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Journal ArticleDOI

A Bioprinted Cardiac Patch Composed of Cardiac-Specific Extracellular Matrix and Progenitor Cells for Heart Repair.

TL;DR: This work shows the successful bioprinting and implementation of cECM‐hCPC patches for potential use in repairing damaged myocardium.
Abstract: Congenital heart defects are present in 8 of 1000 newborns and palliative surgical therapy has increased survival. Despite improved outcomes, many children develop reduced cardiac function and heart failure requiring transplantation. Human cardiac progenitor cell (hCPC) therapy has potential to repair the pediatric myocardium through release of reparative factors, but therapy suffers from limited hCPC retention and functionality. Decellularized cardiac extracellular matrix hydrogel (cECM) improves heart function in animals, and human trials are ongoing. In the present study, a 3D bioprinted patch containing cECM for delivery of pediatric hCPCs is developed. Cardiac patches are printed with bioinks composed of cECM, hCPCs, and gelatin methacrylate (GelMA). GelMA-cECM bioinks print uniformly with a homogeneous distribution of cECM and hCPCs. hCPCs maintain >75% viability and incorporation of cECM within patches results in a 30-fold increase in cardiogenic gene expression of hCPCs compared to hCPCs grown in pure GelMA patches. Conditioned media from GelMA-cECM patches show increased angiogenic potential (>2-fold) over GelMA alone as seen by improved endothelial cell tube formation. Finally, patches are retained on rat hearts and show vascularization over 14 days in vivo. This work shows the successful bioprinting and implementation of cECM-hCPC patches for potential use in repairing damaged myocardium.

Summary (2 min read)

1. Introduction

  • Congenital heart defects affect 35 000 newborns annually, resulting in significant impairments in cardiac function and increased patient morbidity and mortality. [1,2].
  • This chronically elevated load leads to increased fibrosis and hypertrophy, resulting in RV failure. [1].
  • For one, the local structure of the material cannot be controlled during injection, a property that may alter stem cell phenotype.[22].
  • A bioprinting methodology that prints both cells and ECM without using nondegradable components is key in generating functional heart patches with high design control.
  • Ultimately, the patch could be used as therapy for pediatric patients suffering from RV failure, or perhaps even in an allogeneic manner for adult cardiac dysfunction.

2.1.1. Bioprinting Acellular Structures

  • Bioprinting of ECM-based materials has mainly been achieved with the inclusion of a filler polymer to allow for proper printing viscosity.
  • Surprisingly, cECM has been printed directly without the use of filler polymer; however, this approach suffers from two main issues. [26,32].
  • As can be seen in Figure 2D, both GelMA and GelMA-cECM bioinks had printability close to a value of 1.0 and the inclusion of cECM improved the printability of the bioink significantly to achieve a value closest to ideal printing.

2.1.2. Bioprinting hCPC-Laden Structures and Cardiac Patches

  • To perform this, hCPCs were incorporated into the bioinks and evaluated for effectiveness in creating homogeneously distributed cell-laden print structures.
  • As shown in the bright field images in Figure 3, the authors were able to add cells to the print for both GelMA alone and GelMAcECM , where cells were retained in the gels after cross-linking.
  • Figure 3C shows the printed grids after swelling, indicating that the cells appeared homogeneously distributed throughout the filaments.
  • Printing parameters were not modified by the incorporation of cells, maintaining a low printing pressure (0.7–0.8 bar), and thus low shear stress, on the cells.
  • Figure 4A shows the printed patches prior to white light polymerization while Figure 4B shows the CAD models using the patch design.

2.2. hCPC Viability, Differentiation, and Proliferation within Bioprinted Cardiac Patches

  • Evaluating the viability of cells within the cardiac patches is critical to ensure live cells that can participate in producing important proreparative paracrine factors.
  • There was no significant difference between groups or time points when comparing the percent of viable cells.
  • The cell viability overall was most likely not impacted significantly by the printing methodology, or if there were effects to the cells due to the printing, the degree of cell damage was mitigated by the material being an effective environment for cell growth and nutrient diffusion coupled with printing of aligned fibers which may be beneficial to cell function.
  • HCPCs in GelMA-cECM patches showed enhanced cardiac differentiation through increased expression of MEF2C, Cx43, and MYH7, and decreased expression of GATA4, an early differentiation marker, indicating that the hCPCs in GelMA-cECM patches were moving toward later differentiation than hCPCs in GelMA patches.
  • Regardless, it is clear that while hCPCs remained viable in printed patches, the inclusion of cECM improved differentiation and reduced proliferation of hCPCs, which in turn may improve paracrine potential of hCPC-laden GelMA-cECM patches.

