International Journal of

Molecular Sciences

Review
3D Bioprinting of Human Tissues: Biofabrication, Bioinks,
and Bioreactors
Jianhua Zhang , Esther Wehrle, Marina Rubert and Ralph Müller *

Institute for Biomechanics, ETH Zurich, Leopold-Ruzicka-Weg 4, 8093 Zurich, Switzerland;

[email protected] (J.Z.); [email protected] (E.W.); [email protected] (M.R.)
* Correspondence: [email protected]

Abstract: The field of tissue engineering has progressed tremendously over the past few decades
in its ability to fabricate functional tissue substitutes for regenerative medicine and pharmaceutical
research. Conventional scaffold-based approaches are limited in their capacity to produce constructs
with the functionality and complexity of native tissue. Three-dimensional (3D) bioprinting offers
exciting prospects for scaffolds fabrication, as it allows precise placement of cells, biochemical
factors, and biomaterials in a layer-by-layer process. Compared with traditional scaffold fabrication
approaches, 3D bioprinting is better to mimic the complex microstructures of biological tissues
and accurately control the distribution of cells. Here, we describe recent technological advances in
bio-fabrication focusing on 3D bioprinting processes for tissue engineering from data processing to
bioprinting, mainly inkjet, laser, and extrusion-based technique. We then review the associated bioink
formulation for 3D bioprinting of human tissues, including biomaterials, cells, and growth factors
selection. The key bioink properties for successful bioprinting of human tissue were summarized.
After bioprinting, the cells are generally devoid of any exposure to fluid mechanical cues, such as





 fluid shear stress, tension, and compression, which are crucial for tissue development and function in
Citation: Zhang, J.; Wehrle, E.;
health and disease. The bioreactor can serve as a simulator to aid in the development of engineering
Rubert, M.; Müller, R. 3D Bioprinting human tissues from in vitro maturation of 3D cell-laden scaffolds. We then describe some of the most
of Human Tissues: Biofabrication, common bioreactors found in the engineering of several functional tissues, such as bone, cartilage,
Bioinks, and Bioreactors. Int. J. Mol. and cardiovascular applications. In the end, we conclude with a brief insight into present limitations
Sci. 2021, 22, 3971. https://doi.org/ and future developments on the application of 3D bioprinting and bioreactor systems for engineering
10.3390/ijms22083971 human tissue.

Academic Editor: Loredana De Keywords: tissue engineering; 3D bioprinting; bioink; bioreactor

Bartolo

Received: 2 April 2021

Accepted: 9 April 2021
1. Introduction
Published: 12 April 2021
Tissue engineering is a multidisciplinary field that uses a combination of cells, bioma-
Publisher’s Note: MDPI stays neutral
terials, and engineering technologies to develop artificial biological tissue substitutes [1,2].
with regard to jurisdictional claims in
The concept and scope have significantly expanded during the past decades, leading to two
published maps and institutional affil- major areas: (i) developing new methods to repair, regenerate, and replace damaged tissues,
iations. and (ii) creating in vitro tissue models to better understand tissue development, disease
mechanism, and to test and screen drugs [3–5]. Despite significant advancement in the
field of tissue engineering, there is still a continuous shortage of tissues for transplantation
or insufficient tissue regeneration. Besides, there is a lack of tissue models with complex
Copyright: © 2021 by the authors.
multiscale architecture and tissue–tissue interfaces for drug discovery and testing [6]. The
Licensee MDPI, Basel, Switzerland.
conventional tissue engineering approaches use three-dimensional (3D) prefabricated scaf-
This article is an open access article
folds as matrices to load cells [7,8]. The scaffolds serve as 3D templates that support cells to
distributed under the terms and attach, proliferate, differentiate and secrete an extracellular matrix (ECM), which eventually
conditions of the Creative Commons leads to the generation of mature cell-laden grafts with comparable properties to their
Attribution (CC BY) license (https:// native counterparts. However, these conventional scaffold-based approaches are limited
creativecommons.org/licenses/by/ by the intrinsic inability to mimic the complex microstructures of biological tissues and are
4.0/). unable accurately to define the spatial location and distribution of cells [9,10].

Int. J. Mol. Sci. 2021, 22, 3971. https://doi.org/10.3390/ijms22083971 https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2021, 22, 3971 2 of 21

3D bioprinting is an emerging technology expected to revolutionize the field of tissue

engineering and regenerative medicine. As an additive manufacturing technique, 3D
bioprinting shows promise for creating complex composite tissue constructs through
precise placement of living cells and biomaterials in a layer-by-layer fashion [11,12]. The
3D bioprinters’ ability to deposit biomaterials with micrometer precision in cell-friendly
conditions gives it an advantage over conventional scaffold-based approaches since it
shows effective control over scaffold fabrication and cell distribution [13].
Figure 1 shows the typically followed steps for the production of 3D bioprinted human
tissues, which includes pre-processing, processing and post-processing stages. The pre-
processing step is to culture human cells and designs a scaffold model for 3D bioprinting.
Human-specific cells were isolated and expanded to achieve a large number of cells
in vitro. Magnetic resonance (MRI) or computed tomography (CT) imaging technologies
are explored to acquire structure and morphology information of the targeting tissues.
The recorded images are reconstructed to achieve the 3D bioprinting models and then
transfer to the model file, which can be read by the bioprinter, such as gcode [14,15].
Some bioprinting companies also provide professional commercial software (e.g., Axway
TradeSync Integration Manager® , BioAssemblyBot® , and BioCAD® ) to design, draw, and
print multiscale structures ranging from cells to tissue constructs.

Figure 1. The processes of 3D bioprinting of human tissues. (1) In pre-processing: isolation of cells from the human body
and in vitro cell expansion, Magnetic resonance imaging (MRI) or Computed tomography (CT) scanning were used to
achieve the structure information of the target tissue and create the printing model, such as ear, kidney, and bone; (2) In
processing: bioink preparation, 3D bioprinting of 3D cell-laden scaffolds guided by the MRI or CT scanning tissue models;
(3) In post-processing: bioreactor culture system for in vitro scaffold maturation to be 3D functional human tissues, and
potential applications of the 3D bioprinted human tissues. Pictures modified with permission from Reference [16].
Int. J. Mol. Sci. 2021, 22, 3971 3 of 21

The processing step is to fabricate the 3D cell-laden constructs by 3D bioprinting.

