Thermo Scientific), and cut with a cryostat (Microm HM 550) to yield
10 μm sections. The histological sections were stained with hematoxylin
and eosin (H&E) or Sirius red, followed by examination using light
microscope (Zeiss Axio Scope A.1).
4.18. Statistical analysis
Results are expressed as means ± standard deviation. Two-way
ANOVA with Tukey’s multiple comparison tests was performed using
GraphPad Prism ® analysis software (GraphPad Software, La Jolla, CA)
to investigate statistical differences between data populations. Differ­
ences are displayed as statistically significant when p < 0.05.
11
Bioprinting 24 (2021) e00163
CRediT authorship contribution statement
Joanna Idaszek: Conceptualization, Methodology, Investigation,
Writing – original draft, Writing – review & editing, Visualization, Su­
pervision. Marina Volpi: Methodology, Investigation, Formal analysis,
Writing – original draft, Writing – review & editing, Visualization.
Alessia Paradiso: Methodology, Investigation, Formal analysis, Writing
– original draft, Writing – review & editing, Visualization. Martyna
Nguyen Quoc: Investigation. Żaneta Górecka: Investigation. Marta
Klak: Conceptualization, Investigation, Methodology. Grzegorz
Tymicki: Investigation, Methodology. Andrzej Berman: Investigation,
Methodology. Mateusz Wierzbicki: Investigation, Methodology,
Writing – original draft, (CAM model). Sławomir Jaworski: Investiga­
tion, Methodology. Marco Costantini: Methodology. Agnieszka
Kępczyńska: Methodology, Investigation. Ewa Sawosz Chwalibóg:
Supervision, Resources. Michał Wszoła: Conceptualization, Investiga­
tion, Supervision, Resources, Funding acquisition. Wojciech
Święszkowski: Conceptualization, Supervision, Resources, Funding
acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
This work was supported by the National Centre for Research and
Developments in the frame of the STRATEGMED program (contract no.
STRATEGMED3/305813/2/NCBR/2017). The authors would like to
acknowledge Katarzyna Kosowska, Tomasz Bryniarski, Piotr Cywoniuk,
Paweł Turowski, and Magdalena Gomółka, all of Foundation of Research
and Science Development, for their assistance in preparation of
pancreatic dECM, isolation of pancreatic islets, and GSIS assay. We
would also like to thank Mariusz Nyc of the Faculty of Materials Science
and Engineering, Warsaw University of Technology, for his assistance in
rheological measurements.
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