polymers

Review
Emerging Biofabrication Techniques: A Review on Natural
Polymers for Biomedical Applications
María Puertas-Bartolomé 1,2, * , Ana Mora-Boza 3,4, * and Luis García-Fernández 4,5, *

1 INM—Leibniz Institute for New Materials, Campus D2 2, 66123 Saarbrücken, Germany

2 Saarland University, 66123 Saarbrücken, Germany
3 Woodruff School of Mechanical Engineering and Petit Institute for Bioengineering and Bioscience,
Georgia Institute of Technology, 315 Ferst Drive, 2310 IBB Building, Atlanta, GA 30332-0363, USA
4 Institute of Polymer Science and Technology (ICTP-CSIC), Juan de la Cierva 3, 28006 Madrid, Spain
5 Networking Biomedical Research Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN),
Monforte de Lemos 3-5, Pabellón 11, 28029 Madrid, Spain
* Correspondence: [email protected] (M.P.-B.); [email protected] (A.M.-B.);
[email protected] (L.G.-F.); Tel.: +49-(0)681-9300-214 (M.P.-B.); +1-404-894-2000 (A.M.-B.);
+34-915622900 (L.G.-F.)

Abstract: Natural polymers have been widely used for biomedical applications in recent decades.
They offer the advantages of resembling the extracellular matrix of native tissues and retaining
biochemical cues and properties necessary to enhance their biocompatibility, so they usually improve
the cellular attachment and behavior and avoid immunological reactions. Moreover, they offer a
rapid degradability through natural enzymatic or chemical processes. However, natural polymers
present poor mechanical strength, which frequently makes the manipulation processes difficult.
Recent advances in biofabrication, 3D printing, microfluidics, and cell-electrospinning allow the man-





 ufacturing of complex natural polymer matrixes with biophysical and structural properties similar to
Citation: Puertas-Bartolomé, M.;
those of the extracellular matrix. In addition, these techniques offer the possibility of incorporating
Mora-Boza, A.; García-Fernández, L. different cell lines into the fabrication process, a revolutionary strategy broadly explored in recent
Emerging Biofabrication Techniques: years to produce cell-laden scaffolds that can better mimic the properties of functional tissues. In this
A Review on Natural Polymers for review, the use of 3D printing, microfluidics, and electrospinning approaches has been extensively
Biomedical Applications. Polymers investigated for the biofabrication of naturally derived polymer scaffolds with encapsulated cells
2021, 13, 1209. https://doi.org/ intended for biomedical applications (e.g., cell therapies, bone and dental grafts, cardiovascular or
10.3390/polym13081209 musculoskeletal tissue regeneration, and wound healing).

Academic Editor: Paolo Ferruti Keywords: biofabrication; microfluidics; electrospinning; 3D printing; electrospraying; natural
polymers; cell encapsulation
Received: 15 March 2021
Accepted: 3 April 2021
Published: 8 April 2021

1. Introduction
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
Polymeric biomaterials have been developed to provide an artificial matrix that can
published maps and institutional affil- mimic the cell microenvironment. This artificial matrix needs to provide appropriate
iations. biophysical and structural properties (e.g., stiffness, roughness, topography, and alignment)
as well as biochemical cues (e.g., signaling, growth factors, and proteins) in order to
promote the native capacity of cells to adhere, migrate, proliferate, and differentiate towards
the growth of new tissue [1].
Copyright: © 2021 by the authors.
Natural polymers extracted from biological systems such as plants, microorganisms,
Licensee MDPI, Basel, Switzerland.
algae, or animals have been used for decades in the biomedical field. These materials
This article is an open access article
retain the biochemical cues and properties necessary to improve their biocompatibility
distributed under the terms and and present similar structures to the extracellular matrix (ECM) of native tissues [2–5].
conditions of the Creative Commons Therefore, they usually present good cellular attachment, improve cellular behavior, and
Attribution (CC BY) license (https:// avoid immunological reactions, although in some cases, these properties are limited
creativecommons.org/licenses/by/ due to batch variability within production and purification processes. The most com-
4.0/). mon natural polymers used in biomedical applications include polysaccharides (e.g.,

Polymers 2021, 13, 1209. https://doi.org/10.3390/polym13081209 https://www.mdpi.com/journal/polymers

Polymers 2021, 13, 1209 2 of 26

alginate [5–7], hyaluronic acid [3,8], and chitosan [9,10]), proteins (e.g., collagen [11],
silk [12,13], gelatin [14–16], and fibrin [17]), and bacterial polyesters (e.g., bacterial cellu-
lose [18]). However, the poor mechanical strength of natural polymers frequently makes
the manipulation and biofabrication process difficult. For this reason, the use of derivatives
or blends with different polymers are usually required to obtain appropriate mechanical
properties for their use. An example is the modification of gelatin with methacrylamide to
obtain a photopolymerizable biomaterial that can be used for 3D bioprinting and microflu-
idics [19–22].
Actual biomedical challenges require the use of complex polymer matrixes that can
mimic the native ECM and regenerate the lost or damaged tissues [23–25]. Recent advances
in biofabrication techniques allow the production of a polymer matrix with biophysical
and structural properties similar to the ECM, and its combination with different cell
lines is capable of proliferating and differentiating into the desired tissue. Moreover, the
incorporation of different growth factors or other biomolecules can improve the migration,
growth, and differentiation of the cells [3,26].
Currently, numerous research lines for polymer matrix biofabrication follow two
different strategies for the incorporation of the cells: (i) cell implantation on the previously
formed polymer matrix and (ii) fabrication of a polymer matrix with encapsulated cells.
The first strategy was used in the last decade, and it is restricted to the method of
cell implantation. Normally, these systems do not present a good integration between
cells and the polymer matrix, and their efficacy for tissue regeneration depends on the
physical properties of the polymer matrix such as hydrophobicity, degradation rate, or
stiffness [14,27]. Among the most used techniques, we can highlight layer-by-layer [28,29],
melt molding [30], photolithography [31], and self-assembling [32].
The second strategy is the most investigated in recent years, since it allows the fabrica-
tion of advanced cell-laden structures with complex cellular microenvironments. Recently,
some advanced techniques (i.e., microfluidics [33,34], electrospinning [10,35], and 3D print-
ing [36,37]) allow the integration of cells directly into the polymer matrix with the adequate
physical and biological properties to imitate the ECM of the desired tissue.
This review focuses on the biofabrication techniques of microfluidics, electrospinning,
and 3D printing using natural polymers. These techniques have been recently explored to
create polymer matrixes with embedded cells for biomedical applications, and they are in
continuous evolution, as we are going to illustrate in the present review.