2.4. Mechanical Characterization of Bioprinted Patches

  • Mechanical properties of biomaterials play a critical role in modulating cellular function.
  • While the modification increased stiffness over GelMA and GelMA-cECM groups, the patches more readily degraded compared to both groups and did not alter the hCPC viability or paracrine function over GelMA-cECM (data not shown), so this direction was not pursued further, although the properties of the patch could potentially be modified through this method.
  • As seen in Figure 7A, the modulus of pure GelMA was 3000 Pa, similar to published studies, though short of the native myocardium.[56].
  • The authors evaluated the degradation of patches and materials in cell treatment media over 21 d by examining both the change in wet weight and change in stiffness .
  • To evaluate the degradation of the patches in a more physiological relevant environment, hCPC-laden materials were cultured in conditioned media harvested from cardiac fibroblasts (cFBs), which would be present in cases of ventricular remodeling and hypertrophy.

2.5. In Vivo Implantation of GelMA-cECM Patches

  • Attachment of GelMA-cECM patches onto the epicardial surface is critical to ensure the devices can be deployed with minimal manipulation.
  • The authors evaluated the potential of the patches to remain attached to rat hearts after placement on the epicardium.
  • All three methods allowed for patch placement on beating rat hearts, without buckling or patch damage.
  • In either case, the patches were retained throughout 14 d without change to patch shape, fluorescence expression of the cECM-bound dye, or buckling.

3. Conclusions

  • Here, the authors report the development of a novel pediatric hCPC/ cECM cardiac patch that was generated through bioprinting.
  • The inclusion of 5% w/v GelMA allowed for printability of the hCPC/cECM bioink through GelMA polymerization via cooling to 10 °C, followed by white light radical polymerization and incubation at physiological temperatures.
  • Finally, the printed GelMA-cECM patches were effectively attached to rat hearts epicardially, remained on the hearts for 14 d, and showed vascularization.
  • The concentration of cECM used here is similar to previous studies performed in their laboratory and by others that support CPC differentiation and function.
  • Translation of the patch would involve the use to autologous hCPCs derived directly from patients, allowing for generation of patient-specific paracrine release while using commercially available biomaterials such as porcine cECM.