The most commonly used bioprinting systems are based on three major strategies: inkjet,
laser, and extrusion-based bioprinting [16]. In most cases, a three-axis mechanical platform
controls the movements of extruders printing the biomaterials in the required algorithm
and shape based on the 3D tissue models [17]. The biomaterial that is printed is referred to
as a “bioink,” which can be defined as an ink formulation that allows the printing of living
cells and growth factors. The selection of proper bioink is crucial for successful bioprinting.
Because it will provide the required properties for adequate printing fidelity and mechanical
properties to ensure printability and long-term functionality following deposition [12]. Post-
processing involves the maturation of cell-laden constructs to reinforce the development of
desired tissue constructs. The optimal nutrition and oxygen delivery, as well as removal
of wastes, are required to maintain cell viability and functionality. More importantly,
chemical and mechanical cues are of critical importance to direct cellular behaviors and
human tissue development [18,19]. Growth and differentiation factors are commonly used
and carefully chosen as chemical stimulation to drive specific cell responses. Growth
factors play a major role in cell division, matrix synthesis, and tissue differentiation [20].
The need for in vitro mechanical stimulation in tissue engineering is drawn from the
fact that most tissues function under specific biomechanical environments in vivo. These
mechanical environments play a key role in tissue remodeling and regeneration [21].
However, most of the 3D cell-laden constructs are generally devoid of any exposure to fluid
mechanical cues, such as fluid shear stress, tension, and compression in the maturation
process. One potential approach to artificially generating the chemical and mechanical
demands of human tissues is using complexly advanced in vitro culture systems, such
as bioreactors [22]. A bioreactor is described as a simulator, which can be modified and
controlled, including pH, temperature, oxygen tension, and perfusion of the cells, as well
as external stimuli such as shear stress and mechanical forces [23]. Bioreactors have the
ability to aid in the development of engineering human tissues from in vitro maturation of
3D cell-laden scaffolds after bioprinting.
In this review, we describe recent technological advances in 3D bioprinting for human
tissues. The processes are beginning from data processing to bioprinting techniques,
including inkjet, laser and extrusion-based bioprinting. We then review the commonly
bioink formulation, including inks and cell selection, and key bioink properties for the
bioprinting process. Various bioreactors that have been used for the delivery of biochemical
and mechanical cues are discussed. The in vitro bioreactor systems used for the maturation
of human tissues are summarized. Present limitations and future developments on the
application of 3D bioprinting and bioreactor systems for engineering human tissues have
been presented in the end.

2. Technique Approaches
2.1. Data Processing
3D bioprinting starts with a computer-assisted process to design a defined 3D biologi-
cal model. The creation of a 3D model allows using data generated by computer-assisted
design software (CAD, Solidwork) [24] or import data from medical imaging such as MRI
and CT [15]. 3D model data from software design allows greater freedom of design, such
as lattice and circle models. The generation of a 3D model using patient-specific tissue size
and morphology enables the generation of a customized 3D construct that is a closer mimic
of the human tissues. The 3D model is converted to a standard tessellation language (STL)
file and then saved as a file format such as g-code. The file format could be easily followed
by the printer and direct the layer-by-layer depositions of the biological elements [25].

2.2. Bioprinting Techniques

Several additive manufacturing techniques have been used to fabricate 3D scaf-
folds [26]. Among them, inkjet-based, laser-assisted, and extrusion-based bioprinting
are popularly used to fabricate 3D cell-laden scaffolds for human tissues. Each bioprinting
Int. J. Mol. Sci. 2021, 22, 3971 4 of 21

technique has specific strengths, weaknesses and limitations. Table 1 provides a concise
comparison of these three approaches.

Table 1. Comparison of three types of bioprinting techniques. Figures adapted with permission from [27].

Inkjet-Based Bioprinter Laser-Assisted Bioprinter Extrusion-Based Bioprinter

Bioprinters

Cost Low High Moderate

Material viscosities 3.5–12 mPa/s 1–300mPa/s 30 to >6 × 107 mPa/s
Resolution 10–100 µm ~75 µm 100 µm-mm range
Quality of structure Poor Fair High
Print speed Fast Medium Slow
Cell viability >85% >95% 40–80%

Examples

Reference [25,28–31] [32–35] [15,24,36–39]

2.2.1. Inkjet-Based Bioprinting

Inkjet bioprinting was the first bioprinting technology published in 2003 [40] and is
very similar to conventional 2D inkjet printing [41]. Inkjet bioprinters deliver a controlled
amount of bioink to the desired printing surface, forcing the content to flow continuously
(continuous inkjet printing) or drop out from the nozzle (on-demand inkjet printing). The
cell-laden biological solution is stored in the ink cartridge, and the electronically controlled
elevator stage provides the control of the Z-axis in the inkjet printer. During bioprinting,
the printer head was used as a thermal or piezoelectric actuator to generate droplets onto
a substrate, which can support or form part of the final cell-laden construct, as shown in
Table 1. The advantages of inkjet bioprinting include high-throughput capability, high reso-
lution, inexpensiveness, reproducibility, and relatively high cell viability (>90%) [28,42–44].
Furthermore, this technique provides a useful method for depositing multiple cells [29,45]
or proteins [31] onto a targeted spatial position with multiple print heads, therefore allow-
ing the fabrication of complex multicellular constructs. Atala et al. [46] were fabricated
human amniotic fluid-derived stem (AFS) cells-laden alginate/collagen scaffolds by ther-
mal inkjet printing. The printed cell-laden constructs were cultured in the osteogenic
inductive medium for 45 days and resulted in intensely mineralized nodules. Gao et al. [47]
fabricated a bone-like tissue by delivering human mesenchymal stem cells (hMSCs) and
hydroxyapatite or bioactive glass nanoparticles in strong poly(ethylene glycol) gel using a
modified Hewlett-Packard (HP) inkjet printer. Results not only showed high cell viability
post-printing but also hydroxyapatite enhanced the osteogenesis of the bioprinted hMSCs.
However, one major drawback of inkjet bioprinting is the material of choice. The bioink
must be in a liquid state and with appropriate viscosity to be ejected out of the small orifice
of the nozzle [48].
Int. J. Mol. Sci. 2021, 22, 3971 5 of 21