2. Microfluidics
Microfluidics has emerged as a powerful tool for the high throughput generation of
monodisperse microgels [33,34]. Microgels are defined as 3D-crosslinked particles that pro-
vide a porous polymeric network and can recapitulate the cellular microenvironment (i.e.,
ECM), mimicking in vivo conditions and diffusion of nutrients and metabolic waste [38–40]
(Figure 1A,B). Specifically, microgels are fabricated in microfluidic devices by the genera-
tion of polymer droplets (i.e., droplet-based microfluidics) through water/oil emulsions
followed by physical or chemical crosslinking. The most frequently used geometry configu-
rations to generate the droplets in the devices are T-junction, flow-focusing, and co-flowing
(or capillary) laminar streams, which are illustrated in Figure 1C [41–44]. Microgels are
especially attractive as cell carriers, because their large surface-to-volume ratio promotes
efficient mass transport and enhances cell-matrix interactions, but it is important to notice
that cell microencapsulation requires a polymer network that ensures cell viability during
microgel preparation and adequate crosslinking chemistry to form a polymer network [45].
Microfluidics technology provides a tight control over microgel chemical properties and
composition by easily tuning the flow rates and components in the microfluidic channels,
being a versatile biofabrication platform, where different crosslinking strategies can be
applied [41]. As mentioned, the microdroplets generated in the microfluidic devices should
undergo physical (e.g., electrostatic interaction, thermal gelation, and hydrogen bond inter-
action) or chemical crosslinking (photopolymerization, Michael addition, and enzymatic
Polymers 2021, 13, 1209 3 of 26

reaction) to form solidified microgels [38]. Physical or chemical gelation will be chosen
based on different factors like the type of polymer, the strategy for tissue encapsulation, as
well as the final biomedical application. In addition, different crosslinking mechanisms can
be combined to fulfil the desired features of the microgel systems [38,43,46,47], and these
crosslinking processes can take place inside the microfluidic device (in situ crosslinking) or
after microgel collecting [38]. In this review, most recent examples of interesting processes
for the microfluidics generation of cell-laden microgels prepared using natural polymers
and different crosslinking strategies are exposed (Table 1).

Figure 1. Recapitulating the natural cellular microenvironment in biomimetic microgels using

droplet-based microfluidics. (A) The natural cellular microenvironment is composed of different cell
types, ECM, and biomolecules such as growth factors. (B) Droplet-based microfluidics allows for
versatile and high throughput generation of cell-laden microgels that can mimic the natural cellular
environment. By mixing defined amounts of selected cells, ECM, and biomolecules, the microen-
vironment can be designed in a bottom-up approach with defined properties [34]. (C) Schematic
illustration of different types of droplet generators, including T-Junction, flow-focusing, and co-flow
(capillary) configurations. Adapted with permission from John Wiley and Sons Copyright® .
Polymers 2021, 13, 1209 4 of 26

Table 1. Summary of the studies exposed in this review regarding microfluidics generation of cell-laden microgels.

Microfluidics Crosslinking
Polymer Microgel Size Range Additives Cell Type Ref.
Approach Strategy
Ionic crosslinking
Alginate Flow-focusing 10–50 µm No MSCs [48]
(Calcium-EDTA)
Ionic crosslinking
Alginate Flow-focusing 20–50 µm poly-D-lysine bMSCs [49]
(calcium)
Ionic crosslinking
Alginate Flow-focusing ≈140 µm PNiPAM HepG2 [33]
(Calcium-EDTA)
Centrifugal Ionic crosslinking
Alginate Tunable (also fibers) No HepG2 [50]
microfluidics (calcium)
Double emulsion
Ionic crosslinking Hepatocytes and
Alginate (w/o/w) flow ≤200 µm Collagen [11]
(calcium) endothelial cells
focusing
Enzymatic
Acrylamide Human dermal
Flow-focusing reaction and pho- ≈80 µm No [8]
hyaluronic acid fibroblasts
topolymerization
Enzymatic
Furylamine and crosslinking,
tyramine T-junction Diels-Alder click ≈250 µm MAL-PEG-MAL ATDC-5 cells [51]
hyaluronic acid chemistry, or a
combination
N-carboxylic Asymmetric Schiff base NIH-3T3
≈200 µm Oxidized dextran [52]
chitosan cross-section reaction fibroblasts
Ionic crosslinking
Chitosan Lactate Flow-focusing 100–130 µm No hMSCs [45]
(G1 Phy and TPP)
Double
GelMA Photopolymerization 100–200 µm No macrophages [20]
flow-focusing
GelMA Capillary Photopolymerization ≈165 µm no bMSCs [53]
PEGDA
ECFCs
Poly(ethylene
GelMA T-junction Photopolymerization 300–1100 µm breast cancer cells [21]
glycol)-
hiPSCs
fibrinogen
GelNB Capillary Photopolymerization 300–600 µm PEG-SH bMSCs [22]
Thiol-Michael Vinyl sulfonated
Thiolated gelatin T-junction 100–250 µm bMSCs [16]
addition reaction hyaluronic acid
Dextran- enzymatic
Flow-focusing 120–200 µm No hMSCs [54]
tyramine crosslinking
Ionic crosslink- rat pancreatic
Dextran Flow-focusing ≈90 µm PEG and Alginate [55]
ing(calcium) islet
PEG diacrylate
Methacrylated
Flow-focusing Michael addition 60–120 µm monomers with mESCs [56]
heparin
8-arm PEG-thiol