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FULL PAPER
1800672 (1 of 13)
©
2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
A Bioprinted Cardiac Patch Composed of Cardiac-Specific
Extracellular Matrix and Progenitor Cells for Heart Repair
Donald Bejleri, Benjamin W. Streeter, Aline L. Y. Nachlas, Milton E. Brown,
Roberto Gaetani, Karen L. Christman, and Michael E. Davis*
DOI: 10.1002/adhm.201800672
only restorative treatment for patients is
transplantation, which is limited by the
availability of donor hearts and transplant
rejection.
[2,3]
Even in cases where there
is not critical RV dysfunction, RV output
remains reduced, leading to poor quality
of life.
Reparative therapies for improve-
ment of cardiac function are critical, and
although limited in pediatric populations,
new treatments are being explored.
[4–9]
While there have been hundreds of stem
cell trials in adults, very few address
pediatric populations.
[4–6]
A recent study
showed that intracoronary infusion of
cardiosphere-derived cells can improve RV
function in children and follow-up studies
have been promising.
[7]
In addition, bone
marrow stem cells and cord blood-derived
mononuclear cells also improved RV func-
tion following intramyocardial injection.
[8]
We have recently shown that progenitor
cells (CPCs) could improve the failing
RV of juvenile rats subjected to pulmo-
nary banding and a clinical trial is now
underway (NCT03406884).
[9]
Despite this
enthusiasm, similar concerns exist in children as have been
shown in adults. While CPC therapy demonstrated modest
improvements in adult therapy, most CPCs were lost to circu-
lation immediately after injection into the myocardium.
[10,11]
In addition, cells are being injected into a diseased micro-
environment that may not provide healthy cues for optimal
CPC function.
[11]
To increase retention and modify the local microenviron-
ment, researchers have used both synthetic and natural bioma-
terials.
[12,13]
Inclusion of appropriate cues can both direct the
fate of the implanted cells, and improve the release of parac-
rine factors, a main mechanism of cellular therapy.
[14,15]
Several
studies, including ones from our laboratory, have shown that
a decellularized cardiac extracellular matrix hydrogel (cECM) is
a promising biomaterial used in the repair of myocardial dys-
function in adults, as well as for the delivery of stem cells.
[16–21]
In prior studies, cECM increased the differentiation of rat
CPCs compared to either collagen or adipose ECM alone.
[16,20]
Moreover, cECM is currently in clinical trials for adults post-
myocardial infarction (NCT02305602) and thus, combined
with human pediatric CPCs, could rapidly advance to human
testing. In adults, the material is delivered invasively through
Congenital heart defects are present in 8 of 1000 newborns and palliative
surgical therapy has increased survival. Despite improved outcomes, many
children develop reduced cardiac function and heart failure requiring trans-
plantation. Human cardiac progenitor cell (hCPC) therapy has potential to
repair the pediatric myocardium through release of reparative factors, but
therapy suffers from limited hCPC retention and functionality. Decellular-
ized cardiac extracellular matrix hydrogel (cECM) improves heart function in
animals, and human trials are ongoing. In the present study, a 3D-bioprinted
patch containing cECM for delivery of pediatric hCPCs is developed. Cardiac
patches are printed with bioinks composed of cECM, hCPCs, and gelatin
methacrylate (GelMA). GelMA-cECM bioinks print uniformly with a homoge-
neous distribution of cECM and hCPCs. hCPCs maintain >75% viability and
incorporation of cECM within patches results in a 30-fold increase in cardio-
genic gene expression of hCPCs compared to hCPCs grown in pure GelMA
patches. Conditioned media from GelMA-cECM patches show increased
angiogenic potential (>2-fold) over GelMA alone, as seen by improved
endothelial cell tube formation. Finally, patches are retained on rat hearts
and show vascularization over 14 d in vivo. This work shows the successful
bioprinting and implementation of cECM-hCPC patches for potential use in
repairing damaged myocardium.
D. Bejleri, B. W. Streeter, A. L. Y. Nachlas, M. E. Brown, Prof. M. E. Davis
Department of Biomedical Engineering
Georgia Institute of Technology and Emory University
1760 Haygood Dr., Atlanta, GA 30322, USA
E-mail: michael.davis@bme.gatech.edu
Dr. R. Gaetani, Prof. K. L. Christman
Department of Bioengineering and Sanford Consortium
for Regenerative Medicine
University of California, San Diego
2880 Torrey Pines Scenic Dr., La Jolla, CA 92037, USA
Cardiac Bioprinting
1. Introduction
Congenital heart defects affect 35 000 newborns annually,
resulting in significant impairments in cardiac function and
increased patient morbidity and mortality.
[1,2]
Although surgical
treatment methods have improved outcomes, many children
end up with right-ventricular (RV) dysfunction due to increased
load.
[1,2]
This chronically elevated load leads to increased fibrosis
and hypertrophy, resulting in RV failure.
[1]
In cases where RV
dysfunction persists, 18-month survival rates are 35%.
[3]
The
Adv. Healthcare Mater. 2018, 7, 1800672