2.2.2. Laser-Assisted Bioprinting

Laser-assisted bioprinting originated from laser direct-write technology [49] and is a
modified version of the laser-induced forward transform technique, which was developed
to transfer biological material, such as peptides, DNA, and cells [33,35,50]. A typical laser-
assisted bioprinter consists of five elements: (1) pulsed laser beam, (2) a focusing system,
(3) a ‘ribbon’ structure donor layer containing an energy-absorbing layer that responds
to laser stimulation, (4) a layer of liquid bioink solution, and (5) a receiving substrate
for patterning and crosslinking bioink (Table 1). During the laser-assisted bioprinting
process, the absence of direct contact between the bioink and the dispenser prevents cell
stress resulting in high cell viability (>95%) [16]. Besides, laser-assisted bioprinting is
compatible with different bioink types and a wide range of viscosities (1–300 mPa/s) [12].
Laser-assisted bioprinting is thus a promising technique for 3D printing of cell-laden
constructs for human tissues. Catros et al. demonstrated that laser-assisted 3D bioprinting
supported the fabrication of nano-hydroxyapatite (nHA) and human osteoprogenitor
cells (HOP) without altering the physicochemical properties of nHA while keeping the
viability, proliferation, and phenotype of HOPs [34]. Laser-assisted bioprinting has also
been used for the deposition of a high concentration of human osteosarcoma cells (MG63)
on 3D polycaprolactone (PCL) electrospun scaffolds with high cell viability in vitro and
cell proliferation in vivo [51]. Furthermore, in vivo, laser-assisted bioprinting was used
to deposit cell-laden nano-hydroxyapatite in a mouse calvaria 3D defect model. The
preliminary results demonstrated that the 3D cell-laden constructs are possible to fabricated
by laser-assisted bioprinting in in vivo [52]. Although there are many advantages to laser-
assisted bioprinting, the influence of laser exposure on cells is not fully investigated.
Additionally, the high equipment cost and the complexity of the laser printing control
system represent another limitation for the use of this technique [16].

2.2.3. Extrusion-Based Bioprinting

The extrusion-based bioprinting technique combines both a fluid-dispensing system
and an automated robotic system for extrusion and printing, respectively [53]. The fluid-
dispensing system can be driven by a pneumatic- or mechanical-based system as a “power
source”. By applying a continuous force during the bioprinting process, bioink is printed
in uninterrupted cylindrical lines rather than a single bioink droplet. Under the control
of the automated robotic system, the cylindrical filaments can be precisely fabricated to
the desired 3D custom-shaped structures (Table 1). Recently, the development trend of an
extrusion-based bioprinter is multi-head tissue construction. A typical extrusion printer
can be seen in the Dong-Woo Cho’s group, which has a three-axis motion control with six
dispensing heads, supporting up to six different bioinks printing [38].
One of the main advantages of extrusion bioprinters is their ability to print a wider
range of biomaterials with varying viscosity including hydrogels, biocompatible copoly-
mers, and cell spheroids from 30 to 6 × 107 mPa/s [54]. Normally, the higher viscosity
materials often provide structural support for the printed construct and lower viscosity
materials provide a suitable environment for maintaining cell viability and function. One
of the disadvantages is the encapsulated cells are exposed to larger stresses reducing cell
viability during bioprinting [12]. Several researchers have used extrusion-based methods
for bioprinting of human tissues [24,55–58]. Kang et al. have fabricated stable, human-scale
mandible, and calvarial bone tissue construct using an integrated tissue-organ printer
(ITOP). This system was based on extrusion-based bioprinting and 3D human amniotic
fluid-derived stem cells (hAFSC)-laden hydrogels composing scaffolds were fabricated.
In vivo results showed newly formed vascularized bone tissue throughout the implants [15].
Wüst et al. showed that hMSCs encapsulated in alginate and hydroxyapatite constructs
were not damaged during the printing process and the cells showed high viability in
in vitro culture [24]. Fedorovich et al. developed heterogeneous bone constructs containing
endothelial progenitor cells (EPC) and MSCs to promote neovascularization during bone
Int. J. Mol. Sci. 2021, 22, 3971 6 of 21

regeneration. Following in vivo implantation, the EPCs assembled more blood vessel
networks than MSCs during bone formation [55].

3. Bioink Formulation and Key Bioink Properties

3.1. Inks
The scaffolding material used for bioprinting is called bioink. Bioink consists of a
biomaterial solution (ink) and cells in the presence or absence of growth factors. Bioink
formulation is one of the main challenges in the 3D bioprinting of cell-laden scaffolds for
human tissues. One of the reasons for that is the physical and chemical cues of the cell
hosting biomaterials requires an understanding of cell physiology and cell-ECM interaction.
Natural (gelatin, collagen, fibronectin, alginate, chitosan, silk fibroin, and hyaluronic acid)
or synthetic polymer (polyethylene glycol (PEG), PCL, poly (lactic-co-glycolic) (PLGA),
polylactide (PLA)) were used for tissue engineering. The advantages of natural polymers
for tissue engineering applications are their similarity to human ECM and their inherent
bioactivity. Synthetic polymers can be tailored with specific physical properties to suit
particular applications. To combine the advantages of natural and synthetic polymers,
some bioinks are hybrid biomaterials that merge natural and synthetic materials.
Decellularized extracellular matrices (dECM) have been an increasingly promising
material in tissue engineering. Hydrogels made from decellularized tissues including
urinary bladder, heart, liver, dermis, adipose tissue, bone, and lung, among others, were
developed and reported to support growth and function of different cell types. Pati et al.
showed that dECMs from three tissues (cartilage, heart, adipose tissue) could be solubilized
into bioinks and subsequently be bioprinted [59]. dECM bioinks contain the diverse array
of ECM components characteristic of different tissues and, as a result, more closely resemble
the native tissue. Although the low viscosity of dECM bioinks compromise mechanical
properties and shape fidelity of the bioprinted 3D construct, they represent a promising
addition to the bioinks.
A summary of recent outstanding bioprinting studies for tissue engineering is shown
in Table 2, including bioprinting technique, materials, concentration, cell type, cytocom-
patibility, and applications. According to the wide variety of hydrogels that have been
bioprinted, there are three different crosslinking mechanisms: chemical (ion compound [60],
pH [61]), physical (temperature [62], light [63]), and enzymatic [64] crosslinking. The gela-
tion processes of bioink will sometimes include several crosslinking mechanisms to print
stable and complex scaffolds [65,66].

