Journal Pre-Proof: Bioprinting

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Cardiovascular 3D bioprinting: A review on cardiac tissue development
Dianoosh Kalhori, Nima Zakeri, Mahshid Zafar-Jafarzadeh, Lorenzo Moroni, Mehran

Solati-Hashjin
PII: S2405-8866(22)00031-8
DOI: https://doi.org/10.1016/j.bprint.2022.e00221
Reference: BPRINT 221
To appear in: Bioprinting
Received Date: 29 March 2022

Revised Date: 24 May 2022
Accepted Date: 17 June 2022
Please cite this article as: D. Kalhori, N. Zakeri, M. Zafar-Jafarzadeh, L. Moroni, M. Solati-Hashjin,
Cardiovascular 3D bioprinting: A review on cardiac tissue development, Bioprinting (2022), doi: https://
doi.org/10.1016/j.bprint.2022.e00221.
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Cardiovascular 3D Bioprinting: A Review on Cardiac
Tissue Development
Dianoosh Kalhori1, Nima Zakeri1, Mahshid Zafar-Jafarzadeh1, Lorenzo Moroni2,
Mehran Solati-Hashjin1*
1
BioFabrication Lab (BFL), Department of Biomedical Engineering, Amirkabir University of
Technology (Tehran Polytechnic), Tehran, Iran
2
MERLN Institute for Technology-Inspired Regenerative Medicine, Department of Complex Tissue
Regeneration, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, The
Netherlands
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*Corresponding Author: [email protected]
Mehran Solati-Hashjin (PhD):
E-mail: [email protected]
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Postal address: BioFabrication Lab (BFL), Department of Biomedical Engineering, Amirkabir University of
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Dianoosh Kalhori (MSc):
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Nima Zakeri (MSc):

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Mahshid Zafar-Jafarzadeh (MSc):

Lorenzo Moroni (PhD):

Postal address: MERLN Institute for Technology-Inspired Regenerative Medicine, Department of Complex
Tissue Regeneration, Faculty of Health, Medicine and Life Sciences, Maastricht University, Maastricht, The
Netherlands
Table of Contents
Abstract 3
1. Introduction 4
2. Cell Sources 5
2.1. Cardiac Progenitor Cells 6
2.2. Skeletal Myoblasts 7
2.3. Mesenchymal Stem Cells (MSCs) 7
2.4. Human Embryonic Stem Cells (hESCs) 8
2.5. Induced Pluripotent Stem Cells (iPSCs) 8
2.6. Non-parenchymal Cells 9
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3. Hydrogels 10
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3.1. Naturally-derived Polymers 10
3.1.1. Collagen 10
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3.1.2 Gelatin
3.1.3 Alginate
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3.1.4 Fibrin 12
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3.1.5 Decellularized ECM 13

3.2. Synthetic Polymers 14
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3.3. Considerations in Bioink Composition 14

4. Bioprinting Techniques 17
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4.1. Extrusion-based Bioprinting 18

4.2. Scaffold-free Bioprinting 18
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4.3. Bioassembly 19
5. Architectural Designs 20
6. Biophysical Stimulations 21
6.1. Mechanical Stimulation 21
6.2. Electrical Stimulation 23
6.3. Hemodynamic Stimulation 24
7. 3D Bioprinting of Cardiac Tissue 24
8. Conclusion and Future Outlook 38
Acknowledgements 40
References 41
Abstract
Cardiovascular diseases such as myocardial infarction account for millions of worldwide
deaths annually. Cardiovascular tissues constitute a highly organized and complex three-
dimensional (3D) structure that makes them hard to fabricate in a biomimetic manner by
conventional scaffold fabrication methods. 3D bioprinting has been introduced as a novel cell-
based method in the last two decades due to its ability to recapitulate cell density, multicellular
architecture, physiochemical environment, and vascularization of biological constructs with
accurate designs. This review article aims to provide a comprehensive outlook to obtain
cardiovascular functional tissues from the engineering of bioinks comprising cells, hydrogels,
and biofactors to bioprinting techniques and relevant biophysical stimulations responsible for
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maturation and tissue-level functions. Also, cardiac tissue 3D bioprinting investigations and
further discussion over its challenges and perspectives are highlighted in this review article.
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Keywords: 3D Bioprinting; Cardiac Tissue Engineering; Cardiovascular Tissue Engineering;
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Bioink; Cardiomyocytes; Hydrogels
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1. Introduction
Cardiovascular diseases (CVDs) are the leading cause of death globally, involving one-third of
the world’s total death, and accounting for more than 17.9 million deaths each year [1]. CVDs
are cardiac and vessel tissues’ chronic pathological conditions, including coronary heart
disease, rheumatic heart disease, and cerebrovascular disease among which, myocardial
infarction (MI) is accounted as the primary cause of CVDs characterized by hypoxia-induced
cardiomyocyte death and severe inflammation, leading to tissue degeneration and scar
formation [2-4]. Clinically available methods for end-stage CVDs are majorly coronary artery
bypass grafts (CABGs), prosthetic devices, and heart transplantation among which, cardiac
transplantation methods are clinically restricted because of the shortage of donor organs and
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post-transplantation immunogenic rejection [5, 6]. Although there have been many efforts to
improve cell transplantation methods during the last decade, the low local cell retainability due
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to the blood flow and immunogenic inconsistency has remained as main challenges resulting
in low integration with the host tissue [7, 8]. Engineering a transplantable three-dimensional
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(3D) microenvironment for better cell-cell and cell-matrix interaction has the potential to
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replace the native tissue properties [9]. Tissue engineering is an arising alternative for end-stage
CVDs, aiming to alleviate the current therapeutic complications and providing a potential
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platform for improving the efficiency of cell-based therapies. Owing to the vast advantages
tissue engineering suggests, many challenges such as the organization of cardiomyocytes,
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limited self-renewal, electrical and mechanical functions have been addressed in [2]. Aside
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from the extensive research in tissue engineering, several challenges have majorly restricted its
clinical application, including the lack of functional blood supply for cells, non-homogenous
distribution of cells through the construct, and poor control over the 3D architecture precise
structure of the construct, ultimately resulting in lack of integration. To address these
complications, novel bioengineering methods are under investigation to improve the efficacy
of cardiac tissue engineering strategies [10-12].
Among these novel methods, 3D bioprinting, as a powerful biofabrication technology

stemming from additive manufacturing, has an exclusive potential in the fabrication of complex
3D architectures taking advantage of layer-by-layer deposition of biological components (cells,
signaling molecules, etc.) and biomaterials, namely hydrogels known as bioinks [13].
Bioprinting enables precise control over the spatial distribution of cells, architectural
organization, and compositional adjustment of the construct [4, 10]. In fact, there have been
various studies employing different kinds of cells, hydrogels, and architectures in biomimetic
approaches to produce cardiac patches or organoids due to the capability of this method to
fabricate geometrically complex structures in three dimensions and maintain cell proliferation,
maturation, and long-term functions. However, due to the intrinsic structural and compositional
complexity of the cardiac tissue, several significant elements need to be considered in cardiac
biofabrication, including i) suitable manipulation of cells sources having high regenerative
capacity, ii) appropriate biomaterials capable of replicating the native cardiac
microenvironment, stimulating cell fate, providing mechanical stabilization, and non-
immunogenicity, iii) well-defined structural organization, and iv) establishment of relevant
physiological stimulations (mechanical and electrical) for efficient cellular maturity and
functionality. Many bioprinting techniques have been introduced in the last decade based on
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extrusion, inkjet, and laser techniques, which provide a wide range of cell-friendly processes,
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adjustable precision, and compatible crosslinking methods. These techniques have advantages
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and disadvantages for any specific bioinks and crosslinking procedures, making selecting the
method extremely important [4, 10, 14-21].
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There are many significant characteristics and functionalities in genuine cardiac tissue that
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makes the heart pump blood continuously. These specific features have already been attained
in 3D bioprinted constructs designs by implementing biomimetic approaches. Today,
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bioprinted constructs with different organizations of endothelial cells allow vascularization and
stimulate cardiomyocytes growth; anisotropic grid designs allow orientation, contractility; and
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other kinds of mechanical (e.g., external stretching, hemodynamic stress, and the
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microenvironment adjusting) and electrical stimulations (e.g., direct and pulsatile impulses) to
bring maturation and conductivity [22-24]. In this review, we discuss several cell sources and
hydrogels that have been used to prepare bioinks. We also review different bioprinting
techniques, architectural designs, and stimulation methods that increase biological constructs'
functionalities. Finally, we conclude with a discussion of cardiovascular bioprinting challenges
and perspectives based on the last decade’s studies.
2. Cell Sources
3D bioprinting, aside from other cell-based technologies, aims to control the spatial distribution
of cells in high resolutions and achieve a better mimicry of the natural cell microenvironment,
leading to enhanced cell activity and higher tissue-level biological responses [25, 26]. The
introduction of bioprinted functional cells in nonfunctional defect sites is considered to trigger
regeneration (mainly by differentiation of either transplanted stem cells or site-specific
progenitors) and stimulate vascularization [27, 28]. Hence, appropriate cell organization and
tissue-level cell performance are two essential factors that should be strictly regulated for
further tissue regeneration. To this end, cell sources should meet several characteristics,
including i) facile and efficient differentiation of stem cells (increased number of both
differentiated cells and tissue-specific gene expression), and ii) no pathogenic transference
from the source to the host [27-30].
In contrast to non-parenchymal heart cells, parenchymal cardiomyocytes show minimal

proliferative capacity, resulting in a low number of governing cells impairing tissue formation
[31]. Therefore, stem cells, including autologous/allogeneic cardiac progenitor cells, skeletal
myoblasts, mesenchymal stem cells (MSCs), embryonic stem cells (ESCs), and induced
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pluripotent stem cells (iPSCs), are potential sources for both experimental and clinical
regenerative applications [29].
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2.1. Cardiac Progenitor Cells -p
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Based on earlier investigations, neonatal and fetal cardiomyocytes have suggested a favorable
progression rate required for tissue regeneration. However, due to ethical issues, immuno-
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rejection responses related to the allogeneic sources, and short cell survivability, their clinical
application is limited [29, 32, 33]. Since immature early-stage cardiomyocytes possess high
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survivability, they have been exploited extensively in regenerative medicine. Cardiac