2.1. Naturally Derived Polymer Used for the Preparation of Cell-Laden Microgels
Using Microfluidics
2.1.1. Alginate
Alginate is a classic polymer used for the generation of microgels through microflu-
idics [38]. Typically, an aqueous alginate solution is emulsified in an oil phase and
crosslinked ionically with bivalent ions such as Ca2+ , which can be found for example
in CaCl2 or CaCO3 . The ionic crosslinking process occurs immediately upon contact of
alginate chains and Ca2+ ions [48]. Kumacheva and co-workers have reported the most
representative works of alginate-based cell-laden microgels for the last years [43,46,57,58].
Alginate microgels can be prepared through an internal or external gelation approach [38].
In the internal crosslinking, also called in situ crosslinking methodology, alginate is exposed
directly to the crosslinking agent, triggering gelation [59]. In the external approach, alginate
Polymers 2021, 13, 1209 5 of 26

droplets are firstly formed and then put into contact with the crosslinker solution [60]. This
last approach provides a better control over the final morphology of the microgels [38].
Utech et al. [48] reported an interesting method for the fabrication of alginate mi-
crogels using a water-soluble calcium-ethylenediaminetetraacetic acid (calcium-EDTA)
complex as a crosslinking agent. They were able to encapsulate individual MSCs with
high cell viability due to the mild polymerization approach. Moreover, encapsulated MSCs
grew and proliferated over two weeks. Encapsulation of MSCs in polymeric microgels
is an excellent approach to improving cell persistence and immunomodulation [61]. Cell
therapies based on MSCs are particularly interesting in ameliorating immune-related dis-
eases and dysregulations, but they are limited due to short in vivo persistence [44,45,61,62].
Mao et al. [61] reported the encapsulation of MSCs in alginate-polylysine microgels using
a microfluidic device. The encapsulated MSCs in their microgel formulation significantly
increased their in vivo persistence after intravenous injection and responded to inflamma-
tory cytokines, improving immunomodulatory effect of MSCs in a model of allogeneic
transplantation.
Alginate has been also combined with synthetic polymers to fabricate microgels [33,63,64].
Chen et al. [33] synthetized a functional diblock copolymer, alginate-conjugated poly(N-
isopropylacrylamide) (PNiPAM), to fabricate microgels in a flow-focusing device also
using calcium-EDTA complex as a crosslinking agent. The permeability of the as obtained
microgels could be modified by controlling temperature at low critical solution temperature
(LCST), and the encapsulated human hepatocellular carcinoma cell (HepG2) showed high
cell viability thanks to the mild conditions of the crosslinking process.
In recent years, other interesting microfluidic methodologies have been developed to
overcome some limitations of conventional droplet-based microfluidics. This is the case
for the work carried out by Cheng et al. [50], where an efficient centrifugal microfluidic
system for controllable fabrication of simple structured alginate hydrogel beads and fibers
was exposed. Among the advantages of centrifugal microfluidics, it is highlighted by the
use of simple experiment facilities (i.e., a centrifuge) and the absence of an oil continuous
phase and subsequent necessary washing steps. HepG2 cells were encapsulated in the
developed alginate capsules and fibers, demonstrating the high validity of the method, and
showing excellent potential for biomedical applications. Other studies have optimized a
water-in-oil-in-water (W/O/W) double emulsion methodology to encapsulate cells. Chan
et al. [11,35] developed a double emulsion platform to encapsulate rat hepatocytes and
endothelial cells using a combination of alginate and collagen. The developed microgels
provided an excellent physical support for the spheroids.

2.1.2. Hyaluronic Acid

Hyaluronic acid-based building blocks have been prepared by pseudo Michael addi-
tion crosslinking in a microfluidic device [8,38]. Sideris et al. [8] reported a microfluidics
methodology for the fabrication of hyaluronic acid microgels that could self-assemble
to form a biodegradable scaffold through two orthogonal chemistries. Human dermal
fibroblasts were seeded after microgel preparation, demonstrating good cell spreading after
two days of culture. Ma et al. [51] developed a new hyaluronic acid derivative, furylamine
and tyramine hyaluronic acid, that can be crosslinked using enzymatic crosslinking, Diels-
Alder click chemistry, or a combination of both methods. The versatility of this strategy
provided control over crosslinking time and elasticity by simply switching the crosslinking
strategy. After evaluating the mechanical properties, gelation time, microgel size, swelling,
enzymatic degradation, and bioactivity of the obtained microgels, the group concluded
that the microgels synthetized through the combination of both crosslinking methods were
the most promising candidates for cell encapsulation and delivery, since the use of the
strategies alone resulted in low elasticity and poor cell encapsulation performance.
Polymers 2021, 13, 1209 6 of 26

2.1.3. Chitosan
Jang et al. [52] proposed a new in situ crosslinking methodology for the fabrication of
microgels by merging two droplets of different viscosities in an asymmetric cross-junction
microfluidic device. Thus, oxidized dextran (ODX) and N-carboxymethyl chitosan (N-
CEC) were mixed to undergo an in situ crosslinking via a Schiff base reaction, resulting
in microgel formation. This asymmetric cross-junction geometry was an interesting ap-
proach to overcoming the high surface tension of microdroplets of contrasting viscosities,
which usually requires significant surfactant concentrations. In addition, the crosslinking
methodology allowed the encapsulation of NIH-3T3 fibroblasts, which showed high via-
bility after two days of culture, demonstrating the biocompatibility of the entire process.
Mora-Boza et al. [45] reported the fabrication of hMSCs-laden microgels, applying also
an in situ crosslinking approach for chitosan lactate (ChLA), a water-soluble chitosan
derivative. The ionotropic gelation was based on a combination of glycerylphytate (G1 Phy)
and tripolyphosphate (TPP) as ionic crosslinkers, obtaining polymeric microgels with
homogeneous size distribution between 104 and 127 µm. The authors demonstrated that
the presence of G1 Phy, which has been recognized as a potent antioxidant and bioactive
compound [65], supported encapsulated hMSC viability over time and modulated hMSCs
secretome at adverse conditions, resulting in an appealing cell delivery platform for hMSCs
therapy applications.