Table 2. A summary of outstanding recent bioprinting studies for human tissue.

Polymer
Bioprinter
Material Concentration Cell Type Cytocompatibility Application Reference
Types
(w/v)
na/0.1% hAFSC na bone, brain [46]
hAFSC
alginate/collagen
1%/0.3% dSMC 90%, day 7 vascular, bone [11]
Inkjet-based bEC
PEGDMA/HA 20%/2% hMSC 86%, day 21 bone [47]
PEGDMA/
10%/1.5% hMSC 80% bone, cartilage [67]
GelMA
HA 60% MG63 ~100%, day 2 bone [68]
alginate/HA 0.5%/15% HOP high, day 15 bone [34]
Laser- alginate 1% MG63 high, day 4 bone [51]
assisted
alginate 2% hMSC na bone, cartilage [69]
fibrinogen/ adipose,
1.3%/1% ECFC 98%, day 0 [70]
hyaluronic acid vascular
Int. J. Mol. Sci. 2021, 22, 3971 7 of 21

Table 2. Cont.

Polymer
Bioprinter
Material Concentration Cell Type Cytocompatibility Application Reference
Types
(w/v)
3% MG63 80%, day 0 TE (general) [68]
3.5% MSCs 93%, 4 h bone [71]
3% MC3T3 93%, day 1 bone, liver [72]
3.5% MC3T3 94%, day 1 bone [73]
alginate
3.5% MC3T3 85%, day 1 bone, vascular [74]
3% MG63 95%, 4 h bone, TE [75]
10% hMSC 85%, day 7 bone [58]
10% MSC 89%, 5 h bone [56]
alginate/gelatin 0.8%/4.1% hMSC 85%, day 14 bone [76,77]
alginate/gelatin/
0.8%/4.1%/0.1% hMSC 92%, day 42 bone [78]
graphene oxide
alginate/collagen
4%/2%/na MC3T3 88%, day 1 bone, liver [79]
I/GAG
alginate/gelatin 5%/5% SaOS-2 92%, day 1 bone [80]
alginate/gelatin/HA 2%/10%/8% hMSC 85%, day 3 bone [24]
alginate/gelatin/
1%/10%/0.1% hMSC >85%, day 2 TE (general) [81]
carboxymethyl chitosan
alginate/GelMA 4%/4.5% HUVEC 80%, day 1 TE (general) [82]
alginate/matrigel/CaP 10%/na/10% MSC/EPC na bone [83]
Extrusion- alginate/PCL 3.5%/na MSC na bone [84]
based hMSC/
agarose 1.5% excellent, day 21 TE (general) [85]
MG63
collagen I 2% MG63 >90%, day 14 bone, cartilage [86]
GelMA/gellan 10%/1% MSC 90%, day 3 bone [87]
5–20%/
GelMA/collagen MSC 95%, day 28 bone [88]
0.01–0.1%
gelatin/fibrinogen/ bone, ear,
3.5%/2%/0.3% hAFSC 91%, day 1 [15]
hyaluronic acid muscle
matrigel/CaP na/15% MSC 81%, day 1 bone [89]
matrigel na MSC 86%, day 7
alginate 2% MSC >86%, day 14
bone [57]
F127 25% MSC 4%, day 3
agarose 1% MSC 70%, day 7
MeHA 2.5% fibroblast 96%, day 0
GelMA 5% fibroblast 95%, day 7
PEGDA 5% fibroblast >87%, day 7 TE (general) [90]

NorHA 2% fibroblast >87%, day 7

silk fibroin 3% MC3T3 70%, day 2 TE (general) [91]
silk fibroin/gelatin 8%/15% hTMSC 96%, day 1 bone, cartilage [92]
HA: hydroxyapatite; PEGDMA: poly(ethylene glycol)dimethacrylate; GelMA: gelatin methacrylamide; GAG: gly-cosaminoglycan;
MeHA: methacrylated hyaluronic acid; NorHA: norbornene-functionalized hyaluronic acid. hAFS: human amniotic fluid-derived stem
cell; dSMC: canine smooth muscle cell; bEC: bovine aortic endothelial cell; MSC: mesenchymal stem cell; HOP: human osteoprogenitors;
MG63: human osteosarcoma cell; SaOS-2: human osteogenic sarcoma cell; MC3T3: preosteoblast cell; ECFC: endothelial colony-forming
cell; HUVEC: human umbilical vein endo-thelial cell. na: not available; TE: tissue engineering.
Int. J. Mol. Sci. 2021, 22, 3971 8 of 21

3.2. Cell Selection

Tissue regeneration is known to be a well-orchestrated process in which stem cells
play a major role together with growth factors [93]. Stem cells are characterized by their
ability to self-renew and differentiate into a variety of functional specialized cell types.
They are several stem cell types for engineering human tissues used in 3D bioprinting
processes, such as hMSCs [24,67,71,87–89,94], adipose-derived stem cells (ASC) [95], hTM-
SCs [59,92], and human amniotic fluid-derived stem cells (hAFSC) [15,59] (Table 2). In
most cases, it is necessary to add external supplements in the culture medium to induce
differentiation of the stem cells to a targeted cell phenotype. For example, in vitro differ-
entiation of stem cells into the osteoblasts and osteocytes lineage require to supplement
the cells’ culture medium with specific compounds called osteogenic medium (including
β-glycerophosphate, ascorbic acid, and dexamethasone). Riccardo et al. have shown
that the 3D bioprinted MSCs-laden GelMA/gellan gum scaffolds support the osteogenic
differentiation of MSCs and bone matrix deposition when cultured in the osteogenic me-
dia [87]. Another cell type in bioprinting strategies is the usage of tissue-specific cell types,
such as preosteoblasts and osteoblasts for bone tissue [34,73–75,79,80,94], chondrocytes
for cartilage tissue [66] and adipocytes for adipose tissue [96]. Those cells have a stable
tissue phenotype and have been used to regenerate tissues. Neufurth et al. have printed
alginate/gelatin/human osteoblast-like SaOS-2 cells scaffolds, consisting of agarose and
the calcium salt of polyphosphate, which resulted in a highly significant increase in cell
proliferation and mineralization [80].
Meanwhile, to mimic the human tissues does not only require engineered complex
constructs but also represents the cell type diversity of the tissue. The ability of 3D
bioprinting techniques to simultaneously print different cell types with spatial accuracy
has garnered much interest. Co-culture of chondrocytes and MSCs or MG63 cells in
hydrogels have been investigated for improved chondrogenesis and osteogenesis [56,97].
Shim et al. utilized a multi-head tissue building system to separately dispense human
chondrocytes and MG63 cells, which were used to fabricate osteochondral tissue [97].
Human umbilical vein endothelial cells (HUVEC) or endothelial progenitor cells (EPC)
have been shown to produce blood vessels, especially when seeded with osteogenic cells
or MSCs. The combination of HUVECs or EPCs with osteogenic cells has been studied for
angiogenesis and osteogenesis [55,98]. Fedorovich et al. have shown bone formation in
constructs that contain MSCs and EPCs. EPCs derived from peripheral blood contribute to
osteogenic differentiation of MSCs in vitro, and MSCs support the proliferation of EPCs
and stabilize the formed cellular networks. After in vivo implanted in the mice separate
subcutaneous dorsal model, EPCs from peripheral blood assembled into early blood vessel
networks [55].