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progenitor cells are tissue-resident multipotent stem cells that can be harvested by minimally
invasive biopsies and directed toward different myocardium lineages, including
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cardiomyocytes, smooth muscle cells, and endothelial cells [4, 34]. Cardiac progenitor cells
are harvested from two origins; leftover from the embryonic morphogenesis process or
recruited from bone marrow by the circulating system, namely autochthonous and
allochthonous [35]. Compared to other potent stem cells, progenitor cells are closer to the fully
differentiated cardiomyocytes in the cardiac lineage. Therefore, they require fewer
considerations for differentiation toward specified cells in 3D constructs. For instance,
bioprinting of human cardiac-derived cardiomyocyte progenitor cells (hCMPCs) in various
hydrogels has indicated a higher tendency to differentiate toward cardiomyocytes in 3D
constructs [36, 37]. However, the limited availability of these cells has restricted mainly their
clinical implementation. To obtain a cardiogenic construct, Gaetani et al. [38] evaluated
extrusion-based bioprinting of modified alginate laden with hCMPCs. The results showed that
the fetal cardiomyocytes appropriately migrated, formed tubular structure, and could also
express early cardiac transcription factors [38].
2.2. Skeletal Myoblasts
Skeletal myoblasts are among the most commonly used cell sources in cardiac regenerative
medicine, which can be easily harvested by patients’ skeletal tissue biopsies obviating further
immunogenic responses [39]. Several specific characteristics are attributed to the increased
interest in the use of skeletal myoblasts, which majorly include the high rates of cell
proliferation, resistance to hypoxia, and the high potency of these cells in the establishment of
myotubule structures, which can significantly improve cardiac functions [34, 40, 41]. These
particular characteristics are also reported to be accompanied by a decrease in both tissue
fibrosis and cardiomyocyte-hypertrophy [42]. However, after the formation of myotubules, the
electrophysiological coupling was not well-established. This lack of electrophysiological
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coupling was due to the lack of the expression of gap junction-associated genes, including
connexin-43 and N-cadherin, which causes other arrhythmic behaviors [9, 43, 44].
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2.3. Mesenchymal Stem Cells (MSCs) -p
MSCs, also known more recently as medicinal signaling cells, are multipotent stem cells
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isolated from different sources, majorly from bone marrow, adipose tissue, and umbilical cord
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tissue [45]. They can be signaled to differentiate toward mesenchyme-derived cells, including
adipose, osteogenic, and chondrogenic lineages. Various studies have also suggested a different
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potency of MSCs to transdifferentiate towards other lineages, including cardiomyocytes [45-

47].
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However, the differentiation capacity of MSCs toward cardiomyocytes has been reported to be
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limited, producing cardiomyocyte-like cells with low functionality, which makes them less
efficient cells from a therapeutic perspective [48]. Due to this limited differentiation capacity
of MSCs into cardiomyocytes, rather than being exploited as single regenerative cells, they are
mostly implemented in co-culture systems. Several other transplant investigations reported
enhanced survival and regenerative capacity of the MSC-Cardiomyocytes heterogeneous co-
culture system [49, 50]. Aside from the transdifferentiation capacity of MSCs, three other
primary mechanisms were proposed for the enhanced regenerative capacity of MSC-
Cardiomyocytes: i) inducing the latent regenerative capacity by reprogramming the fully
differentiated cardiomyocytes back to the therapeutically potent cardiac progenitor cells [51];
ii) a transient improvement in cardiomyocytes functions in response to the release of several
paracrine factors, especially angiogenic factors [52, 53]; iii) an electrophysiological coupling
between MSCs and cardiomyocytes has been attributed to the formation of gap junctions
between adjacent MSCs and cardiomyocytes [54].
2.4. Human Embryonic Stem Cells (hESCs)

A fundamental challenge to be mentioned in cardiac tissue engineering is cell-cell integration
and paracrine signaling. In other words, incorporated cells remain isolated, and no or low
integration occurs between cells [55]. Cardiomyocytes derived from hESCs have been shown
to form spherical cell aggregates of about 200 cells and up to 300 µm in diameter. They suggest
a strong potential for cardiac tissue engineering [56, 57]. hESCs are pluripotent stem cells
harvested from the blastocyst’s inner cell mass during embryogenesis with high regenerative
potentials. Accordingly, they possess high self-renewal potential and can be differentiated
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toward an extensive choice of tissues with remarkable plasticity [4]. Besides the advantages
hESCs exploitation suggests, several issues need to be mentioned. First, the differentiation of
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these pluripotent stem cells can occur in two different ways, spontaneously and through direct
differentiation, due to the considerable heterogeneity of obtained cells within spontaneous
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differentiation. The direct differentiation of cells toward the myocardium cell lineage is more
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implemented [58]. However, co-cultured hESCs with murine stromal cells have resulted in a
heterogeneous mixture of non-parenchymal and parenchymal cells [34, 59]. Hence, the lack of
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controllability of cell direction, issues related to the immunogenic responses, and the ethical
dilemma associated with their use have restricted these cells’ extensive use in clinical
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applications [60, 61].

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2.5. Induced Pluripotent Stem Cells (iPSCs)

iPSCs are generated directly by reprogramming the patient’s fully differentiated cells to harbor
pluripotency by introducing several stemness transcription factors, mainly through the
exploitation of retroviruses or more recently-deployed, stimulating their expression through
incorporating chemical factors [62]. Like ESCs, iPSCs have a high self-renewal capacity and
high differentiation plasticity toward a wide variety of cell types, including cardiomyocytes
[4]. The autologous origin of iPSCs ensures patient-specific compatibility in four levels of
genomics, transcriptomics, proteomics, and metabolomics [63, 64]. Also, the high
differentiation plasticity of iPSCs provides the potential for spontaneous generation and
direction of different cell lineages, including parenchymal and non-parenchymal cells in
cardiac tissue. However, in spontaneous differentiation, precise protocols are required for the
direction of iPSCs toward intended cell types and suppress non-cardiac cell lineages generation
[58].
There are several complications associated with iPSCs implementation in bioprinting for
regenerative medicine. One of the significant concerns about the iPSCs is the possibility of
teratoma formation and immaturity of governing cells. In this regard, electromechanical
stimulations, introducing differentiative agents (e.g., Activin A) into the culture media or co-
culturing them with endodermal cells, which is responsible for cell-controlled signaling
molecules release, can control further maturation and cellular maintenance [59, 64, 65]. Also,
in vivo transplantation of iPSCs has been reported to result in more mature cells attributed to
the more compatible directing signals and microenvironment. Another significant complication
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is associated with the iPSCs generation, where significant genomic instability, mainly caused
by unusual mutations during the reprogramming procedure, can be resulted [66].
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Recently, there have been several studies on extracellular vesicles derived from stem cells and
their potential effects on tissue restoration and cell viability. Extracellular vesicles derived from
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iPSCs have been shown to contain a wide range of proteins and non-coding RNAs (e.g.,
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miRNAs), strongly influencing cell viability and cell cycle [65-68].

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2.6. Non-parenchymal Cells

In contrast to the parenchymal cardiomyocytes, non-parenchymal cells, including endothelial
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cells (ECs), fibroblasts (FBs), and smooth muscle cells (SMCs), exhibit relatively high
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proliferative capacity [69]. These cells’ phenotype and the source may present important
properties affecting their functionality and parenchymal cardiomyocytes’ survival and
contractility. Both autologous and allogeneic sources of ECs are utilized experimentally to
induce vascular structures in bioprinted tissue, which plays substantial roles in barrier functions
between cardiomyocytes and blood [70, 71]. However, the utilization of primary ECs from the
aorta and human umbilical vein endothelial cells (HUVECs) has been more promising.
FBs are also supportive cells that mainly occupy the space between cardiomyocytes, regulate
ECM synthesis, paracrine signaling, and remodel specific factors, including collagen,
fibronectin, and other glycoproteins. Also, the FBs-cardiomyocytes coupling has effectively
promoted the cardiac tissue mechanoelectrical behaviors, suggesting their utilization in
heterogenic co-culture systems [4, 18, 70, 71].
3. Hydrogels
Various bioinks have been developed for tissue bioprinting. They are mainly categorized into
two groups of scaffold-based (e.g., polymer-cell suspension, microcarriers) and scaffold-free
(e.g., cell/tissue spheroids) bioinks [72]. Scaffold-based bioinks are more common in cardiac
bioprinting since incorporating biopolymers structurally supports tissue elasticity and
myotubules formation, and bioactively can improve cellular fate by maintaining biochemical
and bioelectrical signals [4, 73]. Bioinks are classified as naturally-derived and synthetic
materials. Naturally-derived materials are more commonly used due to their intrinsic
biocompatibility and close ECM-resemblance. Here, we summarize the essential aspects
regarding the majorly used bioinks in cardiac bioprinting comprising naturally-derived
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polymers, synthetic polymers, and also decellularized ECM.
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3.1. Naturally-derived Polymers
3.1.1. Collagen
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The commonly used naturally-derived hydrogels in cardiac bioprinting majorly include
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alginate, gelatin-based hydrogels, collagen, fibrin, and hyaluronic acid-based compounds [18,
74, 75]. Among them, collagen is highly favorable since it is one of the main ECM components.
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It largely contributes to cellular growth and organization. The collagen’s elastic nature has
provided smooth deposition of cells leading to an appropriate cell niche for further myocardium
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formation [76, 77]. Lee et al. [78] proposed a strategy to engineer human heart components at
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specific scales, including heart capillaries and valves. pH-driven gelation enabled a 20-
micrometer resolution that allowed rapid micro-vascularization, cellular infiltration, and
optimized mechanical properties for multiscale vasculature perfusion [74]. In fact, due to the
aligned organization of micro-vessels and cardiomyocytes in a normal heart, the use of
materials with inherent fibril structure is of more importance [78].
Also, collagen possesses poor immunogenic characteristics, which is majorly attributed to a

similarity between different species [79]. However, the lack of mechanical stability after
printing remains a significant complication that requires well-established crosslinking
strategies. Solidification of collagen can be established through either permanent covalent
bonds or physically crosslinked through temperature or pH changes [80].
Crosslinking can also influence the antigenic behavior of collagen. The non-helical telopeptide
regions are majorly known as antibody-recognizing sites (epitopes). Crosslinking has been
found to modify epitopes causing less interaction with antibodies and less antigenic responses
[79, 81]. However, along with collagen’s high cell-friendly properties, its implementation
possesses several complications, including low adjustability, fast degradation, and increased
costs that have limited its wide applications [79].
3.1.2 Gelatin
Gelatin, the collagen partial hydrolysis product, has an uprising biological application as an
alternative for collagen. Besides the high biocompatibility resulting from its peptidyl similarity
to biochemical components of ECM, several significant advantages, including lower
immunogenicity rather than collagen, low costs, and high availability, have attributed to its
increasing application as bioinks [77]. Gelatin can be solidified through either chemical or
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physical crosslinking. Physical pH- or temperature-dependent crosslinking is relatively time-
consuming, which decreases the printability of the bioink [77, 82]. Also, it suffers from low
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rheological properties required for printing. To improve stability and adjustability, blending
gelatin with a readily cross-linkable polymer (e.g., alginate) or implementing chemical
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modifications is proposed. As a widely used gelatin-modified bioink, gelatin methacrylate
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(GelMA), the product of gelatin methacrylation, has often been used. Besides improved shape
fidelity and rheological characteristics, photo-crosslinking of gelatin through permanent
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interaction of methacrylamide groups indicates better biocompatibility and stability in the

biological medium. These characteristics have made it a widely used hydrogel without a need
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for any further modifications [77, 83].