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2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1800672 (2 of 13)
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a catheter, which can present certain challenges.
[19]
For one,
the local structure of the material cannot be controlled during
injection, a property that may alter stem cell phenotype.
[22]
In
addition, while myocardial infarction is a localized disease,
heart failure due to congenital heart defects may be more global
and local delivery may not be sufficient.
[1–3]
One powerful method of generating controlled 3D struc-
tures for cardiac therapy is bioprinting, which has been
used extensively to produce highly defined geometries of
biomaterials and cells.
[23–28]
Bioprinting is effective in gen-
erating polymeric scaffolds, but can be problematic for
naturally derived materials.
[24,25]
For the case of bioprinting
ECM-derived materials, current methods rely on creating
nondegradable polymeric support scaffolds, or require high
concentrations of poorly printed ECM.
[26–28]
The inclu-
sion of polymers produces device-tissue and cell-material
mechanical mismatch, and imposes degradation limita-
tions.
[29]
Further, finding materials that are compatible with
ECM printing is not trivial.
[30,31]
A bioprinting methodology
that prints both cells and ECM without using nondegradable
components is key in generating functional heart patches
with high design control.
This work focuses on developing a bioprinted cardiac patch
composed of native cECM and pediatric human CPCs (hCPCs),
for use as an epicardial device that releases paracrine factors
into the dysfunctional myocardium. The patch may overcome
problems seen in cell therapies by retaining viable hCPCs in
naturally derived cECM, and allowing for improved paracrine
release from hCPCs through the bioactive cECM inducing
guiding effects on cells.
[10,16,20]
Additionally, the bioprinting
approach allows for generation of highly defined patches with
uniform component distribution.
[23]
Ultimately, the patch
could be used as therapy for pediatric patients suffering from
RV failure, or perhaps even in an allogeneic manner for adult
cardiac dysfunction.
2. Results and Discussion
2.1. Bioprinting of hCPC/cECM Cardiac Patches
2.1.1. Bioprinting Acellular Structures
Bioprinting of ECM-based materials has mainly been achieved
with the inclusion of a filler polymer to allow for proper
printing viscosity.
[23,25,26]
ECM solutions at therapeutic concen-
trations (6–10 mg mL
1
) are low viscosity prepolymers, which
do not print effectively due to layers remaining fluid and nono-
verlapping, while polymerized ECM is a fibrous material that,
while more viscous then the prepolymer, comes out in “chunks
rather than a homogenous stream of print filaments.
[18,19,26]
Surprisingly, cECM has been printed directly without the use
of filler polymer; however, this approach suffers from two main
issues.
[26,32]
The first is that the required concentration for
printing pure cECM (20 mg mL
1
) is significantly higher than
has been used in treatment studies with cECM and requires
extensive harvesting from porcine sources for generation of a
limited number of devices. Second, and more pressing, is that
the pure cECM-printed materials are difficult to handle and
risk rupture when potentially used as an epicardial patch, due
to their low mechanical modulus and fibrous nature.
[18,33]
In
order to address this, cECM was printed with the use of filler
polymers such as polycaprolactone in alternating layers, which
then produced mechanical mismatch with the patch and the
native myocardium, also rendering the patch with a degrada-
tion time much longer than a natural biomaterial system.
[32]
Although methods have been employed to modify pure cECM
mechanical properties in printed constructs, such as by inclu-
sion of vitamin B2, it is unclear if this method can be employed
as a cell-laden patch without the use of supporting polymer
layers.
[27,32]
To generate a cECM patch that has a high degree of printa-
bility, proper mechanical properties for myocardial therapy, and
allows for cell viability and paracrine release, we used gelatin
methacrylate (GelMA) as a support material. GelMA is a natural
biomaterial based on collagen, which has methacrylate groups
grafted onto the gelatin structure so that the material can
undergo radical polymerization.
[34,35]
GelMA is used extensively
as a bioactive and resorbable material for regenerative medicine
applications, and in a multitude of tissues such as muscle, liver,
and bone.
[34–36]
In order to limit cell damage, we employed a
white light system for gel polymerization after structure forma-
tion. This white light system has advantages over UV systems
that otherwise induce increased cell death and stress.
[37–39]
We
investigated the use of various cross-linking systems, such as
ruthenium-sodium persulfate or Irgacure 2959, but found that
an Eosin Y system allowed for the most effective formation of
structurally reliant and viable patches.
[35,38,39]
Most importantly
for bioprinting, GelMA undergoes a polymerization when
cooled from physiological temperatures to below 10 °C, and is
viscous even at room temperature with concentrations of 10%
weight/volume (w/v) and above. This phase transition makes
it suitable for bioprinting as a natural material, and has been
used often for this application.
[39]
This work utilized 5% w/v
GelMA in the bioink formulations so that the bioink was still a
significant portion cECM (8 mg mL
1
), compared to increasing
the concentration of GelMA to 10% or higher, which would
have produced a bioink that is mostly GelMA with some cECM
added. In addition, low w/v % GelMA supports more effective
cellular outcomes such as viability and proliferation.
[40]
Our
printing strategy involved cooling 5% w/v GelMA to 10 °C for
10 min to allow for gelation and enhanced printing viscosity of
the cECM/hCPC bioink. An overview of the printing strategy is
seen in Figure 1.
The printing methodology allowed for clean and defined
extruded filaments when printing either GelMA or GelMA-
cECM (Figure 2A). To ensure that the cECM fibers were uni-
formly distributed in the printed structures, we stained the
cECM with AF568, which forms a strong bond to primary
amines on the cECM proteins. The red staining in Figure 2B
is the cECM fibers, indicating that the cECM was distributed
homogeneously throughout the entire printed structure, rather
than in clumped locations such as filament junctions. A higher
magnification in 3D of a printed filament in Figure 2C shows
that the cECM formed homogeneously distributed dense fibers
after polymerization at physiological pH and temperature. We
quantified the printability of the structures using a parameter
based on the extent to which the holes between filaments
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match a square shape, as previously described and discussed in
the Experimental Section.
[41]
A value of printability close to 1.0
demonstrates ideal gelation, and thus the printing property, of
the bioink. This value shows that the holes are close to a perfect
square shape due to the filaments being uniform in thickness,
homogeneous, and rigidly defined with multiple layers stacking
on one another. As can be seen in Figure 2D, both GelMA
and GelMA-cECM bioinks had printability close to a value of
1.0 and the inclusion of cECM improved the printability of the
bioink significantly to achieve a value closest to ideal printing.
2.1.2. Bioprinting hCPC-Laden Structures and Cardiac Patches
Following incorporation of cECM in to the printed structure,
we next sought to determine if primary cells could be added
to the printing mix. To perform this, hCPCs were incorporated
into the bioinks and evaluated for effectiveness in creating
homogeneously distributed cell-laden print structures. Nonex-
trusion-based bioprinting methods require high printing pres-
sures that render cells nonviable or methodologies that result
in dispersion of cells toward the edges of printed constructs,
rather than homogeneously distributed throughout.
[42,43]
In
addition, cells can leach out of printed hydrogel constructs
if the materials are soft and not effectively polymerized,
resulting in a loose network.
[44]
As shown in the bright field
images in Figure 3, we were able to add cells to the print for
both GelMA alone (Figure 3A) and GelMA-
cECM (Figure 3B), where cells were retained
in the gels after cross-linking. To obtain a
clearer image of hCPCs throughout the test
grids, cells were stained with a lipophilic dye
(DiD) prior to printing. Figure 3C shows the
printed grids after swelling, indicating that
the cells appeared homogeneously distrib-
uted throughout the filaments. To quantify
distribution, an averaged fluorescence line
scan along filaments showed that the fluo-
rescence intensity throughout the filaments
was uniform and that the cells were homo-
geneously distributed (Figure 3D). Cells were
incorporated throughout the filaments, and
GelMA-cECM grids once again appeared
to have better printability, as indicated by
the hole geometry, where the GelMA-cECM
grids had more square holes than GelMA
grids. Printing parameters were not modified
by the incorporation of cells, maintaining a
low printing pressure (0.7–0.8 bar), and thus
low shear stress, on the cells. In addition,
cells remained firmly supported within the
printed constructs, with no cells leaching out
of the grids or sifting to the bottom of the
filaments.
The hCPC/cECM bioink was shown
to have ideal printability with homoge-
neous distribution of both cECM and
hCPCs throughout the printed structures,
as described in the above sections. Moving
on, we were able to create cardiac patches using the cell-laden
bioink, based on a cylindrical shape, as indicated in Figure 4.
Figure 4A shows the printed patches prior to white light poly-
merization while Figure 4B shows the CAD models using the
patch design. The patches were pink due to the Eosin Y photo-
initiator and change to clear after polymerization. The printed
patches maintained the same shape and structure as the CAD
model, due to the high printability bioink. The infill pattern of
the patches were perpendicular aligned filaments generated
through multiple print layers, indicating further degrees of
printing control and structure fidelity.
2.2. hCPC Viability, Differentiation, and Proliferation
within Bioprinted Cardiac Patches
Evaluating the viability of cells within the cardiac patches is
critical to ensure live cells that can participate in producing
important proreparative paracrine factors.
[12,15]
Evaluation of
cell viability directly is also critical within bioprinted scaffolds,
particularly because bioprinting has been shown to reduce cell
viability in printed constructs due to high shear stresses on the
cells from small diameter needle tips, such as the tips used in
this study.
[42,43]
In addition, cells grown in thick 3D structures
can suffer death due to lack of nutrient diffusion, particularly
at the center of the structures, producing a necrotic core.
[42,45]
As shown in Figure 5, hCPCs within printed cardiac patches
Adv. Healthcare Mater. 2018, 7, 1800672
Figure 1. Printing overview. A) Bioink preparation involved combining cECM, hCPCs, and
GelMA to form naturally derived and cell-laden materials for printing. B) Printing methodology
involved cooling the bioink to 10 °C in the 3D bioprinter barrels to allow GelMA polymeriza-
tion for improved printability. Patches were printed with infill patterns of 90° intersecting fila-
ments and contour. Patches were polymerized via white light to induce radical polymerization
of GelMA, followed by incubation at 37 °C for at least 1 h to induce cECM polymerization.
C) Patch implementation will involve pericardially inserting the patch to the RV of pediatric
patients, where the patch will release key proregenerative paracrine factors.