2.1.4. Gelatin
Photocrosslinkable gelatin derivative, methacrylated gelatin (GelMA), has been widely
applied for the preparation of cell-laden microgels through microfluidics [15,38]. Lee
et al. [20] developed microtissues containing macrophages through flow-focusing mi-
crofluidics using GelMA as a macromer solution. The macrophages’ viability was well
maintained, and mechanical properties of the microgels could be controlled through GelMA
concentration, which had a strong influence on the proliferation and polarization of the
encapsulated cells. A similar methodology was applied by Weitz’s group to encapsulate
bone marrow derived MSCs. The authors demonstrated that the encapsulated cells mi-
grated to the surface of the microgels after four weeks of culture, indicating their capacity
to participate in regenerative processes. Moreover, they demonstrated in vivo osteogenic
potential by increasing the percentage of calcium deposits and expression of bone-related
proteins like BMP-2 [53].
Photocrosslinkable gelatin has also been applied in combination with other synthetic
polymers like polyethyleneglycol (PEG) [21,22]. Seeto et al. [21] used a custom designed
microfluidic device with a T-junction geometry that allowed the production of microgels
with a wide range of diameters from 300 to 1100 µm. The group used a combination
of poly(ethylene glycol) diacrylate (PEGDA), poly(ethylene glycol)-fibrinogen (PF), and
GelMA, which underwent fast photocrosslinking using a full spectrum light source and
Eosin Y as a light photoinitiator. High cellular densities of different cell lines, including
horse endothelial colony forming cells (ECFCs), breast cancer cells, or human induced
pluripotent stem cells (hiPSCs), were encapsulated in the microspheres, showing good cell
distribution, high viability, and functional cellular activities. Forsythe’s group combined
PEG with another photocrosslinkable derivative of gelatin, gelatin norbornene (GelNB),
to fabricate cell-laden microgels using visible light. The encapsulated hBMSCs in the
GelNB-PEG microspheres demonstrated chondrogenesis properties when incubated with
chondroinductive media, including significant upregulation of collagen-II expression in
comparison to bulk hydrogels [22]. Cartilage repair properties have also been observed in
the stem cell-laden microgels reported by Feng et al. [16]. In their work, thiolated gelatin
and vinyl sulfonated hyaluronic acid were mixed in a microfluidic device to generate mi-
crogels through a thiol-Michael addition reaction. Encapsulated bMSCs showed excellent
viability, proliferation, and chondrogenic properties. Furthermore, the in vivo experiments
demonstrated that the cell-laden microgels were injectable and could self-assemble into
Polymers 2021, 13, 1209 7 of 26

cartilage-like structures, providing an effective method for cartilage tissue regeneration,

since they were able to inhibit vascularization and hypertrophy.

2.1.5. Dextran
Dextran application in microfluidics technology has also been explored [38,54,55].
Henke et al. [54] developed very stable dextran-tyramine microgels through enzymatic
crosslinking for hMSCs encapsulation, which demonstrated significantly higher cell viabil-
ity in comparison to PEGDA and alginate microgels. Dextran-based microgels supported
cells’ metabolic activity and allowed cell analysis for 28 days of culture. Liu et al. [55]
combined dextran with PEG to generate water-in-water droplets in a cross-flow microflu-
idic device. This strategy allowed avoiding the use of organic solvent and its subsequent
removal. The droplets could be also used as templates for the fabrication of alginate
microgels. Moreover, the platform was demonstrated to be a promising system for tissue
engineering applications, since the encapsulated rat pancreatic islets maintained high
viability and the function of insulin secretion after seven days of culture.

2.1.6. Heparin
Heparin has been combined with PEG to generate bioactive microgels via Michael
addition to encapsulate and enhance the differentiation of mESCs [38,56]. Siltanen et al. [56]
mixed heparin methacrylate and PEG diacrylate monomers with 8-arm PEG-thiol to fab-
ricate bioactive microgels that provided a suitable environment for endodermal differ-
entiation. The authors also incorporated growth factors FGF-2 and Nodal to evaluate
the differentiation processes of the encapsulated cells, showing that 3D differentiation
processes significantly upregulated the expression levels of endoderm markers.

2.2. Future Perspectives in Fabrication of Cell-Laden Microgels through Microfluidics

Microfluidics is a versatile technology to generate monodisperse cell-laden microgels,
whose properties can be easily tuned if a sensible selection of biomaterials and crosslink-
ing strategies is applied [38,47,66]. These microgels can be applied as building blocks
that can self-assemble into mesoscale tissue structures and replicate structures of native
tissues [8,67,68]. Nevertheless, some limitations must be overcome before clinical imple-
mentation of microfluidic microgels see a bright future [38]. One of the foremost concerns is
related to the scalability of current microfluidic strategies. A higher and more robust mass
production of cell-laden microgels is necessary to scale-up this technology and be able to
obtain macroscale tissue assemblies that can be implemented in the clinic [38,67]. Therefore,
new devices that can support large-scale production of microgels with complex geometries,
such as core-shell morphology, are needed [38]. Another limitation of current cell-laden
microgels is the lack of proper vascularization. Vascularization is essential for effective
tissue implantation. Thus, a biomimetic tissue construct should contain essential elements
like different cell lines, ECM components, and a vasculature network to maintain cellular
interaction and normal tissue function [38,69–71]. Regarding this issue, many efforts are
being made in recent years to develop devices and strategies to fabricate vasculature in
the hydrogels or incorporate well-perfused vasculature networks through microfluidics
systems [38].