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3.1.3 Alginate
Alginate is a water-soluble polysaccharide composed of glucuronic (G blocks) and mannuronic
(M blocks) residues, which can be readily crosslinked upon exposure to divalent cations
(especially Ca2+) through the establishment of electrostatic bonds between cations and G block
residues [84].
Hence, G blocks’ varying densities through the polymer chain can result in different
crosslinking densities and mechanical strengths [84, 85]. However, the solidification time
should be optimized to prevent further cell mortality [86]. In a study conducted by Gao,
extrusion-based bioprinting and organ weaving were combined to bioprint vascular conduits
containing multilevel fluidic channels based on alginate. Multilevel micro-channels, with two
FBs and SMCs levels, were fabricated through coaxial extruding hollow cell-laden alginate
filaments forming a tubular conduit concentric to the main macro-channel. Endothelial cells
were subsequently seeded into the inner wall of the main channel [87]. Although, larger
densities of G blocks, known as hard segments, have been shown to possess less
biocompatibility.
For this reason, higher M/G ratios are favorable in biological applications [86]. As a significant
drawback, alginate is a biologically inert material and supports low cell adhesion. Nevertheless,
rapid solidification and its ability to maintain structural and mechanical stability have made it
an indispensable component in various bioinks [84-86]. However, as a post-printing procedure,
the post-crosslinking time of alginate should be strictly taken into consideration since it can
significantly affect the viability of cells. To overcome the low cell-friendliness of alginate,
chemical modifications and also functionalizing alginate with specific polypeptides supporting
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cell adhesion (such as RGD polypeptide) have been majorly investigated [88, 89]. In this
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regard, alginate is mainly accompanied by gelatin to guarantee appropriate rheological,
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thermoresponsive, and cell-friendliness characteristics. However, in most bioengineered
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alginate-based bioinks, alginate serves as a sacrificial template component of the bioink,
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allowing the well-controlled deposition of hydrogel fibers. This is generally due to the presence
of chelating EDTA in the medium, which de-crosslinks the alginate hydrogel by releasing the
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Ca2+ cations. Also, to enhance this sacrificial characteristic, alginate polymer chains have been
oxidized to increase the degradation rate of the polymer thanks to the presence of more reactive
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groups [18, 90].

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3.1.4 Fibrin
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Fibrin, a biodegradable protein that naturally exists in the blood, suggests significant potentials
for cardiac regeneration. It is noteworthy to mention the mimicry potential of fibrin’s
incorporation with various peptides, which acts as a local reservoir for specific growth factors.
This reservoir behavior provides an appropriate microenvironment for instigating cellular fate
[80, 91]. This intrinsic bioactivity, along with fibrin fibers’ structural strength and physical
characteristics, has made it a potential matrix for endothelial cell adherence and angiogenesis
[92]. Fibrin fibers undergo degradation and backbone cleavage through the activity of protease
enzymes. Aprotinin is a protease inhibitor typically incorporated with varying concentrations
in the medium of fibrin-based constructs to control the degradation rate [80]. However,
diffusion of aprotinin in the medium leads to the loss of fibrinolysis protection. It was found
that the aprotinin-conjugated fibrinogen was able to prevent the plasmin-mediated fibrinolysis,
and its functionality was found to be as significant as the non-conjugated soluble form [93].
Also, the potential of autologous isolation of fibrin from patients is vital in eradicating the
immunogenic responses. Nevertheless, weak mechanical stability, structural shrinkage, and the
possibility of disintegration are the main complications associated with fibrin fibers, which can
be adjusted to a great extent by varying concentrations of Ca2+, buffers, crosslinking agents, or
combining them with other supportive materials [80, 91].
3.1.5 Decellularized ECM

ECM decellularization is currently being investigated as another method to prepare bioinspired
bioinks. Potentially, decellularized ECM is an appropriate scaffold for regenerative medicine
applications since it removes cells from the tissue’s ultrastructure while preserving its
mechanical and biological properties. Using 3D bioprinting technology, cell-laden structures
representing the intrinsic cues of natural ECM can be fabricated by layering ECM and
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autologous cells as a reproducible and accurate method [94, 95].
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Decellularization specifically aims to detach cells from their ECM to remove potential antigens
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that may cause inflammation or further immune reactions. Hence, ECM’s ultrastructure
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induces tissue repair, whereas the host tissue does not develop antigenicity, inflammation, or
immunological response, increasing the implantation success [94, 95]. Furthermore, the intact
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decellularized ECM structure is a potential reservoir for various biomolecules found in native
tissue, including proteins and growth factors [94, 95].
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According to different studies conducted on heart-derived decellularized ECM, one of the main
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challenges within this field is the mismatch between the mechanical properties of the
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decellularized ECM and native cardiac tissue. However, there have been studies aimed to
improve the mechanical properties through different methods of two-step crosslinking or the
inclusion of methacrylated natural polymers [96, 97]. More specifically, Jang et al. [96], in an
attempt to improve the mechanical properties of heart bio-constructs, employed Vitamin B2
(VB2) into pepsin digested decellularized-ECM as a photoinitiator. This construct was further
exposed to UV light to pursue photo-crosslinking. This first crosslinking step was followed by
a thermal crosslinking, ensuring the stabilization of the final construct. The results indicated
appropriate printability, comparable mechanical properties, and significantly higher
cardiogenic differentiation [96]. In another study conducted by Yu et al. [97], GelMA was
introduced into the decellularized-ECM with which the mechanical properties could be
adjusted with the post-printing exposure time of UV light [97].
3.2. Synthetic Polymers
Although naturally-derived materials possess more cell compatibility and relatively improved
bioactivity, several associated disadvantages, including low mechanical stability,
immunogenicity, and less reproducibility due to the batch-to-batch variations, have led to the
uprising incorporation of synthetic polymers in bioinks [4]. Generally, synthetic materials such
as polyethylene glycol (PEG) and polyethylene glycol-diacrylate (PEG-DA) suggest adjustable
molecular and physicochemical properties, better physical integrity, and enhanced printability.
However, they do not inherently support well-established cell-matrix interactions, do not
closely mimic cardiac ECM, and possess less bioactivity [4]. The utilization of blends or
composites of synthetic and naturally-derived polymers such as PEGylated gelatin
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methacrylate (PEGgelMA) is mostly under investigation in which synthetic polymers majorly
contribute to the physical support of biologically advanced bioinks. Also, there are various
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thermoplastic polymers under investigation which are designed to be utilized in frameworks
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(e.g., polycaprolactone (PCL)) or as sacrificial polymers (e.g., polyvinyl alcohol (PVA)) [10].
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3.3. Considerations in Bioink Composition
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To achieve an appropriate cell-cell and cell-matrix interaction required for tissue regeneration,
the bioink should mainly support cell adhesion, cell alignment, oxygen transport, and,
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specifically for cardiac tissue, electromechanical synchronization through successful

establishment of gap junctions [98]. To this end, the bioink should possess several significant
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characteristics, including appropriate viscosity and shear thinning for enhanced printability,
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well-established solidification kinetics, high hydration ability, viscoelastic behavior close to

the native cardiac ECM, and enough diffusivity to oxygen and cellular bioactivity to increase
physiological synchronization (Figure.1) [98, 99]. In this regard, researchers are highly inclined
to incorporate blended bioinks. Composite bioinks reported to be successful in biological
characterizations are mainly Alginate-GelMA, GelMA-cardiac extracellular matrix, alginate-
PEG-fibrinogen, and collagen-fibrin compositions. However, aside from bioink intrinsic
properties and architectural considerations, adjusting composition and rheology can
significantly influence the printability and biological characteristics of bioinks (Figure.1) [90].
One of the deterministic factors influencing the rheology of bioinks is the concentration of the
components. Although higher concentration may result in better mechanical stability and shape
fidelity, it can significantly affect oxygen transportation [100, 101]. An important parameter
impacting the transport kinetics is the ratio of applied concentration (C) to the critical
overlapping concentration (C*) of the polymer [102]. C* is the concentration in which the
polymer chains start to overlap regarding their radius of gyration. Hence, the more C/C * ratio,
the more densified solution results, which causes higher compaction and less porosity. This
can significantly affect the transportation of biomolecules, especially oxygen [86, 102, 103],
thus resulting in impaired maintenance of cell viability.
Another parameter that can be influenced by concentration is the shear-thinning behavior of

the bioink [104]. Typically, shear stress is experienced by cells during bioprinting, while the
biomaterial compartment of the bioink acts as a shield to mitigate the exerted shear stress on
the cells. The shear-thinning characteristic is responsible for the mentioned behavior by which
the exerted shear stress is damped by lowering the hydrogel viscosity. Therefore, a
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scientifically reasonable trade-off between printability and transport phenomena regarding the
concentration of components is required [105, 106].
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Along with scaffold-dependent parameters, cell density can considerably influence the further
regeneration potential of bioinks since key elements in regeneration, such as gene regulation,
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differentiation, and cell progression, are highly density-dependent elements [107]. Hence,
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optimizing the cell density is a crucial step toward developing a clinically applicable bioink.
This can be discussed by accounting for two primary biological and mechanical considerations.
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Regarding the former, the cell type and associated proliferative capacity are determining factors
in optimizing initial cell density [108-110]. The initial low density of highly proliferating cells
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may be reasonable, while the initial low densities can inhibit cell population regarding the other
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cells with no or moderate proliferative capacity. This is due to the lack of required cell-cell
interactions and the release of directing agents [110]. However, considerably high densities can
also contribute to the formation of hypoxic regions due to an imbalance between uptake and
intake rates of oxygen and nutrients. Inharmonious degradation of the hydrogels may also result
in higher ECM production due to the utilization of high densities [100, 110, 111].
Regarding the mechanical considerations, higher cell densities increase the bioink viscosity
requiring higher loads for printing. This can negatively influence cell viability to a great extent.
Also, cells act as disintegration sites in the hydrogel in high densities, lowering deposited
tissue’s mechanical stability, influencing long-term maintenance [100, 111, 112].
Modifying the internal structure of hydrogels through crosslinking is almost a post-printing