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were stained to determine the total number of dead (red) to
live (green) cells for either GelMA (Figure 5A) or GelMA-cECM
(Figure 5B). Cell viability was quantified by measuring the
number of live and dead cells at different locations and heights
within the cardiac patch at days 1, 3, and 6 after formation and
showed high viability, from 70 to 80% live cells on average
Adv. Healthcare Mater. 2018, 7, 1800672
Figure 3. Printing hCPC-containing bioinks. A) Bright-field image of printed test grids of GelMA bioinks containing hCPCs, taken 1 h after printing. B) Bright-
field image of printed test grids of GelMA-cECM bioinks containing hCPCs. C) Fluorescence image of printed test grids of GelMA-cECM with hCPCs stained
with DiD. D) Normalized fluorescence intensity of line scans performed on stained hCPC test grids. Line scans were performed across several filaments.
Figure 2. Printability analysis of GelMA-cECM bioinks. A) Bright-field image of printed test grids of GelMA. B) Fluorescence image of printed test grids
of GelMA-cECM with staining for cECM by AF568. C) 3D fluorescence close-up view of printed filament of GelMA-cECM, with staining for cECM by
AF568. D) Printability comparison between GelMA and GelMA-cECM bioinks. * = p-value < 0.03, given by paired t-test, n = 5.