3. Cell-Electrospinning (CE) and Bio-Electrospraying (BES)

Electrospinning is a well-known technology that allows the fabrication of micro/nanofiber
scaffolds using different synthetic and natural polymers [72–75]. A typical electrospinning
set up requires a nozzle tip, a high voltage supply, a pump to control flow rate, and a
grounded collector. The process is based on the application of an electric field between the
metallic syringe needle and the grounded collector, while the polymer solution is pumped
out from the needle at a controlled rate. A conical shape called a “Taylor cone” is generated
at the end of the nozzle. When the electrostatic forces within the cone are higher than the
Polymers 2021, 13, 1209 8 of 26

surface tension of the solution, the polymer generates a jet, and it is accelerated toward the
collector plate, forming a randomly oriented nanofibers mat [75,76].
The concept of electrospinning was first introduced by Anton Formhals in the 1930s.
Since then, it has been widely applied in numerous fields such as textiles, agriculture,
filtration, sensors, and the biomedical area [24,77–83]. Specially, this technique has had a
great impact on the area of tissue engineering, since it presents several advantages: the
nanofiber mats can create complex structures that can simulate the native structure of the
ECM, promoting the normal functions of cells; it has an easy manufacture and availability;
the high porosity and high surface area due to the nano size of the fibers enhance cellular
activities such as cell attachment proliferation and differentiation [84–89].
Electrospinning has been a great advancement in the context of biomedical and tissue
engineering applications. However, this technique presents some limitations, i.e., the use
of cytotoxic solvents, and poor cell infiltration and distribution, since the cell seeding
and incubation take place after the substrate processing. In order to overcome these
limitations, a new methodology was developed called cell-electrospinning (CE), which
differs from the conventional electrospinning on the use of living cells. CE consists of the
application of the electrospinning process to a polymer solution combined with living
cells, generating electrospun fibers with embedded cells. Figure 2a shows the schematic
setup of the CE technique [90,91]. Jayasinghe et al. introduced this methodology for the
first time in 2006. In this study, the authors were able to encapsulate living astrocytoma
(1321N1) cells into polydimethylsiloxane electrospun fibers using a coaxial methodology.
Cell viability, metabolic activity, and cell proliferation were examined and proven to be
maintained for six days [90,92]. After this innovative study, the concept of CE was extended
to different cell lines (stem cells, osteoblasts, cardiac myocytes, or neuroblastoma) and
materials (polyvinyl alcohol (PVA), alginate, or Matrigel) [6,91,93–97]. Today, CE presents
a breakthrough in polymer scaffolds processing, and offers remarkable opportunities in
the area of biomedicine.
On the other hand, electrospray is a technique analogous to electrospinning that can be
performed using the same device. The main difference between both techniques relies on
the jet of the polymer generated after the high voltage application. In this case, the resulting
jet suffers continuous break-ups, and the aerosolization of the solution takes place, resulting
in the production of polymeric nanoparticles [98,99]. The properties and size of the particles
will depend on the material and processing parameters. Particularly the viscosity of the
polymer solution is a crucial parameter that can act as a switch between electrospinning
and electrospraying [100]. Electrospray has been broadly used in different fields such as
sensors, food processing, and biomedical applications due to its simplicity and ability to
process different polymers. Even though it was developed before ES, today the use of
electrospray is less common than electrospinning for the processing of polymer solutions.
Similarly to CE, the technique bio-electrospray (BES) was developed for the prepara-
tion of nano/microgels encapsulating living cells (images of living structures fabricated
using BES are shown in Figure 2b,d). Jayasinghe et al. were also pioneers in using this
technique in 2006 [92]. In this work, the group processed Jurkat cells obtaining deposited
droplets in the range of tens of micrometers. BES does not influence cell viability, which
has been verified on cell lines such as sperm or stem cells [101–105]. This methodology has
been proven to be a useful tool for cell encapsulation, the controlled deposition of cells on
planar surfaces, drug delivery, and immunotherapy [102,106].
The successful application of these techniques requires that the viability and bifunc-
tionality of the encapsulated cells be not negatively affected during the process. Several
conditions such as the solution’s viscosity, electric field applied, distance to the collector, or
feed rate can influence the fiber size and shape, as well as the viability of the loaded cells.
Therefore, controlling both material and processing parameters is essential to avoid stress
damage of cells during the process, and therefore to provide high cell viability values of
encapsulated cells. In this review, we extensively investigate the most important research
studies carried out to fabricate cell-laden scaffolds using selected natural polymers and
Polymers 2021, 13, 1209 9 of 26

applying CE and BES technologies. It is expected that this review can serve as a reference
tool and can give a better understanding of the methodologies for future research works.

Figure 2. (a) Cell-electrospinning process with the basic components. Adapted from [79,91] with permission from MDPI.
(b–d) Images of living structures fabricated using either BES or (e–g) CE. (b) Characteristic optical image illustrating a four
cell culture system created in three dimensions (the image depicts cellular networks in three dimensions). (c) Confocal
microscopy image of a three-dimensional culture prepared with the three major cell types of the myocardium (cardiac
myocytes, endothelial cells, and fibroblasts). (d) Immobilized cells as composite living beads. (e) A vessel formed with cells
embedded in the individual fibers. (f,g) The fiber configurations that could be altered from containing a single cell to a
heterogeneous cell population. Adapted from [103] with permission from Wiley Materials.