process, which is determined with respect to the chemical composition of the hydrogel. The
reaction can be generally established through physical and chemical routes. In each
crosslinking method, specific considerations should be taken into account to obtain appropriate
mechanobiological properties. Obviously, in chemical crosslinking, along with effective
structural hardening, the crosslinker should elicit no cytotoxicity; in this regard, agents like
genipin and EDC/NHS have suggested an appropriate biological response. Generally, in
thermally and pH-dependent cross-linkable hydrogels like gelatin, the ideal plasticizing
temperature is a point that has the minimum difference with the physiologic condition (37ᵒC
and pH=7.4) accompanying with appropriate mechanical properties. Hence, the cells are less
likely to undergo thermal or pH shocks [113].
In irradiation crosslinking, three particular parameters should be strictly controlled for

enhanced and appropriate mechanobiological characteristics: the photoinitiator used for
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physical crosslinking, exposure time, and the irradiation wavelength. The commonly used
photoinitiators are Irgacure 2959, Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP),
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and ruthenium-sodium persulfate. However, a study on cardiac patch bioprinting found that the
Eosin Y system can more effectively act as a crosslinking agent in methacrylated collagen
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(MeCol) hydrogel accompanied by higher viable biological constituents. Regarding the
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exposure, it is critically important to adjust the composition to minimize the exposure time
[114]. Accordingly, as reported in a study conducted by Izadifar et al. [115], cell viability was
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characterized for varying UV exposure times of 45s, 120s, and 270s. Quantified results
indicated an inverse relationship between exposure time and cell viability [115]. These results
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were consistent with the alterations in cellular morphology in a way that HUVECs were found
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to be able to preserve their stretchable morphology in MeCol hydrogel at 45s and 120s
exposures. In comparison, in the 270s exposure time, the cells mainly were stayed round in
shape. The utilized irradiation wavelengths mainly were in the range of 300 to 500 nm.
Nevertheless, in the same study used the Eosin Y system, white light irradiation was used over
UV exposure to decrease the induced cell death [113, 114].
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Figure.1 Engineering considerations in Bioink compositions and correlated cellular and print fidelity
challenges. Cardiovascular bioprinting is mostly correlated with several specific challenges
subcategorizing in cellular challenges and those associated with print fidelity. To overcome these
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challenges, particular engineering considerations come into the role of establishing a well-organized
bioink able to satisfy correlated challenges.
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4. Bioprinting Techniques
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Cells, biological macromolecules, and structural moieties are three main components that aim
to provide a native environment to repair heart tissue injuries. However, fabrication and
mimicking the complex 3D architecture of normal tissue is a challenging issue. Novel
bioprinting systems have been developed to overcome this issue to a great extent. Aside from
scaffold-free methods of bioprinting, other methods such as extrusion-based bioprinting
(EBB), inkjet-based bioprinting, and light processing-based bioprinting (including laser-
assisted bioprinting, stereolithography, and laser-Induced forward transfer (LIFT)) are three
genuine scaffold-based techniques that exhibit high potentials in fabricating complex
architectures (Figure.2). [116-118]. However, among these various strategies, the extrusion-
based bioprinting method, due to the specific characteristics suggested, is the most
considerably applied method in cardiac bioprinting application, while to a great extent, no
studies have been reported the utilization of other approaches in cardiac bioprinting.
Accordingly, here in this section, the focus has been established on extrusion-based and
scaffold-free methods.
4.1. Extrusion-based Bioprinting

EBB is a widely-applied method used to fabricate cardiovascular tissues by simultaneously
dispensing cells and materials matrix. In this method, complex structures are biofabricated with
various cells precisely. Mechanistically, EBB technology is very similar to the fused deposition
modeling among 3D printing systems. It is based on extruding viscoelastic polymeric bioinks
via an automatic robotic system to form 3D constructs layer by layer. An automated mechanical
system can move in x, y, and z directions controlled and adjusted perfectly by a computer [72,
117, 119]. The dispensing system, which is pneumatic or mechanical (piston or screw-based),
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extrudes bioinks through nozzles and dispenses them on build-bed. Hence, a 2D pattern is
primarily printed. After printing this 2D pattern, different curing methods solidify the bioink,
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and subsequently, the second layer is printed on the first layer to fabricate the 3D pattern layer
by layer. Cell viability and cell density are critical factors in bioprinting. In EBB techniques,
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the effect of shear stress on cell viability is one of the parameters that should be controlled
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besides thermal stresses, which both can cause cell death. On the other hand, printing bioinks
with high cell densities require higher shear stress. So, the printing pressure should be adjusted
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due to these two factors [72, 116, 118, 120-126].

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4.2. Scaffold-free Bioprinting

No biomaterials are used to print cells and generate a 3D structure in scaffold-free bioprinting.
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Cell pellets are fused in a 3D printed mold to secrete the extracellular matrix and to be held
together. Through this method, the cells should go under a series of cell recapitulations to be
further employed in bioprinting tissues. Since many cell expansions would be required to
obtain a tissue-level regeneration, this method may be slower than the scaffold-based methods.
Despite cell proliferation inside the scaffold, many cells are poured into the scaffold to form
the ECM in the shape of a cylinder, torus, spheroids, and honeycomb. Various developed
methods are used to biofabricate scaffold-free structures, including hanging drop, pellet (re-
aggregation) culture or conical tube, micro-molding, microfluidics (hydrodynamic cell
trapping), liquid overlay, spinner flask, and rotating wall vessel techniques. [116, 127, 128].
Scaffold-free bioprinting is a method that fabricates structures through bioprinting of living

cells with designed patterns. Cells are deposited on a substrate or a spheroid mold to derive
them in a specific module and are bioprinted layer by layer to form the final 3D structure. Using
this method, many kinds of cells can be implemented to biofabricate tissue-like structures [116,
127]. Compared to scaffold-based bioprinting methods, scaffold-free practices suggest a higher
range of efficiencies, primarily attributed to the cells’ self-assembly and high-speed bioprinting
process. Furthermore, in this strategy, cell retention is high. Post-printing maturation time is
comparable with other methods. One of the acute adverse effects of using these constructs is
the possibility of immunogenic rejection unless the patient-derived cells would be employed
[116, 124, 127, 128].
4.3. Bioassembly
Self-assembly bioprinting is a subclass of a scaffold-free method to bioprint desired
microstructures. Several processes such as self-assembly, robotic assembly, Faraday acoustic
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assembly, bio-acoustic levitational assembly, magnetic assembly enable the user to organize,
reorganize and regenerate basic units to form the 3D tissue architecture used in scaffold-free
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based bioprinting [120, 129]. In this method, the micro-tissue bioinks are deposited in a closed
area, fused, and further structured layer by layer to biofabricate the final construct. Flexibility,
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high scalability of desired architecture, reducing cardiac hypertrophy and fibrosis, high cell
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densities, and paracrine signaling are promising properties that have made this method more
and more desirable. Nevertheless, low architectural resolution and lack of control are two main
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disadvantages of using the self-assembly technique [126, 128, 129].

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Figure.2 Schematic illustration of 3D bioprinting technologies: a) Piston-based, pneumatic-based, and
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screw-based micro-extrusion technique b) Thermal and piezoelectric inkjet printing c) Pulsed-laser-

assisted bioprinting d) Stereolithography.
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5. Architectural Designs
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The biological construct’s architectural design is one factor that can be considered to provide
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cell-signaling features and affects cell-biomaterial interaction such as adhesion, cell growth,
and especially migration, which directly leads to higher cell functions. Specifically, cell
alignment, conductivity, and contractility are the functions that can be controlled under the
influence of the design of the bioprinted construct [24, 130]. Several architectural designs such
as honeycombs, grids, strings, and even anatomical models have been investigated to induce
higher cell viability and functions. In a study conducted by Zhang and colleagues, a biological
construct was printed in an anisotropic honeycomb design, which provided higher connexin-
43 (Cx-43) expression and cell alignment in the direction of anisotropy compared to the
isotropic design [24]. In another study, Noor et al. [131] used decellularized omentum tissue-
based bioink to bioprint a vascularized cardiac patch in an anatomical design. The study
indicated that the cardiac patch is contractile, bioprinted cardiomyocytes are aligned and
elongated, and endothelial cells formed lumens, which shows that biomimetic structural design
can influence the morphology and functions of cells [131].
Vascularization is a significant challenge that should be overcome in 3D bioprinting due to
delivering nutrients and oxygen to the constructs’ inner space [3]. Different designs are
considered for endothelial cells and cardiomyocytes co-culture, mainly grids and layer by layer
co-culture. In another study, Maiullari et al. used a bioink consisting of cardiomyocytes and
endothelial cells in a grid design. In this design, a cardiomyocyte layer and endothelial layer
were bioprinted respectively through a microfluidic printing head. Higher cell viability and
tissue-level functions were provided by this design compared to other bioinks containing only
cardiomyocytes [18].
Regarding converted 2D images of the human heart to 3D models, designed models could be
produced and reproduced safely and in a patient-specific manner with complex architectures.
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In most designed structures, X-ray computed tomography, or MRI (magnetic resonance
imaging), is used to capture the human heart’s complex anatomy, and subsequently, 2D images
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are converted to a 3D image. The 3D image will be processed in Biomimics software to
optimize the desired architecture. Thus, the final design will be converted to a G.code file to
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bioprint designed human heart model [132-134].
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6. Biophysical Stimulations
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The heart is a highly active organ in which, in both developing and adult cardiac tissue,
cardiomyocytes are subjected to various kinds of stimulations, including contractile
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mechanical forces, electrical forces, and hemodynamic stresses [16, 135]. Numerous studies
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have revealed that these intracellular and extracellular stresses can have deterministic effects
in cardiomyocyte maturation, mechanoelectrical coupling, and maintaining the differentiated
phenotype of cells. Traditional bioengineering systems have shown immaturity in
cardiomyocytes derived from stem cells such as iPSCs and skeletal myoblasts [136, 137]. This
immature characteristic of cardiomyocytes results in impaired synchronization and poor
mechanoelectrical integration with the host tissue [2, 137]. Hence, novel bioreactors and
progressive methods are developed to provide relevant physiological states and deliver guiding
stimuli to the bioengineered tissue to direct the cardiomyocytes’ maturation through the
construct. Here, we discuss major contributing stimuli in cardiac regeneration.
6.1. Mechanical Stimulation