3.3. Growth Factor Selection

Growth factors are soluble signaling molecules that control a wide variety of cellular
responses, such as cell growth, proliferation, and differentiation through specific binding
of transmembrane receptors on target cells [99]. The idea to use growth factors to promote
tissue regeneration is intuitive, as growth factors are highly related to the repair of damaged
human tissues. For example, transforming growth factor-β (TGF-β) [100,101], insulin-like
growth factors (IGF) [102], bone morphogenetic proteins (BMP) [103], vascular endothelial
growth factor (VEGF) [104], and parathyroid hormone (PTH) [105] are among the most
extensively used growth factors and hormones to stimulate the differentiation of stem cells
(Table 3). TGF-β superfamily plays an important role in embryonic development, tissue
morphogenesis, cell proliferation and cell differentiation. TGF-β1 and TGF-β3 have been
used for chondrogenic differentiation and chondrogenic phenotype maintenance of MSCs
for cartilage and osteochondral tissue regeneration. However, TGF-β has only provided
limited success for endochondral bone formation in adult non-human primates [106].
BMPs, particularly BMP-2, BMP-4, and BMP-7, are the most extensively studied osteogenic
molecules for inducing de novo bone formation in ectopic and orthotropic sites, including
Int. J. Mol. Sci. 2021, 22, 3971 9 of 21

critical size defects [107]. VEGF and IGF can regulate angiogenesis, and bone research
with angiogenic factors has primarily focused on VEGF’s role in neovascularization and
osteogenic recruitment [108]. Although PTH mechanisms for directing osteogenic activity
are not well understood, studies have shown that periodic exposure of PTH can stimulate
bone formation in rats and humans [109].

Table 3. A summary of growth factors in tissue regeneration. Adapted with permission from Reference [37].

Growth Factor Tissues Treated Representative Function

Proliferation and differentiation of bone-forming cells
TGF-β3 Bone, cartilage The antiproliferative factor for epithelial cells
Enhances hyaline cartilage formation in vivo
Cell proliferation and differentiation of osteoprogenitor cells, inhibition of
IGF-1 Muscle, bone, cartilage
cell apoptosis
Differentiation and migration of osteoblasts
BMP(-2, -7) Bone, cartilage Enhanced bone healing and increased bone mechanical strength in the
majority of patients
VEGF Bone, blood vessel Enhanced vasculogenesis and angiogenesis
Migration, proliferation and survival of endothelial cells, inhibition of
Blood vessel,
FGF (-1, -2, -18) differentiation of embryonic stem cells, increased osteogenic differentiation
bone, muscle
of mesenchymal stem cells
HGF Bone, muscle Proliferation, migration and differentiation of mesenchymal stem cells
Blood vessel, muscle, Proliferation, migration, growth of endothelial cells
PDGF-AB (or -BB)
bone, cartilage Osteoblast replication and collagen I synthesis in vitro
Accelerated bone healing through upregulation of bone markers and
resultant bone tissue
PTH, PTHrP Bone
Bone formation was activated in postmenopausal females but inhibited in
healthy adults

Among all the delivery methods, such as freedom adding in culture medium and
microsphere delivery, 3D bioprinting provides a promising approach to incorporate growth
factors into hydrogel scaffolds more easily compared to in a spatiotemporal distribution.
Du et al. created a collagen-binding domain (CBD), which collagen microfibers bound
BMP-2. The results show that BMP-2 was able to be controllably released in vitro. The
CBD-BMP2-collagen microfibers induced the differentiation of MSCs into osteocytes within
14 days more efficiently than the osteogenic media [88]. Spatial patterns of BMP-2 with
a 10 mg/mL concentration were printed on a fibrin-coated glass surface using an inkjet
bioprinter. Murine muscle-derived stem cells seeded onto the BMP-2 pattern exhibited
alkaline phosphatase (ALP) activity, indicating osteogenic differentiation [110].

3.4. Key Bioink Properties for 3D Bioprinting of Human Tissues

During the selection of foundation components (hydrogel, cell, growth factor), we
need to consider the bioink’s printability. The suitability of a bioink for the bioprinting
process mainly depends on its physicochemical properties under the conditionals imparted
by the specific bioprinting parameters. One of the specific bioprinting parameters is nozzle
gauge, which will consequently determine the resolution of the scaffold, fabrication speed,
and time, as well as the shear stress at which embedded cells are exposed to during the
printing process. The major physicochemical properties that determine the printability
of a bioink are its rheological properties and crosslinking mechanisms. In the rheological
parameters, viscosity is the resistance of a fluid to flow upon the application of stress. The
polymer type, concentration, and molecular weight determine the viscosity of a polymer
solution. Printing fidelity generally increases with increasing viscosity [111]. However, an
increase in viscosity implies an increase in applied shear stress, which may be harmful to
the suspended cells [112]. Gelation of crosslinking of a printed bioink structure is necessary
Int. J. Mol. Sci. 2021, 22, 3971 10 of 21