3.1. Naturally Derived Polymers for CE and BES

Electrospinning and electrospraying techniques have shown great processability
using both naturally and synthetically derived polymers. Synthetic polymers such as
poly(dimethylsiloxane), polycaprolactone, or polylactic acid have been widely used for
electrospinning and electrospray because of their versatility in the selection of the solvent
that can provide adequate viscosity, conductivity, and surface tension of the polymer solu-
tion [91,106,107]. However, viability of encapsulated cells is highly affected by the use of
the organic solvents commonly used, such as tetrahydrofuran, acetone, or chloroform [108].
Therefore, natural polymers such as alginate, collagen, and cellulose, compatible with
non-toxic solvents, are frequently used for achieving biofabrication using living cells by
cell-electrospinning and bio-electrospraying methodologies. Naturally derived polymers
possess numerous advantages such as high cell affinity, low immunogenicity, or ECM
biomimetic properties [109]. However, their low mechanical strength makes them challeng-
ing to process by electrospinning or electrospray [110]. These limitations can be overcome
by blending natural polymers with synthetic polymers as well as modifying the processing
methodology by employing a core-shell nozzle [109,111,112].
In this review, we will make an overview of the different biomaterials based on natural
polymers and blends used for CE and BES techniques and the processing procedure used
to obtain mechanically stable systems.

3.1.1. Alginate
Alginate is a low-cost biodegradable polymer biocompatible with numerous cell
lines, but it also presents a poor mechanical strength. Alginate was used by Xie et al.
for entrapment of living cells by BES technology. In this study, the droplet formation
was analyzed to optimize the production of monodisperse cell-laden microcapsules with
controllable size. The electrospray procedure was performed in dripping mode, and
an additional ring electrode was used to improve stabilization. This modified set up
Polymers 2021, 13, 1209 10 of 26

allowed the successful encapsulation of Hep G2 cells into calcium alginate microbeads
with narrow size distribution and more controlled conditions compared to conventional
electrospray [113].
Several works have prepared blends of alginate with other polymers to increase its
electrospinnability. For example, Yeo et al. described a cell-electrospun system based on a
blend of alginate, poly(ethylene oxide) (PEO), and lecithin to encapsulate MG63 osteoblast
cells for their application in bone regeneration [6]. The concentration of the polymers as
well as the electric field applied were optimized to ensure adequate cell viability as well as
mechanically stable nanofiber mats. As a result, the highest cell viability for encapsulated
osteoblasts (around 80%) was obtained with 2 × 105 MG63 cells/mL, 2 wt% alginate,
2 wt% poly(ethylene oxide), and 0.7 wt% lecithin subjected to a 0.16 kV/mm electric field.
Moreover, osteogenic differentiation of the cells was confirmed after 10 days of culture.
Subsequently, hybrid scaffolds with high mechanical strength were prepared combining
the cell-laden electrospun fibers and poly(e-caprolactone) microstructures prepared by 3D
printing. It can be said that the cell-laden electrospun scaffolds enhanced the potential of
3D structures for bone regeneration, providing a high surface area and ECM-like structure.
Alginate/PEO blend was also used by Yeo et al. for encapsulating C2C12 myoblast
cells by CE for skeletal muscle regeneration [114]. In this work, a high cell viability (around
90%) was obtained for encapsulated cells with an applied electric field of 0.075 kV/mm. It
must be highlighted that alignment of the cell-laden fibers in the mat during the CE process
allowed the achievement of highly aligned cells, which is proven to facilitate myogenic
differentiation. Therefore, this study provides a new tool for achieving cell topographical
cues by controlling fibers’ orientation, which can be very advantageous, especially for
muscle regeneration.
In order to go a step further in this direction, this group proposed a method to
prepare a hierarchical platform with a topographical cue for co-culture of human umbilical
vein endothelial cells (HUVECs) and C2C12 myoblasts cells [115]. An alginate/PEO
blend was again used to develop aligned HUVECs-laden fibers by uniaxial CE, and cell
viability at different electric fields was studied (schemes of native skeletal muscle structure
and CE process, SEM and live/dead images, and quantitative analysis of orientation
cell viability are presented in Figure 3a–e). Encapsulated HUVECs presented high cell
viability (around 90%) at 10.5 kV of electrical field, homogeneous cell distribution, and
efficient cell growth. The mat was combined with PCL/collagen struts prepared by 3D
printing as a physical support. C2C12 cells were then seeded on the cell-laden fibers and
co-cultured to facilitate myoblast regeneration. As a result, scaffolds containing HUVECs-
laden electrospun fibers with a highly aligned topographical cue were able to enhance the
myogenic-specific gene expressions.

3.1.2. Gelatin
Gelatin is a collagen derivative with great biodegradability and biocompatibility, but
low mechanical strength, which limits its fiber-forming ability [14]. Nosoudi et al. have
demonstrated the successful production of cell-laden nanofibers using a gelatin/pullulan
blend [116]. In this work, the electrospinnability of gelatin is enhanced by the presence of
pullulan that increases the tensile strength of the blend. An 8 kV voltage and a concentration
of 5 mg/mL gelatin/pullulan were used during the process, and adipose-derived stem
cells (ADSCs) encapsulated within the fibers presented a 90% viability. This work offers a
new area to be studied, since the use of gelatin for CE has been restricted until now by its
mechanical properties.
Polymers 2021, 13, 1209 11 of 26

Figure 3. Schematics of (a) a native skeletal muscle structure with a vascular network and (b) the cell electrospinning
process using human umbilical vein endothelial cells (HUVECs). (c) SEM and live/dead images of HUVECs-laden fibers
fabricated using various electric fields. A quantitative analysis of (d) orientation factor of nanofibers and (e) cell viability
where the analysis of variance (ANOVA) was used for the multiple comparisons and p * < 0.05, p ** < 0.01, and p *** < 0.001
indicate the statistical significance. Adapted from [115] with permission from Elsevier.