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Adv. Healthcare Mater. 2018, 7, 1800672
Figure 4. Printed patches. A) Printed patches of 10 mm diameter and 0.6 mm height. Patches are printed uniformly from patch to patch, and the
grid infill pattern can be seen. Patches are pink postprinting due to inclusion of photoinitiator Eosin Y, and become clear postpolymerization. B) CAD
model sketch used for patch printing.
Figure 5. hCPC functionality within printed patches. A) Characteristic live/dead fluorescence image of hCPCs in GelMA patches, with live cells marked
green (Calcien AM) and dead cells marked red (EtD) at 1 d after formation. B) Characteristic live/dead fluorescence image of hCPCs in GelMA-cECM
patches at 1 d after formation. C) Viability of hCPCs in printed patches at 1, 3, and 6 d. D) Proliferation of hCPCs in printed patches at 3 and 7 d, where
absorbance intensity is normalized to the measured absorbance of hCPCs in GelMA patches in all experiments. E) Fold change gene expression over
hCPCs in GelMA patches for Cx43, GATA4, MEF2C, MYH7, VE-Cad, CD31, FLT-1, and ACTA-2 at day 3. F) Fold change gene expression over hCPCs in
GelMA patches for Cx43, GATA4, MEF2C, MYH7, VE-Cad, CD31, FLT-1, and ACTA-2 at day 7. * = p-value < 0.05, ** = p-value < 0.005, given by ANOVA
with Tukey’s post-test, n = 3–6 for all samples at all time points.