to preserve its 3D construct with structural integrity. The crosslinking mechanisms are
determined by the hydrogels chosen for printing, and normally it can be either physical
or chemical or a combination of both mechanisms. Physical crosslinking mechanisms rely
on non-chemical interactions, including ionic [74,94], stereo complex [113], and thermal
crosslinking [114]. Physically crosslinked hydrogels are the most prominent hydrogel
class used for bioprinting, but a significant drawback is their poor mechanical properties.
Chemical crosslinking forms newly covalent bonds to connect gel precursors. Chemical
crosslinking may provide the hydrogel with good handling properties and high mechanical
strength but needs a very stringent control of crosslinking kinetics. The readers are referred
to the paper by Malda et al. [27] for detailed information about how rheological properties
and crosslinking mechanisms affect 3D bioprinting processes and structure fidelity. Except
for the printability, the ink features, such as biocompatibility, biodegradability, mechanical
property, and material biomimicry, were important for scaffold maturation to achieve the
functional human tissues (Table 4).

Table 4. Ink features for 3D bioprinting of human tissue. Table modified with permission from reference [12].

Physicochemical properties (surface tension, viscosity, crosslinking) of the ink that allows its
Printability
spatial and temporal deposition with high precision and accuracy during the printing process.
The ability of the ink to support normal cellular activity (cell attachments and proliferation)
Biocompatibility
without causing an inflammatory or immune response to the host tissue.
The ideal degradation rate of ink is matching the ability of cells to replace the ink material with
Biodegradability their extracellular matrix proteins. Degradation by-products should be harmless and easily
metabolized from the host.
Bioinks should provide the required tensile strength, stiffness, and elasticity for mimicking the
Mechanical property mechanical properties of native bone tissues and provide the cells with a stable environment for
attachment, proliferation, and differentiation.
Engineering bioink material with specific physiological functions requires mimicking the
Material biomimicry naturally tissue-specific composition and localization of extracellular matrix components in the
human tissue.

4. In Vitro Bioreactor Systems for Scaffold Maturation

After bioprinting, static cultivation is the main culturing approach for scaffold matura-
tion. 3D bioprinted cell-laden scaffolds are statically cultivated in the incubators, covered
with media that has to be exchanged manually. Several disadvantages follow the static
cultivation, like mass-transfer limitations of nutrients and oxygen or waste removal. Dif-
ferent types of bioreactors have been designed for scaffold maturation allowing dynamic
cultivations adapted to the requirements of individual cells or tissues. The dynamic biore-
actor system enables the control and monitoring of pH value, O2 saturation, flow rate, and
temperature, as well as mechanical stimulation.
The choice of a bioreactor to cultivate 3D cell-laden scaffolds after bioprinting depends
upon the tissue to be engineered and its functional biomechanical environment. For
example, Wolf’s law indicates that bone strength increases and decreases as the mechanical
forces on the bone increase and decrease [115]. Mechanical loading is applied to the
bone causes fluid to flow through the lacuna-canalicular system in bone, and osteocytes
sense to the shear stress and induce osteoblasts to form bone or osteoclasts to resorb
bone [116]. Therefore, bioreactors designed for bone tissue, compression, shear stress
and perfusion are constantly highlighted. Emulation of physiological conditions has been
addressed in different ways and the incorporation of convective forces has become a
common characteristic among most bioreactors. In this section, we describe some of the
most common bioreactors found in the engineering of several functional tissues such as
bone, cartilage, skin, and kidney applications.
Int. J. Mol. Sci. 2021, 22, 3971 11 of 21

4.1. Spinner Flask Bioreactor

The spinner flask was first designed with the idea to use convection in order to
maintain a well-mixed system. It consists of a dual-side arm cylindrical flask with a
rubber stopper serving as a cover, which is shown in Figure 2A. 3D scaffolds are threaded
into needles connected to the cover of the flask and submerged in the culture medium. A
magnetic stir bar or a shaft is used to generate cell culture media convection, which provides
a homogeneous distribution of oxygen and nutrients surrounding the scaffolds [117].
Spinner flasks bioreactors have been shown as an effective method for large-scale in vitro
chondrogenic differentiation and the subsequent in vivo cartilage formation of human
adipose-derived stem cells (ADSCs) [118]. Mygind et al. [119] have found that dynamic
spinner flask cultivation of hMSCs-laden hydroxyapatite scaffold constructs resulted in
increased proliferation, differentiation, and distribution of cells in scaffolds compared to
static controls. Stiehler et al. [120] worked on the same dynamic spinner flask system and
repeated the experiment on PLGA scaffolds for up to 3 weeks. They demonstrated a 20%
increase in DNA content (day 21), enhanced ALP specific activity (7 days and 21 days),
a more than tenfold higher Ca2+ content (21 days), and significantly increased transcript
levels of early osteogenesis markers (e.g., COL1A1, BMP2, RUNX2) in spinner flask culture
compared to static culture. However, the reasons for their success in the spinning flask
culturing remain inconclusive and the mechanical stimuli induced by the magnetic stir
bar or the shaft may contribute to the functional tissue formation. Melke et al. [121] have
demonstrated that the complex flow within the spinner flask and mechanical stimulation on
the scaffold were different when culturing at 60 and 300 RPM. Results show that culturing
at 300 RPM led to a more homogeneously distributed ECM than at 60 RPM, which is mainly
at the bottom of the scaffold. Those results were in agreement with the computational
simulations that predicted maximal scaffold mineralization based on different wall shear
stress stimulation. Despite these advantages, the disadvantages of spinner flask cultures
showed that the magnitude of the shear stresses could vary significantly between different
locations; therefore, not all the cells are exposed to the same shear stresses.