There is an increasing body of evidence that mechanical stimulations have profound impacts
on cell physiology modulation and may, as a result, promote biosynthetic activities in cells
residing in bioartificial matrices, thereby facilitating or speeding up tissue regeneration in vitro.
Mechanical forces experienced by cardiomyocytes in vivo are exerted mainly by contractile
forces, blood flow, shear stress, and pulsatile blood pressure [16]. These mechanical
stimulations have been helpful in the anisotropic alignment of cells and directing functional
and structural maturity of cells by regulating the genes and proteins expression [138]. In vitro
Mechanical stimulations can be established by adjusting the mechanical properties of the
biomaterial compartment of the bioink or exerting external stretching forces on the construct
[2, 16, 138]. However, external stretching is the simplest way to exert mechanical stimulus.
Since it can be adjusted to establish a broad range of stimulations, there is a significant focus
on different modules of stretching forces in the literature [4].
Although various studies indicate the potential impact of mechanical stimulation on cells’
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functional and morphological organization, there is still little knowledge regarding particular
specifications of mechanical stimulations like or regimes of application (i.e., magnitude,
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continuous or sporadic, frequency). The mainly applied mechanical stimulation studied in the
literature is the stretch force exerted in different modalities. Suspending engineered cardiac
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tissue between fixed holders is the most straightforward approach to load mechanical forces.
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In addition to being easy to implement, static loading protocols can also be performed for
extended periods without causing tissue rupture. However, as another highly demanding
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approach, cyclic stretching has been effective while only feasible for a limited period (7 to 10
days) without premature rupture. In this approach, the stimulation period can be adjusted within
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two specific ways of (i) introducing advanced materials through which the viscoelastic
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properties of engineered tissue are changed and (ii) by adjusting the stretching algorithm to the
intrinsic characteristic of engineered tissue [139]. It is required to mention that in the second
approach, in optimizing the cycle length, it should be in harmony with the preexisting
pacemaker cells within the bioengineered cardiac tissue. Ultimately, the tissue is suspended
between resilient mounts to optimize auxotonic contractions of engineered cardiac tissue. This
appears to mimic the physiologic contraction cycle substantially, but it is also the most
challenging to generate in a way that corresponds to the constantly evolving contractile
characteristics [139, 140].
These extracellular mechanical stresses can be transmitted by transmembrane integrins

binding, such as the tyrosine kinase receptor, which further activates particular intercellular
pathways including Rho/ROCK, MAPK/ERK, FAK, and AKT [138-143]. These pathways and
cellular fate largely contribute to regulating specific cellular activities, including hypertrophy,
mitochondrial oxidative stress, calcium handling, and remodeling, causing anisotropic cell
alignment and both phenotypical and functional maturation [144]. Hence, the bioink should be
well-optimized to harbor dynamic mechanical stretch-relaxation stresses for long periods,
while architectural and compositional considerations can establish phenotypical and functional
maturation.
6.2. Electrical Stimulation

The heart’s electrical activity is attributed to the well-organized patterns of voltage-gated ion
channels responsible for action potential formation and gap junction proteins contributing to
the propagation of electrical signals through the cardiac syncytium. The electrical signals are
converted to the contractile force by the excitation-contraction coupling (ECC) [2, 145]. Hence,
cardiac tissue’s coordinated contraction primarily depends on gap junction proteins’ presence
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and expression patterns, specifically Connexins (Cxs) [146, 147]. Both direct and pulsatile
electrical impulses are crucial in developing hearts regulating the expression of connexins and
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voltage-gated channels ions [138]. This is consistent with the finding that the conventional
culturing iPSC-derived cardiomyocytes have shown to lack voltage-gated ion channel
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expression, which leads o immature nonfunctional cardiomyocytes [148]. The bioengineered
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tissues may also possess electrically and mechanically active but occur in varying rates and
less spontaneous behavior. Therefore, cells’ electrophysiological activity in bioengineered
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tissues can indicate cardiac maturity [138, 149]. Numerous studies have developed biomimetic
systems to deliver electrical impulses to bioengineered cardiac tissue to harbor control over the
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tissue function and to develop mechanoelectrical properties, especially cell alignment,

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increased electrical coupling through up-regulation of junction proteins, amplified contractions

concurrently, less arrhythmia, and highly organized ultrastructural organization [138, 144,
150]. For instance, through a study conducted by Asulin et al. [151] on one-step 3D bioprinting
of cardiac patches with built-in soft and stretchable electronic systems, it is demonstrated that
electrodes built into the engineered tissue enable the monitoring of extracellular potentials,
which gives a clearer picture of the tissue’s function and more controllability [151]. In another
investigation, Ruan et al. [136] studied the effect of electrical stimulation coupled with
mechanical conditioning on the force maturation and contractility of iPSC-Derived human
cardiac tissue. They indicated that the electric pacing cooperated with static stress conditioning
has resulted in increased force production (1.34±0.19 mN/mm2), increased RYR2 (Ryanodine
Receptor 2), and SERCA2 expression and hence, promoted maturation of excitation-
contraction coupling [136].
6.3. Hemodynamic Stimulation
Aside from the conventional strategies of applying mechanical stresses, hemodynamic-induced
stress, as an epigenetic factor, can significantly influence cardiomyocytes’ maturation and
functionality [4, 152]. The native shear stress experienced by cells due to the dynamic blood
flow contributes to a highly organized gene expression modulation, resulting in particular
morphological and functional maturity [4]. A significant result is the increased viability of cells
attributed to the highly improved transport of nutrients, oxygen, and regulatory molecules.
Static conditions are majorly involved with the diffusion exchange of biomolecules, a short-
term phenomenon that does not efficiently contribute to macroscale constructs. Perfusion
allows the more homogenous spatial distribution of vital biomolecules such as oxygen through
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the dynamic convection of mass, crucial in large-scale constructs [4, 138, 153, 154]. It has also
been shown that the shear stress induced by perfusion results in improved compliance to the
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burst pressures, cellular polarization, angiogenic responses, and more organized ECM
production [4, 155].
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Perfusion can be exerted in two ways; steady-state flow and pulsatile flow [138]. Based on the
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results from a study by Brown et al. [156], it has been shown that the pulsatile perfusion in
high flow rates leads to a higher contraction strength and lower excitation thresholds required
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for coordinated stimulations. However, in low flow rates, the morphological changes are
accompanied by hypertrophy of the biological construct [156]. In another study about the
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perfusion impact, compared to the static conditions, a six-fold increase in the ERK1/2 signaling
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pathway has been reported, highlighting the crucial role of ERK1/2 contributing to the high
expression of contractile and cellular junction proteins in cardiomyocytes. These modulations
in the protein expression induced by pulsatile perfusion showed enhanced viability, cellularity,
and ultrastructural organizations [157, 158].
7. 3D Bioprinting of Cardiac Tissue

Recently, many research groups have been working on 3D bioprinting of cardiac tissue. They
have investigated many approaches to form different biological constructs such as patches,
organoids, and other scaffolds to study cardiac tissue behaviors, drug tests, and differentiation
of several stem cells [159].
Zhang et al. [24] fabricated a biological construct emphasizing the impact of HUVECs on
neonatal rat cardiomyocytes and anisotropic design using an extrusion-based 3D bioprinter.
Different types of bioinks were prepared so as to contain different GelMA and alginate
concentrations, and dual crosslinking was employed for alginate and GelMA. In this research,
prepared bioinks were extruded via coaxial nozzles, which have been used to crosslink alginate
during the printing process and provide higher shape fidelity before the second step of GelMA
crosslinking by UV exposure. HUVECs with a cell density of 1×107 cells/ml were encapsulated
in alginate. The bioink were further bioprinted in a grid design with different aspect ratios of
unit grids (2×2, 2×3, 2×4, and 2×5) to form an accordion-like honeycomb structure, to mimic
genuine endothelium, and stimulate cell orientation in the direction of bioprinting. The
anisotropic design and surface-to-volume ratio of the construct was found to affect the
migration of HUVECs forming vascularized structures [24, 160]. After 15 days, neonatal rat
cardiomyocytes were cultured on the confluent layer of endothelial cells, which was prepared
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from the same bioink without encapsulated cells to investigate the impact of endothelium layer
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on cardiomyocytes viability and functions for three days. Cardiomyocytes were matured and
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expressed proteins necessary for conductivity, contractility, and synchronous beating rate. It
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was also demonstrated that the biological constructs with anisotropy design (2×5 sample)
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expressed Cx-43 with a coverage area of about 8.02 ± 0.54%, which provided a higher
synchronous beating rate and a higher percentage of aligned cardiomyocytes. Zhang et al. [24]
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employed the CD31 surface marker and GFP-HUVECs to demonstrate the formation of lumen-
like endothelial layers on the surface of microfibers and the migration of endothelial cells from
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the core of microfibers to the surface of them respectively. The results indicated that the
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endothelial cells migrated through microfibers to form the endothelium-like layer because of
the high HUVECs density (10×106 cells/ml). To evaluate cardiac tissue functions, Cx-43 and
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sarcomeric actinin were used to compare cardiomyocyte contractility, orientation, and beating
in anisotropic constructions with other 2×2 and 3×3 isotropic samples. Eventually, the
organization of cardiomyocytes was evaluated after culturing them on the endothelium-like
bioprinted construct. It was illustrated that the existence of VGEF, which was secreted from
endothelial cells, enhanced the tissue-specific functions of cardiomyocytes [24, 160, 161].
Preparing GelMA-based bioink and using alginate as a sacrificial material is one of the
excellent methods for bioprinting; however, using photoinitiation in the UV range is still
harmful to cells. Besides, preparing a construct with anisotropic structure helps cardiomyocytes
elongate significantly in the long term and encourages contractility.
Wang et al. [162] have studied another composite bioink containing neonatal ventricular
cardiomyocytes with a density of 10×106 cells/ml and fibrin-based hydrogel, which was
prepared out of fibrinogen, gelatin, glycerol, aprotinin and, hyaluronic acid with different ratios
to fabricate a contractile and functional cardiac tissue. The bioink incorporated a sacrificial
hydrogel, which contained the same bioink without fibrinogen and aprotinin, to a reinforced
cell-laden hydrogel. They used an extrusion-based tri-nozzle bioprinter to deposit two types of
constructs, including string and patch form out of the bioink and the sacrificial hydrogel,
surrounded by a PCL frame. First, the frame was printed to anchor the biological construct
from two sides to enable intrinsic forces. After 30 minutes of resting in the chamber, the
sacrificial hydrogel and cell-laden hydrogel were bioprinted at 18ºC. Subsequently, functional
characterization of contractility, cell alignment and, electromechanical coupling of the
construct was investigated for three weeks [162]. The construct’s contractility was localized
and limited after three days; however, the synchronous and spontaneous beating was detected
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after four weeks. By detecting α-actinin and connexin, elongation of cardiomyocytes was
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observed, without appreciable differences in both designs. A contractility and maturation
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positive feedback loops were observed, allowing the maturation of the constructs.
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Cardiomyocytes from single cells to a dense tissue-like structure were assessed with the same
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immunofluorescence markers to evaluate string and patch forms in 3 weeks. The results
showed that both designs supported cardiomyocytes’ functions properly. After four days,
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cardiomyocytes were just aggregated in the bioprinted construct; however, there were more
elongated and denser populations of cells after three weeks in cardiac patch form. This study
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showed that design plays a significant role in cardiac tissue development and contractility.
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After three weeks, the patch sample provided higher concurrent contractility, and
cardiomyocytes proliferated more than the string sample [162].
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One of the bioprinting applications is to support 3D culture and the differentiation of stem cells.
Tijore et al. [23] utilized this capability to differentiate human mesenchymal stem cells
(hMSCs) toward cardiomyocytes and further investigated the impact of micro-channeled
hydrogel design on differentiation. In this study, neonatal rat cardiomyocytes with a density of
2×105 cells/cm2 were seeded on similar micro-channeled scaffolds to investigate the impact of
micro-channeled design on elongation, contraction, and alignment of cardiomyocytes. The
gelatin-based hydrogel was further crosslinked with microbial transglutaminase (mTgase)
overnight at 37 ºC to form a rectangular hydrogel sheet with a microchannel structure. After
the crosslinking process, the micro-channeled construct was soaked in culture media for 24 h,
hMSCs with cardiomyocytes were seeded on the plain and microchannels of printed hydrogels.
Cell viability, cardiomyogenic lineage commitment, and cell alignment were evaluated over
nine days. The difference between the impact of plains, micro-channels, and spacings between
them was discussed after evaluation. The results showed that the micro-channels facilitated
elongation of cellular morphology, well-established F-actin anisotropy, and a significant
increase in mature cardiac markers. Also, seeded cardiomyocytes exhibited synchronized
beating and more alignment. Tijore et al. [23] used β-mhc along with DAPI and Ph to compare
the influence of spacing between microchannels and plains on the hMSC morphology and
demonstrated that stem cells cultured on microchannels with 500 µm spacings were stretched
in the direction of gelatin pattern more than samples with 1000 µm spacings. The morphology
of cultured stem cells on micro-channels and plains was assessed with Ph and DAPI. The
assessments showed that 40% of hMSCs cultured on microchannels were orientated 0 to 10
degrees; however, about 7% of plain cultured cells were oriented in the same degree interval
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[23].
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Izadifar et al. [163] prepared an alginate-based bioink mixed with Human coronary artery
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endothelial cells (HCAECs). The constructs were printed in grid designs with different strand
alignment angles (15/165°, 0/90°, and 0/45/90/135°) to demonstrate the effect of mechanical
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properties and design on viability and functions of endothelial cells. It was indicated that the
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construct with 0/45/90/135° strand alignment angle provided higher compressive modulus,
stiffness, and conductivity, leading to higher cell viability and cell functions [163].
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In another study, Izadifar et al. [115] introduced a new hydrogel mixture prepared out of
MeCol, alginate, which was reinforced with functionalized carbon nanotubes (CNTs) to
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improve mechanical, electrical, and biological performance properties. CNTs were