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Journal ArticleDOI
TL;DR: The continuous convergence of the experts in the fields of material sciences, cell biology, engineering, and many other disciplines will gradually allow us to overcome the barriers identified on the demanding path toward manufacturing and adoption of tissue and organ replacements.
Abstract: Bioprinting techniques have been flourishing in the field of biofabrication with pronounced and exponential developments in the past years. Novel biomaterial inks used for the formation of bioinks have been developed, allowing the manufacturing of in vitro models and implants tested preclinically with a certain degree of success. Furthermore, incredible advances in cell biology, namely, in pluripotent stem cells, have also contributed to the latest milestones where more relevant tissues or organ-like constructs with a certain degree of functionality can already be obtained. These incredible strides have been possible with a multitude of multidisciplinary teams around the world, working to make bioprinted tissues and organs more relevant and functional. Yet, there is still a long way to go until these biofabricated constructs will be able to reach the clinics. In this review, we summarize the main bioprinting activities linking them to tissue and organ development and physiology. Most bioprinting approaches focus on mimicking fully matured tissues. Future bioprinting strategies might pursue earlier developmental stages of tissues and organs. The continuous convergence of the experts in the fields of material sciences, cell biology, engineering, and many other disciplines will gradually allow us to overcome the barriers identified on the demanding path toward manufacturing and adoption of tissue and organ replacements.

157 citations

Journal ArticleDOI
TL;DR: A bioink capable of promoting human induced pluripotent stem cell proliferation and cardiomyocyte differentiation to 3-dimensionally print electromechanically functional, chambered organoids composed of contiguous cardiac muscle is developed.
Abstract: Rationale: One goal of cardiac tissue engineering is the generation of a living, human pump in vitro that could replace animal models and eventually serve as an in vivo therapeutic. Models that rep...

147 citations

References
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Journal ArticleDOI
TL;DR: Although modern synthetic biomaterials represent oversimplified mimics of natural ECMs lacking the essential natural temporal and spatial complexity, a growing symbiosis of materials engineering and cell biology may ultimately result in synthetic materials that contain the necessary signals to recapitulate developmental processes in tissue- and organ-specific differentiation and morphogenesis.
Abstract: New generations of synthetic biomaterials are being developed at a rapid pace for use as three-dimensional extracellular microenvironments to mimic the regulatory characteristics of natural extracellular matrices (ECMs) and ECM-bound growth factors, both for therapeutic applications and basic biological studies. Recent advances include nanofibrillar networks formed by self-assembly of small building blocks, artificial ECM networks from protein polymers or peptide-conjugated synthetic polymers that present bioactive ligands and respond to cell-secreted signals to enable proteolytic remodeling. These materials have already found application in differentiating stem cells into neurons, repairing bone and inducing angiogenesis. Although modern synthetic biomaterials represent oversimplified mimics of natural ECMs lacking the essential natural temporal and spatial complexity, a growing symbiosis of materials engineering and cell biology may ultimately result in synthetic materials that contain the necessary signals to recapitulate developmental processes in tissue- and organ-specific differentiation and morphogenesis.

4,288 citations

Journal ArticleDOI
TL;DR: GelMA hydrogels could be useful for creating complex, cell- responsive microtissues, such as endothelialized microvasculature, or for other applications that require cell-responsive microengineered hydrogELs.

1,871 citations

Journal ArticleDOI
TL;DR: The potential paracrine mechanisms involved in adult stem cell signaling and therapy are reviewed: cytokines and growth factors can induce cytoprotection and neovascularization, and cardiac remodeling, contractility, and metabolism may also be influenced in aParacrine fashion.
Abstract: Animal and preliminary human studies of adult cell therapy following acute myocardial infarction have shown an overall improvement of cardiac function. Myocardial and vascular regeneration have been initially proposed as mechanisms of stem cell action. However, in many cases, the frequency of stem cell engraftment and the number of newly generated cardiomyocytes and vascular cells, either by transdifferentiation or cell fusion, appear too low to explain the significant cardiac improvement described. Accordingly, we and others have advanced an alternative hypothesis: the transplanted stem cells release soluble factors that, acting in a paracrine fashion, contribute to cardiac repair and regeneration. Indeed, cytokines and growth factors can induce cytoprotection and neovascularization. It has also been postulated that paracrine factors may mediate endogenous regeneration via activation of resident cardiac stem cells. Furthermore, cardiac remodeling, contractility, and metabolism may also be influenced in a paracrine fashion. This article reviews the potential paracrine mechanisms involved in adult stem cell signaling and therapy.