4.2. Rotating-Wall Vessel Bioreactor

The rotating-wall bioreactor consists of two concentric cylinders whose annular space
contains the cell culture medium, which is shown in Figure 2B. The inner cylinder is
static and permeable to allow CO2 gas exchange for oxygen supply. The outer cylinder
is impermeable and horizontally rotates at a speed that causes centrifugal forces that can
balance the gravitational forces. Continuous rotation of the outer cylinder results in the
gentle falling of cells through the medium while remaining in suspension. The rotating-wall
vessel bioreactor is an optimized suspension culture system in which cells are grown in a
physiological low fluid shear environment in 3D. To date, more than 50 rotating-wall vessel-
derived tissue models have been engineered, including bone, cartilage, liver, neuronal
tissue, cardiac muscle, adipose tissue, and epithelial tissues [126–130]. Song et al. [131]
demonstrated that rat osteoblasts cultured in rotating wall vessel bioreactors expanded by
more than 10 times compared to osteoblasts in spinner flasks and static controls, and they
presented better morphology, viability, and stronger ability to form bone tissue. Human
cartilage progenitor cells have also been shown to differentiate into mature chondrocytes
using a combination of scaffold and rotating-wall vessel cultivation [132]. Cardiac tissue
has also been engineered using rotating-wall vessel cultivation, which gave rise to a highly
differentiated tissue that exhibited normal anisotropic electrophysiological properties [126].
However, the transport of nutrients to the center of the scaffold was still limited because
the convective forces could not extend to the interior of the large-scale constructs. Large
rotation speeds of the outer wall will increase mass transport, whereas an increase in the
differential rotation enhances shear stresses on scaffolds [133].
Int. J. Mol. Sci. 2021, 22, 3971 12 of 21

Figure 2. The four most prevalent bioreactors used in tissue engineering. (A) Spinner flask bioreactor using a magnetic
stir bar (1) or a shaft (2). (B) Rotating wall vessel bioreactor in different views (1,2). (C) Compression bioreactor: a piston
applies a direct dynamic compression load on the scaffold construct, (1) release, (2) load. (3) A bioreactor system of dynamic
mechanical stimulation for engineered cartilage constructs, (a) at the bioreactor base is a culture medium reservoir, (b)
the lid of the bioreactor, (c) the engineered constructs are molded into cylindrical plugs. (D) Perfusion bioreactor: (1) the
perfusion system consists of a pump, a media reservoir and the bioreactor housing the scaffold; (2) the scaffold is press-fitted
into a perfusion chamber to ensure medium flow through the scaffold. (3) Schematic bioreactor drawings of an in-house
designed perfusion bioreactor for engineered bone tissue. (a) 3D computer-aided design model, (b) inverted volume of
perfusion bioreactor, (c) material definitions for computational fluid dynamics model. Picture modified with permission
from reference [122–125].

4.3. Compression Bioreactor

Compression bioreactors are made of a compression chamber with one or more pis-
tons, which applies compressive loads directly to the scaffolds [123,124,134] (Figure 2C).
Generally, the supporting facilities such as the mechanical stimulation unit allow control
on the loading frequency, strain, force, and time. Compression bioreactors are intended
to mimic the natural physiological loading of tissues in vivo and they are becoming more
widely used in bone and cartilage tissue engineering. Mauck et al. [135] demonstrated that
the application of dynamic deformational loading at physiological strain levels enhances
chondrocyte matrix elaboration in cell-seeded agarose scaffolds to produce a more func-
tional engineered cartilage tissue construct than in free swelling controls. Compression
bioreactors can improve glycosaminoglycan and hydroxyproline formation and increase
the elastic modulus of the cartilage formed to approach that of native cartilage [136,137].
Meanwhile, Sittichockechaiwut et al. [138] have shown that osteoblasts were highly sensi-
tive to mechanical loading. The compression loading at 1 Hz, 5% strain has a strong effect
Int. J. Mol. Sci. 2021, 22, 3971 13 of 21

on mineralized matrix production and osteogenic-related gene expression in comparison

to static conditions. Compression bioreactor design and mechanical loading protocols are
varied in different tissues to be engineered.

4.4. Perfusion Bioreactor

Flow perfusion bioreactors provide continuous culture medium flow through scaffold,
which generates shear stress on cells. The culture medium is continually recirculated
through the chamber by directly pumping, thus improving the transport of nutrients and
oxygen to the constructs. Perfusion bioreactors are useful in large tissue mass constructs
as it allows for more precise control of the culturing environment. Meanwhile, structural
parameters of the scaffold-like porosity or permeability have a significant influence on
the experimental outcome in perfusion cultures. Vetsch et al. [125,139] have developed a
perfusion bioreactor system (Figure 2D) to produce shear stress forces on the engineered
bone-like tissues scaffolds. The designed bioreactors enable to non-invasively and tem-
porally monitor the development of mineralized ECM by the monitoring of micro-CT.
Vetsch et al. [139] have investigated the influence of curvature on three-dimensional min-
eralized matrix formation under static and perfused conditions. The results showed that
the ingrowth of mineralized tissue into the channels was dependent on curvature and
was higher under perfusion. Large channels were not closed in any group compared with
partially (static) or fully (perfused) closed medium and small channels. Mineralized tissue
morphology was cortical-like in static samples and trabecular-like in perfused samples.
The flow rate in the perfusion bioreactor system is one of the most essential parameters
for tissue engineering. The optimal range varies according to the design of the bioreactor
and the cell type used. For instance, increasing flow rates of 0.075–0.2 mL/min to human
chondrocytes seeded on PLGA scaffolds for up to 5 weeks increased the percentage of
glycosaminoglycan retained in the ECM [140]. Vetsch et al. [125] showed that mineralized
extracellular matrix formation was completely inhibited at a low flow rate (0.001 m/s)
compared to a high flow rate (0.061 m/s) and the static group. Biochemical assays and
histology confirmed these results and showed enhanced osteogenic differentiation in the
high flow rate group. Meanwhile, Zhao et al. [141] demonstrated that the various ranges
of optimal flow rates to induce mineralization are within 0.5–5 mL/min among different
hMSCs-laden silk fibroins scaffolds in the perfusion bioreactor by combining computational
fluid dynamics and mechano-regulation theory.