3.1.3. Fibrin
Fibrin matrix is formed by the polymerization of fibrinogen and thrombin in blood
plasma. Due to good biocompatibility and fast biodegradability, it has been widely investi-
gated for tissue engineering applications such as skin, cardiovascular, or musculoskeletal
tissue regeneration. However, the mechanical properties of the fibrin matrix are very low.
In a recent study by Guo et al., a fibrin matrix was used for cell encapsulation using CE
technology [17]. C2C12s murine myoblasts were loaded as cellular aggregates (80–90 µm in
diameter) into a fibrin/PEO polymer solution (schematic of CE process and cell suspension,
cell-laden scaffold, and live/dead images are shown in Figure 4a–e). PEO was used to
improve the mechanical properties of fibrin, as previously observed with alginate [6,114].
Electrospinning parameters were optimized to obtain homogeneous cells distribution in-
side the fibers and good proliferation after exposure to a 4.5 kV electric field and seven days
of incubation. Moreover, myogenically induction provided elongated and multinucleated
cells, demonstrating that encapsulated cells remained reactive to biological cues.
Polymers 2021, 13, 1209 12 of 26

Figure 4. C2C12 can be electrospun into fibrin scaffolds. (A) Schematic of cell-laden wet-
electrospinning setup identifying key parameters. (B) Bright field image of an aggregated cell
suspension. (C) Cell-laden scaffold wrapped around ABS frame. (D) Cross-section of a cell-laden
scaffold on Day 0 stained with DAPI (blue, nuclei). (E) High (20×) magnification of cell-laden mi-
crofiber bundles showing live (green) cells and dead (red) cells. For interpretation of the references to
color in this figure caption, the reader is referred to the web version of this paper. Adapted from [17]
with permission from Elsevier.

3.1.4. Collagen
Collagen is one of the most abundant proteins in mammals and a main protein
of the ECM. It is highly biocompatible and relatively non-immunogenic. Matrigel™ is
derived from extracts of Engelbreth-Holm-Swarm mouse tumors and consists principally
of collagen type IV, entactin, perlecan (heparan sulfate proteoglycan), and laminin.
CE technology was applied to a Matrigel-rich collagen biopolymer to encapsulate
primary cardiomyocytes within fibers for the first time [117]. In this case, the applied
voltage was 230 V, which resulted in cell viability values of around 80%, similar to the
controls. Immunofluorescence staining exposed that the integrity of the encapsulated
cells was maintained after the CE process. Combination of CE methodology with this
biopolymer system allowed creating 3D cardiac patches that were demonstrated to enhance
the cardiac tissue regeneration.
Matrigel with a high concentration of laminin was used in another work by Sampson
et al. to encapsulate N2A mouse neuroblastoma cells [95]. In this study, CE technology was
compared to aerodynamically assisted bio-threading (AABT). Samples prepared by CE
presented cell viability values from 60% to 85% until three days of incubation. In vivo eval-
uation in mice was performed, demonstrating a good biocompatibility of the electrospun
samples and the CE technique, compared to the control by AABT.

3.2. Future Trends

CE and BES are emerging biomedical techniques with great capabilities for living cells’
encapsulation into nano/microscale fibers. They allow the preparation of cell-laden scaf-
folds with high surface area and ECM-like structure using a simple methodology. Variation
of both material’s and processing parameters can be controlled, which has demonstrated
to have a direct effect on cell viability. Embedded cells have been proven to present good
cell viability values and proliferation, and are responsive to cell cues. Moreover, alignment
of the fibers can guide the cells to grow in the fiber direction. Therefore, these technologies
have been demonstrated not to have a negative effect on cells for optimized processing
conditions. However, some challenges still need to be addressed. Preliminary in vivo
studies tested on animal models have demonstrated a good biocompatibility of CE, but
more research studies are necessary to assess the efficacy and true applicability for tissue
regeneration. Improvement of the mechanical properties of the mats and the cell density is
still required. In addition, restrictions to developing 3D structures must be solved. These
limitations can be overcome by combining these techniques with other biofabrication
methodologies such as 3D printing. In this way, more complex scaffolds that are able to
Polymers 2021, 13, 1209 13 of 26

better simulate the complexity of native tissues can be achieved and the range of potential
biomedical applications can be expanded.

4. 3D Printing
3D printing is a technology with the capacity to create objects by adding materials
layer by layer using computer aided design (CAD) software [109,118]. This technology
converts an object into sliced horizontal cross-sections that can be printed layer by layer
to sort out the complete object in 3D (Figure 5). This technology allows the preparation
of complex scaffolds for tissue engineering in a fast and low-cost way without the use of
other expensive techniques [119]. One of the main advantages is the capacity to prepare
low-volume scaffolds with the appropriate geometry to use in tissue engineering, allowing
huge advances in implant materials and personalized scaffolds.

Figure 5. Differences between 2D, 3D, and 4D printing.

The development of new bioinks for 3D bioprinting has attracted attention in recent
years. A bioink for biomedical applications can be defined as a formulation that can contain
biologically active components and cells and is suitable for processing by an automated
biofabrication technology [120]. The limitations of polymer-inspired bioink are toxicity,
presence of toxic degradation products, and immune response between others. Recent
studies have tried to develop new biocompatible bioinks and also polymer-free “bioink”
consisting only of cells [121].
In recent years, 3D printing has been evolving into 4D printing. This technology is
based on a shape transformation of the printed object in response to external stimulus,
such as light, humidity, magnetic fields, enzymatic reactions, pH changes, or peptide
detection [122,123] (Figure 5).
Therefore, functional 3D objects with the capacity to respond to biological conditions
have been reported [123,124]. One of the main functions of 4D printing is the production
of flexible-wearable biosensors with the capacity to detect small metabolites. In this sense,
Nesaei et al. develop a bioink based on Prussian Blue and glucose oxidase enzyme solution
to print two different microelectrodes that detect glucose in a concentration range between
100 and 1000 µM [125].
4D bioprinting also tries to include the use of cells to print living cellular structures
with the capacity to evolve over time. The capacity to change the structure after receiving
a stimulus could modify cell behavior and allow the formation of complex structures for
tissue engineering [122]. For example, Kirillova et al. reported and advanced 4D bioprinting
that allowed the fabrication of self-folding tubes based on hyaluronic acid and alginate.
In this system, bone marrow stromal cells were encapsulated in a methacrylated alginate
bioink and printed in different layers in combination with methacrylated hyaluronic
acid layers. The system was crosslinked using a green light that is safe for the cells.
Due to the difference in crosslinking degree between layers, the 3D bioprinted scaffolds
have the capacity to fold forming tubes with the cells homogeneously distributed on the
surface [126].
Polymers 2021, 13, 1209 14 of 26

The technology used for 3D printing is an important factor to determine the resolution
capacity, velocity, and cell viability. The most important technologies used in the biomedical
field are inkjet, extrusion, laser-assisted, and stereolithography bioprinting (Figure 6) [127].