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functionalized with carboxylic groups and were incorporated into crosslinked alginate and
MeCol. A photopolymer (verowhite fullcure 835) was printed in parallel strands micro-mold.
Before printing the bioink, HCAECs were encapsulated in MeCol and mixed with alginate
hydrogel with a density of 0.8 – 1×106 cells/ml. The cell-laden hydrogel mixture was bioprinted
on the micro-mold, and alginate was crosslinked through Ca2+. Subsequently, the MeCol was
photo-crosslinked with methacrylic anhydride chemistry through UV exposure. The micro
mold was removed, and a micro-patterned bioink after crosslinking was left. Patches were
soaked in calcium-free DMEM (Dulbecco’s modified eagle media), and the impact of the CNT
nanotubes density on mechanical, electrical, and biological behavior was evaluated in several
days. The results displayed that CNT-incorporated alginate enabled a highly interconnected
meshwork with robust viscoelastic and electrical conductivity of photo-crosslinked MeCol and
alginate (Figure.3 a-b) [115]. Izadifar et al. [115] assessed cardiac patches’ cell viability and
compared the influence of UV exposure on cell viability. Cells were colored with Calcein-AM
and Hoechst dyes, and it was shown that with an increase of time of the UV exposure, cell
death would be higher after three days. Izadifar et al. [115] demonstrated the impact of CNTs
with the biomimetic view from Purkinje fibers on the morphology and orientation of
cardiomyocytes. The results showed that HCAECs entrapped in the CNT-incorporated hybrid
were more elongated and aligned in the direction of CNTs than the alginate-HCAEC bioprinted
construct (Figure.4 a) [115].
It is reported that cardiac fibroblasts affect the maturation and function of cardiomyocytes.
However, because of the high proliferation of fibroblasts compared to cardiomyocytes,
conductivity and contractility of cardiomyocytes could be impaired if fibroblast proliferation
is not properly controlled. To overcome this challenge, CNTs, gold-based nanomaterials such
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as gold nanorods, nanowires, and nanospheres can be used to induce higher electrical
conductivity. Zhu et al. [164] prepared gold nanorods and coated them with GelMA to fabricate
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a cardiac patch with higher conductivity and contractility. In this study, neonatal rat ventricular
fibroblasts and cardiomyocytes were suspended in Alginate and gold nanorod (G-GNR)-
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incorporated GelMA hydrogel and printed in a grid pattern. Higher beating rate, elongation,
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and contraction rate were reported compared to Alginate-GelMA bioprinted constructs [164].
To this end, it was shown that a bioink consisting of CNTs or GNR could provide higher
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electrical conductivity, leading to a more developed cardiac tissue that can perform a repetitive
and concurrent beating.
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The cardiac patch is a proper substitute for the cell therapy method due to the limited retention
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and low efficiency of cell-based approaches. Bejleri et al. [113] fabricated a cardiac patch using
extrusion bioprinting. Human cardiac progenitor cells (hCPCs) have been suspended in the
cardiac extracellular matrix (cECM) to prepare a novel bioink. However, due to the low
mechanical properties of cECM, GelMA was added to the bioink composition. The biological
construct was bioprinted in a grid design, and white light was used to crosslink GelMA instead
of UV radiation to decrease cell death. The cECM-GelMA bioink provided a higher mechanical
storage modulus and cell viability [113]. Mechanical compressive or tensile strength of a
construct is one characteristic that usually has not been considered in cardiac 3D bioprinting;
however, it was demonstrated that it could impact cardiomyocytes’ viability and functions.
Investigation of mechanical properties of the cardiac patches alongside electrical properties can
lead to a better understanding of cardiac patch development.
Printing hydrogel-free bioink is another method that has been used to fabricate cardiac patches.
This method was called the cardiosphere by Ong et al. [159]. It was made out of 3300 primary
cells in total, including human cardiac fibroblasts, human iPSC-derived cardiomyocytes, and
HUVECs, which were co-cultured with different ratios (70:15:15, 70:0:30, 45:40:15) to
produce mixed-cell aggregate. They were placed on a needle array by an extrusion-based
bioprinter. It was then put on a shaker in an incubator for 72h to allow the fusing of spheroids
before removing the needle array. Printing spheroids with different cell densities in precise
positions is the critical factor of this method, enabling the fabrication of a whole cardiac patch.
In another study, Yeung et al. [165] used the 70:15:15 sample and showed higher contractility,
ejection fraction, and vessel count, provided by the higher concentration of fibroblasts and
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endothelial cells [159, 165].
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Lee et al. [74] have introduced a novel reversible freeform embedding of suspended hydrogels
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(FRESH v2.0), a buffer system consisting of a thermo-reversible bath of gelatin microparticles
that unmodified collagen can be extruded in it. This method enables collagen with high
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concentration to self-assemble in a gelatin bath by rapid pH change of the gelatin bath and
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allows fabricating structures with high resolution. Gelatin baths act as a support to the
biological construct and can be uncrosslinked at 37ᵒC. Another improvement of FRESH v2.0
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is using morphological uniform microparticles with a diameter of 20 µm, which provides a

support with higher mechanical properties. In this study, different advantages of this method
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to bioprint components of the cardiovascular system were evaluated. Collagen type I was
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bioprinted with this method, and gelatin microparticles were purposely incorporated in the
construct and melted away to provide a porous structure. The porous and non-porous constructs
were implanted in vivo to evaluate cell infiltration. This comparison showed that porous
uniform structure provides higher cell-infiltration because of uniform 25 µm pores prepared by
the FRESH v2.0 method. Both constructs were later incorporated with VEGF and fibronectin
to provide a microenvironment for angiogenesis and were compared again after subcutaneous
implantation. FRESH-printed construct provided enhanced vascularization after ten days. The
same method was used to print the left ventricle model with two nozzles. One of them printed
collagen bioink in two shells, and another bioprinted a bioink consisting of human embryonic
stem cell-derived cardiomyocytes and cardiac fibroblast in the spaces between two shells. After
four days, the construct contracted, and after seven days, the contraction was synchronous
throughout the entire construct. Eventually, a structural model of the human heart was printed
out of a bioink consisting of neonatal rat cardiomyocytes and collagen hydrogel to show the
capability of this approach to bioprint micro-scale structure [74]. Mirdamadi et al. [166]
repeated this method using the same bath, but GelMA was substituted by alginate [166].
Collagen is one of the functional proteins of cardiac tissue ECM; However, fabricating a
construct from only one type of protein might not lead to a complex structure with tissue-level
functions. Due to the different microenvironments of cell types, different types of proteins and
polysaccharides should be considered in preparing a bioink to bioprint a complex structure
from a biomimetic point of view.
Co-cultured cardiomyocytes and endothelial cells have been commonly used in cardiac tissue
engineering; Maliauri et al. [18] researched the same topic by combining microfluidic and
bioprinting approaches to print more precise structures. They introduced a microfluidic printing
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head made of polycarbonate with a Y-junction microchannel structure connected to a coaxial
syringe. Alginate and polyethylene glycol monoacrylate-fibrinogen (PF) bioink containing
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iPSC-derived cardiomyocytes with an initial concentration of 8×106 cells/ml and HUVECs
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with the density of 6×106 cells/ml were bioprinted in a grid design. Alginate hydrogel was
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crosslinked ionically while the bioink was printed through a coaxial microfluidic nozzle. After
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bioprinting the construct, PF was crosslinked by photochemical crosslinkers (Figure.3 c). Cell
viability, several functional gene expressions, including cardiac early and late genes, and
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different tissue evaluations like orientation and contractility were studied in 14 days. It showed
printed cardiomyocytes’ orientation and organization is significantly similar to genuine
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myocardium compared to the casted construct.