5. Present Limitations and Future Perspectives

The challenges facing the 3D bioprinting of cell-laden scaffolds for tissue regeneration
field relate to specific technical, material and cellular aspects of the bioprinting process.
Although 3D bioprinting techniques offer a precise and structured approach for tissue
engineering, there are some significant challenges to create tissue constructs of clinical
relevance. For example, bone is a metabolically active tissue supplied by an intraosseous
vasculature [142]. Angiogenesis occurs spontaneously upon implantation of a bone graft,
but host-derived neovascularization of the implant is slow (<1 mm/day) [37], and thus
insufficient for constructs. As complex engineered 3D constructs of clinically relevant
size cannot be sustained by the diffusion of nutrients alone, creating a functional vascular
network is necessary for ensuring nutrient supply and waste removal [37]. Approaches to
potentially improve vascularization bioprinting include computer simulation, microscale
technology, and sacrificial printing. Computer modeling is a powerful tool in designing
engineered tissue with the desired properties, such as gradient porosity and mechanical
properties [143,144]. The use of computer-simulated models to optimize the design of the
vasculature network will be an empowering tool to increase nutrient and waste efficiency.
Microscale technology offers flexibility in creating precise 3D architectures with embedded
vascularized and capillary networks, with a layer-by-layer assembly. This approach creates
a trench that is molded into one layer before a second layer is aligned and deposited,
forming laminated channels or grooves in an iterative manner [145] (Figure 3A). Although
Int. J. Mol. Sci. 2021, 22, 3971 14 of 21

promising, this method is slow. Small bioprinted tissue may take only minutes or hours
to print, but the question of cell viability both within a pre-polymer bioink and within
the polymerized early regions of a large multi-day print must be addressed. Another
alternative strategy has been generated a vascular network using 3D printing sacrificial
biomaterials, such as gelatin, Pluronic F-127 (Figure 3B), and carbohydrate glass [145].
The sacrificial materials print vascularization channels and provide mechanical support
at each layer during fabrication, and then are removed from the completed object in a
post-processing step. This method will increase the complexity of the printing process
and the method of removal and breakdown products must be cytocompatible. Although
all these methods are not reliable approaches to print pre-vascularized tissues, a faster
bioprinter with higher resolution would be poised to solve some of the problems.
The ink selection remains a major concern and limitation in 3D bioprinting cell-laden
scaffolds for tissue regeneration, as the selected materials should consider both their com-
patibility with cell growth and functions and their printability characteristics. For this
reason, many published studies select a limited range of materials, including alginate,
gelatin, collagen, silk fibroin, chitosan, PEG and agarose. Meanwhile, each type of biomate-
rial has specific advantages and disadvantages. The common approach is multi-material
printing. It can not only better mimic native organic and inorganic hybrid components
of bone tissue, but also provide a way to improve shape fidelity and mechanical strength.
Kang et al. have fabricated a human-sized structural integrity calvarial scaffold with
printing hAFSCs-laden composite hydrogel with poly(ε-caprolactone) (PCL)/tricalcium
phosphate (TCP) framework using an integrated tissue-organ printer. The results showed
large blood vessel formation within newly formed bone tissue throughout the bioprinted
bone constructs, including the central portion [15]. Incorporating multiple materials also
remains a challenge in creating gradients of cells or growth factors due to the need to
prepare many independent solutions [16]. Further smart biomaterials need to be devel-
oped. Promising developments are the generation of self-assembly materials and stimulus-
responsive hydrogels. Self-assembly is the way to originate materials (nanoparticle or
hydrogel), cells, and proteins to produce novel supramolecular architecture at micro-levels,
which will provide a way to produce complex combinations and gradients of native bone
ECM components [146]. Stimulus responsive hydrogels can be classified into mechano-,
chemo-, heat, pH, and light-responsive hydrogels. Bioprinted constructs with shapeshift-
ing ability can be formed through placing hydrogels with different stimulus responses
strategically [147]. Moreover, the degradation of hydrogel scaffolds can be tailed through
incorporating cell-responsive sites.
In the end, different kinds of forces, such as shear stress and compression loading
in bone tissue, have a synergism effect in the native tissue development and remodeling
processes. To mimic the natural microenvironment of tissue, bioreactors are developed to
apply combined mechanical force on 3D cell-laden scaffolds. Shahin et al. [149] illustrated
that human chondrocytes benefit from the combined application of intermittent unconfined
shear and compressive loading at a frequency of 0.05 Hz using a peak-to-peak compressive
strain amplitude of 2.2% superimposed on a static axial compressive strain of 6.5% for
2.5 weeks. Glycosaminoglycan and collagen type II productions were enhanced between
5.3- and 10-fold after simultaneous stimulation. We foresee that future research would be
centered on a more complex mechanic system that mimics the in vivo mechanical loading
condition of natural tissue. Figure 3E,F shows a future trend of 3D bioprinting multiple
cells-laden scaffolds for cardiac tissue engineering combined with complex bioreactors
with different stimulations, such as biochemical, mechanical, electric, and perfusion. After
in vitro scaffold maturation in a bioreactor system, a whole functional heart is formed.
Int. J. Mol. Sci. 2021, 22, 3971 15 of 21

Figure 3. Examples of future perspectives of 3D bioprinting cell-laden scaffolds for tissue regeneration. (A) Carbohydrate
glass to cast vascular features into a variety of hydrogels, forming perusable vessels that support cell growth. Schematic
illustrations and optical images, of 2D (B,C), and 3D (D,E) embedded vascular networks that are printed, evacuated, and
perfused with a water-soluble fluorescent dye. (F–H) A whole functional heart is formed from 3D bioprinted multiple
cells-laden scaffolds cultured in the complex bioreactor combined with different stimulations. Pictures modified with
permission from references [39,122,139,145,148].

6. Conclusions
In this review, we focus on the 3D bioprinting of cell-laden scaffolds for human tissue
engineering applications. Due to advantages in micro-scale, high-throughput, and cell
deposition, bioprinting has become a strong fabrication tool to create complex micro-
and macro-scale biomedical systems. The recent advances in different 3D bioprinting
techniques, bioink consideration for 3D bioprinting, including hydrogel, cells, growth
factors selection, and ink properties were systematically summarized. Advanced bioreactor
systems providing the dynamic cultivation and mechanical stimulation to mimic the native
human tissues have promising applications for scaffold maturation in vitro. Limitations of
the technology and outlines promising directions for future prospects are further addressed.
Overall, 3D bioprinting is an advanced fabrication technique for the fabrication of 3D cell-
laden constructs for human tissue, with a bright future but encompassing numerous
challenges and problems.

Funding: This research was funded by Chinese Scholarship Council (grant no. 201508310116) and
Swiss Federal Institute of Technology in Zurich (ETH Zurich) Postdoctoral Fellowship Program
(grant no. MSCA-COFUND, FEL-25_15-1).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Int. J. Mol. Sci. 2021, 22, 3971 16 of 21

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