1,855 citations

Journal ArticleDOI
TL;DR: The versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function are shown.
Abstract: The ability to print and pattern all the components that make up a tissue (cells and matrix materials) in three dimensions to generate structures similar to tissues is an exciting prospect of bioprinting. However, the majority of the matrix materials used so far for bioprinting cannot represent the complexity of natural extracellular matrix (ECM) and thus are unable to reconstitute the intrinsic cellular morphologies and functions. Here, we develop a method for the bioprinting of cell-laden constructs with novel decellularized extracellular matrix (dECM) bioink capable of providing an optimized microenvironment conducive to the growth of three-dimensional structured tissue. We show the versatility and flexibility of the developed bioprinting process using tissue-specific dECM bioinks, including adipose, cartilage and heart tissues, capable of providing crucial cues for cells engraftment, survival and long-term function. We achieve high cell viability and functionality of the printed dECM structures using our bioprinting method.

1,401 citations

Journal ArticleDOI
TL;DR: It is concluded that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions, and are regulated by the elastic modulus of PAAm.
Abstract: To investigate how substrate properties influence stem-cell fate, we cultured single human epidermal stem cells on polydimethylsiloxane (PDMS) and polyacrylamide (PAAm) hydrogel surfaces, 0.1 kPa-2.3 MPa in stiffness, with a covalently attached collagen coating. Cell spreading and differentiation were unaffected by polydimethylsiloxane stiffness. However, cells on polyacrylamide of low elastic modulus (0.5 kPa) could not form stable focal adhesions and differentiated as a result of decreased activation of the extracellular-signal-related kinase (ERK)/mitogen-activated protein kinase (MAPK) signalling pathway. The differentiation of human mesenchymal stem cells was also unaffected by PDMS stiffness but regulated by the elastic modulus of PAAm. Dextran penetration measurements indicated that polyacrylamide substrates of low elastic modulus were more porous than stiff substrates, suggesting that the collagen anchoring points would be further apart. We then changed collagen crosslink concentration and used hydrogel-nanoparticle substrates to vary anchoring distance at constant substrate stiffness. Lower collagen anchoring density resulted in increased differentiation. We conclude that stem cells exert a mechanical force on collagen fibres and gauge the feedback to make cell-fate decisions.

1,393 citations

Frequently Asked Questions (2)
Q1. What contributions have the authors mentioned in the paper "A bioprinted cardiac patch composed of cardiac-specific extracellular matrix and progenitor cells for heart repair" ?

A recent study showed that intracoronary infusion of cardiosphere-derived cells can improve RV function in children and follow-up studies have been promising. [ 7 ] The authors have recently shown that progenitor cells ( CPCs ) could improve the failing RV of juvenile rats subjected to pulmonary banding and a clinical trial is now underway ( NCT03406884 ). [ 9 ] In the present study, a 3D-bioprinted patch containing cECM for delivery of pediatric hCPCs is developed. This work shows the successful bioprinting and implementation of cECM-hCPC patches for potential use in repairing damaged myocardium. Several studies, including ones from their laboratory, have shown that a decellularized cardiac extracellular matrix hydrogel ( cECM ) is a promising biomaterial used in the repair of myocardial dysfunction in adults, as well as for the delivery of stem cells. [ 16–21 ] Human cardiac progenitor cell ( hCPC ) therapy has potential to repair the pediatric myocardium through release of reparative factors, but therapy suffers from limited hCPC retention and functionality. Conditioned media from GelMA-cECM patches show increased angiogenic potential ( > 2-fold ) over GelMA alone, as seen by improved endothelial cell tube formation. 

With printability achieved, future directions include incorporation of increased amounts of proregenerative ECM components such Adv. Healthcare Mater. Following hCPC expansion, patch manufacture can be expedited and customized through the 3D bioprinting methodology, which allows for incorporation of additional commercially available cell sources such as mesenchymal stem cells, and the tailored patch can be delivered directly to the patient for surgical attachment.