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On the other hand, HUVECs were stained with von Willebrand factor (vWF) and DAPI to
indicate the influence of vascularization in different geometries. The orientation, organization,
and microenvironment of cardiomyocytes affect the functionality of these cells extensively.
Hence, cardiomyocytes’ orientation was compared in three mentioned designs, bulk hydrogel,
and 3d bioprinted hydrogel. Cardiomyocytes demonstrated more alignments in the direction of
printing and more maturation in the Janus geometry (one layer of cardiomyocytes : one layer
of HUVECs) compared to two other constructs, which were 4:2:4 (four layers of
cardiomyocytes : two layers of HUVECs : four layers of cardiomyocytes) and 2:2:2:2:2 (two
layers of cardiomyocytes : two layers of HUVECs : two layers of cardiomyocytes : two layers
of HUVECs : two layers of cardiomyocytes) (Figure.3 d) (Figure.4 b). besides, it is believed
that the maximum oxygen and nutrients diffusion distance to obtain the highest cell viability
and functionality without vascularization is approximately 100-200 µm. Hence, more layers of
endothelial cells in the Janus design compared to two other biological constructs helped
cardiomyocytes be more aligned and organized (Figure.4 b) [18].
Decellularized human tissues are one of the most promising resources to develop hydrogels as
a component of a bioink. Noor et al. [131] used decellularized omenta from humans or pigs
mixed with iPSC-derived cardiomyocytes for the main bioink to bioprint vascularized cardiac
patches as a proof-of-concept for the patient-specific treatment (figure.5 a-c). Gelatin was
mixed with iPSC-derived endothelial cells and bioprinted as a sacrificial bioink to induce
vascularization. To this end, the orientation and 3D structure of a patient’s heart vasculature
were identified through a computational tomography image and Computer-aided design that
can be attached to the patient’s heart with the same vasculature structure (figure.5 d-f). Blood
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vessels and cardiomyocytes were identified by CD31 and actinin, respectively (figure.5 g), and
the contraction of the cardiac patch was observed through transient calcium. Elongated and
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aligned cardiomyocytes were detected (figure.5 h). It was demonstrated that a contracting
cardiac patch could be prepared by a patient’s own cells with similar cell activity. To assess
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this method’s ability to bioprint larger constructs with more complexity, Noor et al. [131]
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mixed neonatal cardiomyocytes with the same personalized bioink to bioprint an anatomical
model of a whole human heart (figure.5 i-j). HUVECs were combined with gelatin sacrificial
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bioink like the previous step to print complex vasculatures. The whole heart tissue had close
mechanical properties to the rat’s heart. According to the bioprinting plan, a confocal image of
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the bioprinted heart showed that the same spatial organization of cardiomyocytes and
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endothelial cells was achieved (figure.5 k). Sarcomeric actinins were observed after one day,
which showed an internal compartmental structure close to the rat’s heart (figure.5 l) [131].
The limited electrical function is one of the most critical issues that cause the disability of
cardiac patches to treat the diseased areas of the genuine cardiac tissue. Asulin et al. [151]
studied the fabrication of stretchable and flexible planar electronic systems by lithography and
integration of it in a bioprinted construct to produce controlled electrical function. Three types
of bioink were prepared, a cell-laden bioink to encapsulate cardiac cells and other two PDMS
bioinks to act as electrodes and dielectric, respectively. Cellular bioink was prepared by
decellularization of pigs’ omenta. Two other bioinks were prepared by mixing graphite
synthetic powder and span 80 with PDMS to conduct and passivate electrical signals,
respectively. First, the electrical and mechanical properties of the bioinks were investigated.
To determine the optimum conductivity, bioinks with different concentrations of graphite
flakes were prepared, and the conductivity was measured, and it was demonstrated that 45%
(wt) showed the highest conductivity. Subsequently, stress-strain behavior of the ink was
evaluated that showed robustness about 50% and elongation under 20%, which is close to the
mechanical behavior of cardiac tissue. Change of resistance by degradation and mechanical
evaluation were assessed for the printed passivation bioink that showed no significant change
of resistance and 135% elongation. Next, the main construct was printed to assess the electrical
and mechanical ability of the cardiac patch. Neonatal rat cardiomyocyte containing bioink was
printed with eight electrodes, including six core electrodes which had a passivation layer
around them; however, the end of each electrode was left without a passivation layer for
stimulation and point sensing. Two outer electrodes were exposed for field stimulation. The
cardiac patch showed high levels of actinin and synchronized contractions through four regions
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of the patch after 12 days [151]. To this end, bioprinting of hydrogels from decellularized
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tissues alongside conductive biomaterials in a denser hydrogel bath is one of the most accurate
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methods which can be used to biofabricate a complex micro- and macrostructure with specific
tissue-level functions.
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Human native cardiac tissue has vital abilities such as contractility, excitability, conductivity,
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and automaticity, which should be considered in cardiac tissue bioprinting. Preparing an

appropriate microenvironment encourages cells to develop a tissue-like structure and operate
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tissue-level functions. Researchers working on cardiac tissue bioprinting have used many
natural and synthetic hydrogels; however, hydrogels based on decellularized tissues have
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provided a more effective environment than other natural hydrogels. Proteins in cardiac tissue
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ECM such as collagen and fibrin alongside hydrogels based on decellularized tissue have been
used extensively to meet the expectation of a genuine microenvironment from a biomimetic
point of view. Crosslinking the hydrogel composition is one of the challenges that should be
overcome in cardiac tissue bioprinting. UV crosslinking is one of the crosslinking methods that
have been used repeatedly in studies. It has been reported that UV radiation cause cell death
and decreases cell viability which is the first step to having functional tissue. Hence,
photoinitiators that work in the visible range with higher length wave has been utilized to
overcome this challenge. Using gold nanorods or CNTs is the next step in this field to provide
higher tissue-level functions. These nanorods or nanotubes can stimulate contractions and
alignment of the cardiomyocytes, leading to better contractile cardiac tissue development.
Besides, investigating the differentiation and growth of MSCs toward cardiomyocytes during
embryo development could be crucial in achieving and eventually developing more similar
constructs to genuine cardiac tissue. The presence of endothelial cells or endothelium lumen-
like structure, fibroblasts, and VEGF are significant factors alongside printing constructs with
anisotropic architecture, hydrogel’s mechanical, chemical and electrical properties that help
cardiomyocytes cell growth and functions such as elongation and orientation; however, it might
not be enough to obtain a tissue formation and tissue-level functions [167, 168]. Another
challenge that should be considered is the maturation of bioprinted constructs. Microfluidic
bioreactors with a biomimetic point of view are one of the elegant designs proposed by Zhang
et al. [24] to mature bioprinted constructs with perfusion of culture medium and showed that
more mature tissue-like structures could be obtained compared to static cultures. Overall, to
achieve similar bioprinted constructs with native cardiac tissue, many aspects should be studied
to have a biomimetic point of view, from the embryo development in the early stages of seeding
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to different bioink compositions, designs, and maturation techniques [72, 133].
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Figure.3 Illustration of methods and designs used in cardiac 3D bioprinting: a) cell-laden CNT-
incorporated MeCol and alginate bioink 3D bioprinting and crosslinking with Ca2+ and UV exposure
[115] b) architectural design of cell-laden CNT-incorporated MeCol and alginate [115] c) preparation
of bioprinted construct with grid design out of cardiomyocyte- and endothelial-laden alginate,
polyethylene glycol monoacrylate-fibrinogen through microfluidic printing head and processes of
crosslinking with Ca2+ and UV exposure [18] d) different architectural designs of bioprinted constructs
(Janus, 4:2:4, 2:2:2:2:2) [18].
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Figure.4 Immunofluorescence and immunocytochemistry images of cardiomyocytes and endothelial
cells: a) Investigation of cardiomyocytes and endothelial cells behaviors (orientation, alignment, and
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migration) in CNT-incorporated MeCol and alginate 3D bioprinted constructs [115] b) Images of
explants, orientation of cardiomyocytes and vascularization of the 3D bioprinted constructs prepared
out of cardiomyocyte- and endothelial-laden alginate, polyethylene glycol monoacrylate-fibrinogen
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bioink [18].
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Figure.5 Biofabrication of cardiac patch and whole heart anatomical model: a) decellularized human
omentum tissue b) SEM image of a personalized hydrogel structure based on decellularized omentum
c) A personalized hydrogel before gelation at room temperature (left) and after gelation at 37 °C (right)
d) Schematic steps of free-form 3D bioprinting of the personalized hydrogel in the support material,
crosslinking at 37 °C, extraction of the biological construct by an enzymatic or chemical degradation
process of the support material, and transferring into culture medium e) A 3D model of a vascularized
cardiac patch f) The concept which was used to bioprint the patch g) A bioprinted iPSCs-derived cardiac
patch where the blood vessels are marked by CD31 (green) and cardiomyocytes are marked by actinin
(pink) h) Sarcomeric actinin (red) and nuclei (blue) staining of sections from the explanted patch i-j)
biofabricated anatomical model of a human heart k) 3D confocal image of the bioprinted heart
(Cardiomyocytes in pink, endothelial cells in orange) l) Cross-sections of the anatomical model of
human heart immunostained against sarcomeric actinin (green) nuclei (blue). Scale bars: (b) = 10 µm,
(g) = 500 µm, (h) = 25 µm, (j) = 0.5 cm, (k) = 1 mm (l) = 50 µm [131].
Bioprinting Architectural Cell activities and/or
No. Hydrogels Cells (density) References
Techniques Designs Tissue-level Activities
-Human umbilical vein
endothelial cell A higher percentage of
(HUVEC) (1×107 area covering,
-GelMA (low and high Extrusion based Anisotropic
cells/ml) synchronous beating,
1 molecular weight) bioprinting accordion-like [24]
and alignment were
-Alginate (pneumatic) honeycomb
-Neonatal rat seen in the (2×5)
cardiomyocytes (1×106 sample.
cells/ml)
Contractility, cell
Fibrin-based hydrogel, gelatin, Neonatal ventricular Extrusion based alignment and,
String and patch
2 glycerol and, hyaluronic acid cardiomyocytes bioprinting electromechanical [162]
form
(10×106 cells/ml) (pneumatic) coupling were observed
in both samples.
Higher elongation,
-Neonatal rat
contraction, and
cardiomyocytes (2×105 Extrusion based
Gelatin Micro-channeled alignment of
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3 cells/cm2) bioprinting [23]
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(crosslinked with mTgase) sheet cardiomyocytes were
-Human mesenchymal (pneumatic)
seen in micro-channeled
stem cell (h-MSC)
sample.
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-Cecm
Human cardiac
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Extrusion based
Higher mechanical
modulus and cell
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4 progenitor cell (hCPC) bioprinting Grid viability were provided [113]
-GelMA
(3×106 cells/ml) (pneumatic) in cECM-GelMA
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sample.
The biological construct
Human coronary artery Grid with different with 0/45/90/135°
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Extrusion based
endothelial cells angles (15/165°, pattern provided the
5 Alginate bioprinting [163]
HCAEC (0.6×106 0/90°, and most cell viability,
(pneumatic)
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cells/mL) 0/45/90/135°) compression modulus,

and conductivity.
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More organized vital

Extrusion based gap junctions and
-CNT incorporated MeCol HCAEC (0.8 – 1×106 Accordion-like
6 bioprinting migration of cells were [115]
-CNT incorporated alginate cells/ml) honeycomb
(pneumatic) observed between
meshes.
The gold nanorod based
Extrusion based
-GelMA Neonatal rat bioink provided a
bioprinting (co-
7 -Alginate cardiomyocytes Grid higher beating rate, [164]
axial and
-Gold nanorods (7.5×105 cells/well) elongation, and
pneumatic)
contraction rate.
The perfusion sample
-Collagen type I -h-MSC derived
Extrusion based provided a higher
-Gelatin cardiomyocytes
8 bioprinting Grid percentage of cell [74]
-Fibrinogen -HUVEC
viability and a more
-Alginate -Cardiac fibroblasts
vascularized structure.
- iPSC-derived Higher alignment and
Extrusion based
-Alginate cardiomyocytes vascularization were
bioprinting
(8×106 cells/ml) Grid obtained in the Janus
9 (co-axial [18]
-Polyethylene glycol sample, but none of
microfluidic
monoacrylate-fibrinogen -HUVEC (6×106 them showed any tissue
printing head)
cells/ml) formation.
-iPSC-derived Higher conduction
cardiomyocytes velocities, longer action
-Human cardiac Extrusion based potential durations, and
10 - fibroblasts bioprinting Sphere vascularization were [159]
- HUVECs observed in 70:0:30
(5-60×103 sample.
cells/Cardiosphere)
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- iPSC-derived
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Cardiomyocytes (1×108
cells/ml)
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-iPSC-derived
endothelial cells (2×107
cells/ml)
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-Patient’s heart
-Contracting cardiac
patch with aligned and
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-Decellularized humans/pigs Extrusion based
-Neonatal vasculature elongated
11 omenta tissue bioprinting [131]
cardiomyocytes (1×108 (anatomical model) cardiomyocytes
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-Gelatin as a sacrificial layer

cells/ml) (Whole heart - Whole rat’s heart -Close internal structure
study) (anatomical model) to rat’s heart
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-HUVEC (1.5×107
cells/ml) (whole heart
study)
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-High levels of actinin

- Decellularized pigs omenta
Extrusion based -Synchronized
tissue -Neonatal rat -spiral
12 bioprinting contraction in four [151]
-Synthetic graphite mixed in cardiomyocyte
regions of the patch
PDMS
Table.1 Cardiac 3D bioprinting: bioink composition, bioprinting technique, the architectural design
used in cardiac 3D bioprinting case studies, and highlighted result of cell activity and tissue formation.
8. Conclusion and Future Outlook
Cardiovascular bioprinting has drawn remarkable attention during the last decade, providing
precise control over fabricating 3D biological constructs. The significant progress in
cardiovascular bioprinting includes improved cell-cell integration, vasculature incorporation,
well-organized cell alignment, better management of complex architectures, and better
functions. These are majorly attributed to the development of bioprinting strategies and the
incorporation of more biologically responsive bioinks. The latter has resulted from more
diversified natural and synthetic hydrogels and the utilization of cells with regenerative
potential. Despite the remarkable advances, specific challenges restrict the fabrication of
functional large-scale cardiac tissue. Full maturation of cardiomyocytes has remained a
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significant challenge limiting the tissue-specific functions, which further impedes integrating
the host tissue. There have been remarkable attempts toward employing physiologically
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relevant stimulations inducing tissue-specific gene expression. However, reaching an
appropriate population of fully mature cardiomyocytes remains a substantial challenge. The
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lack of vasculature with high density in the bioprinted construct is another challenge that has
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significantly restricted the clinical application of bioprinted cardiac tissues. Efficient mass
transfer in the construct is a critical issue that allows uniform distribution of heterogeneous cell
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types through the scaffold, improved cell viability, and better cellular activity. Although
bioprinting allows precise control in fabricating complex architectures, supplying a vasculature
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network with the approximate density of 3000 cells/mm2 requires novel bioprinting systems
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with higher resolutions and improved modeling properties. The novel systems should pave the
way for improved mimicking of the cardiac tissue complex heterogeneous architecture.
Also, novel and closely tissue-mimicking bioink systems should be developed to improve the
construct cellular functionality and mechanical stability. Mechanical characteristics of the
construct could strongly influence both the integration with the host system and the direction
of cells toward cardiomyocytes. Thus, there should be a trade-off between the mechanical
properties and the processability of the bioink.
Bioprinting of bioinks with different architectures influences cardiomyocytes’ organization in

cardiac patches and leads to higher proliferation, biological functions such as notch signaling.
On the other hand, bioprinting of bioinks made out of hydrogels with high conductivity like
CNTs is another way to stimulate cardiomyocytes to higher cell growth and enhance functional
gene expression leading to contractility. New techniques like bioprinting scaffold-free cells
have been used to fabricate cardiac patches with determining cell densities of different cells
along cardiomyocytes, which produce their own ECM similar to genuine tissue in composition
structure after in vivo implantation. Fabricating vascularized biological constructs with
different designs that lead to more tissue-like structures and organoids is another application
of this technology. Bioprinting layers of endothelial cells between cardiomyocytes layers is
one of the approaches that highlight endothelial cells’ impact on cardiomyocytes’ viability and
function. Similar strategies like culturing cardiomyocytes after fabricating endothelialized
constructs have been employed to mimic cardiac tissue structure. It has been shown that
anisotropic endothelialized structure leads to more oriented cardiomyocytes with higher
frequencies of contractions. The combination of hydrogels with additives with high
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conductivity, the architecture of 3D bioprinted constructs, and the co-culture of cardiomyocytes
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and endothelial cells are the major strategies that have been used to improve cardiac tissue
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functions, which influence cell aggregation, proliferation, and differentiation to achieve tissue-
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like contractile structures with beating areas. Besides, there have been endeavors to fabricate
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chambered cardiac organoids with macroscale beating and the ability to pump blood through
in situ differentiation of hiPSCs to overcome the challenge of high cell density fabrication.
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As an exciting perspective in cardiovascular bioprinting, the fabrication of bioprinted tissues

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under zero- or microgravity conditions can promise other abilities in the field. Contrary to the
current situation on earth, the microgravity conditions suggest implementing lower viscosity
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bioinks and more satisfactory resolutions in printing at a single-cell scale. These conditions can
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remarkably improve the current mentioned challenges in cardiac bioprinting.

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Figure.6 Schematic representation of bioink preparation, bioprinting process, and maturation of the
cardiac construct (Created with BioRender.com).
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Acknowledgements
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The authors would also like to thank Dr. Mitra Asadi-Eydivand, Department of Biomedical
Engineering, Amirkabir University of Technology (Tehran Polytechnic), Iran and Dr. Sasan
Jalili, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology,
Cambridge, MA 02139, USA for assisting in designing and preparation of the graphical
abstract.
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit
sectors.
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Declaration of interests
☒ 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.
☐ The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
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Cardiovascular 3D bioprinting: A review on cardiac tissue development

Dianoosh Kalhori, Nima Zakeri, Mahshid Zafar-Jafarzadeh, Lorenzo Moroni, Mehran

To appear in: Bioprinting

Received Date: 29 March 2022

© 2022 Published by Elsevier B.V.

Nima Zakeri (MSc):

Technology (Tehran Polytechnic), Tehran, Iran

Mahshid Zafar-Jafarzadeh (MSc):

Lorenzo Moroni (PhD):

3.1.5 Decellularized ECM 13

3.3. Considerations in Bioink Composition 14

4.1. Extrusion-based Bioprinting 18

Among these novel methods, 3D bioprinting, as a powerful biofabrication technology

In contrast to non-parenchymal heart cells, parenchymal cardiomyocytes show minimal

survivability, they have been exploited extensively in regenerative medicine. Cardiac

potency of MSCs to transdifferentiate towards other lineages, including cardiomyocytes [45-

2.4. Human Embryonic Stem Cells (hESCs)

applications [60, 61].

2.5. Induced Pluripotent Stem Cells (iPSCs)

miRNAs), strongly influencing cell viability and cell cycle [65-68].

2.6. Non-parenchymal Cells

Also, collagen possesses poor immunogenic characteristics, which is majorly attributed to a

interaction of methacrylamide groups indicates better biocompatibility and stability in the

for any further modifications [77, 83].

groups [18, 90].

3.1.5 Decellularized ECM

specifically for cardiac tissue, electromechanical synchronization through successful

well-established solidification kinetics, high hydration ability, viscoelastic behavior close to

Another parameter that can be influenced by concentration is the shear-thinning behavior of

Modifying the internal structure of hydrogels through crosslinking is almost a post-printing

In irradiation crosslinking, three particular parameters should be strictly controlled for

4.1. Extrusion-based Bioprinting

due to these two factors [72, 116, 118, 120-126].

4.2. Scaffold-free Bioprinting

Scaffold-free bioprinting is a method that fabricates structures through bioprinting of living

disadvantages of using the self-assembly technique [126, 128, 129].

screw-based micro-extrusion technique b) Thermal and piezoelectric inkjet printing c) Pulsed-laser-

6.1. Mechanical Stimulation

These extracellular mechanical stresses can be transmitted by transmembrane integrins

6.2. Electrical Stimulation

tissue function and to develop mechanoelectrical properties, especially cell alignment,

increased electrical coupling through up-regulation of junction proteins, amplified contractions

7. 3D Bioprinting of Cardiac Tissue

improve mechanical, electrical, and biological performance properties. CNTs were

is using morphological uniform microparticles with a diameter of 20 µm, which provides a

myocardium compared to the casted construct.

and automaticity, which should be considered in cardiac tissue bioprinting. Preparing an

cells/mL) 0/45/90/135°) compression modulus,

More organized vital

-Gelatin as a sacrificial layer

-High levels of actinin

Bioprinting of bioinks with different architectures influences cardiomyocytes’ organization in

As an exciting perspective in cardiovascular bioprinting, the fabrication of bioprinted tissues

remarkably improve the current mentioned challenges in cardiac bioprinting.

"Bioengineering cardiac constructs using 3D printing." Journal of 3D printing in medicine 1, no.

thoracic research 9, no. 3 (2017): 127.

no. 8 (2015): 1503-1521.

repair." Biomaterials 29, no. 26 (2008): 3547-3556.