Figure 6. Schematic representation of bioprinting technologies.

Inkjet bioprinting is a droplet-based bioprinting system where the polymer solution

in the chamber is extruded through a nozzle and droplets are generated on demand by
the breaking of surface tension. The droplet can be generated using a thermal actuator, a
piezoelectric actuator, or electrostatic forces. This technique only works with low viscosity
liquids with low cell density [128].
Extrusion-based bioprinting is the most common and inexpensive technique. It is
able to produce structures by staking multiple layers of a bioink by extrusion of a polymer
solution through a micro-nozzle using continued pressure (pneumatic or mechanical) [129].
The properties of this technique are the capacity to deliver multiple cells and materials, i.e.,
high viscosity polymers with high cell densities, with a high cell viability.
Laser-induced forward transfer bioprinting consists of the deposition of a bioink layer
that is in contact with a donor layer with the capacity to respond to a laser stimulation.
During printing, a laser pulse is applied on the donor layer, and the bioink is propelled
to the underneath substrate and immediately crosslinked [130]. This technique presents
problems of cell viability due to the heating from the laser.
Finally, stereolithography uses light or laser to photolytically crosslink the bioinks
layer by layer. This technique presents the highest resolution possible and high cell
viability [128].
The reason for the increasing popularity of 3D bioprinting is the tremendous potential
of the technique, which allows the production of tissues and other biological systems that
mimic the in vivo tissue to repair. We are going to focus on the key points in 3D printing
technology for biomedical applications: the development of new bioink and 3D printing
for biomedical applications.

4.1. Recent Advances in Bioinks

Normally, the material used as bioink consists of natural polymers, cells, drugs,
growth factors, and other materials that can be deposited in a controlled way. Bioinks
should be non-toxic, easily printable, biocompatible, and biodegradable. We can define
different families of natural polymers, as described in Table 2, that are commonly used for
the preparation of bioinks.
Polymers 2021, 13, 1209 15 of 26

Table 2. Most common natural polymers used for the preparation of bioinks.

Bioprinting
Compound Advantages Disadvantages Ref.
Technique
Low cytotoxicity,
Alginate biodegradable, allow cell Low mechanical properties Extrusion [19,130,131]
adhesion
Low cytotoxicity, Low mechanical properties
Chitosan biodegradable, antibacterial and depends on the origin Extrusion [131,132]
activity, allow cell adhesion and MW
Natural Polymers

Poor mechanical properties

Lox cytotoxicity, improved Extrusion, Inkjet,
Gelatin and depends on the [133–135]
cell adhesion, biodegradable Laser-assisted
temperature. Low viscosity
Similar to the ECM,
Hyaluronic Low mechanical strength
biocompatible and Extrusion, Inkjet [136–138]
acid and rapid degradation
biodegradable
Improved cell adhesion, good Low mechanical strength Extrusion, Inkjet,
Collagen [139–141]
biocompatibility and low viscosity Laser-assisted
Good mechanical properties,
Agarose Low cell adhesion Extrusion [142]
biodegradable
Biocompatible, improved cell Low mechanical properties,
Fibrin Extrusion, Inkjet [143]
adhesion, non-cytotoxic rapid degradation

The main problems of the commonly used bioinks are their low cell affinity and their
limited mechanical properties. Recent advances in the development of bioinks are focused
on the synthesis of derivatives that improve the mechanical properties and cell affinity and
that provide signals to promote cell growth, adhesion, and differentiation.

4.1.1. Alginate Based Bioinks

Alginate hydrogels have been extensively used as bioinks due to their good biocom-
patibility and similitude with the ECM. The easy way to use alginate as a bioink is by
crosslinking with a solution of calcium chloride [144]. One of the main problems of alginates
is their low printability and geometry accuracy due to their limited mechanical properties.
To improve these properties, a variety of covalent crosslinking methods have been used.
For example, Aldana et al. developed an alginate-based bioink with tunable mechanical
properties using blends of alginate and gelatin methacrylamide (GelMA), obtaining a
photopolymerizable biomaterial with different printability, accuracy, and mechanical and
biological properties, depending on the ratio of alginate:GelMA [19].
A similar approximation was used by Soltan et al. The authors investigated the
use of oxidized alginate (alginate dialdehyde, ADA) to obtain covalently crosslinked
hydrogels with gelatin [145]. The mechanical properties of the hydrogel and therefore
their printability and cell viability depend on the degree of oxidation and the ratio of
ADA:gelatin. The authors printed different layers using two different cell types, human
umbilical vein endothelial cells and rat Schwann cells, checking their viability over time.
In addition, the cell adhesion could be modified by the development of new blends.
For example, the group of Boccaccini developed a hybrid hydrogel composed of alginate
and keratin. This hydrogel promotes cell attachment, proliferation, spreading, and viability,
being a good candidate for biomedical applications [146].

4.1.2. Chitosan Based Bioinks

Chitosan has been widely employed in tissue engineering and biomedical applica-
tions due to its biocompatibility, biodegradability, and antimicrobial activity [147–150].
Normally, chitosan is crosslinked using genipin or glutaraldehyde by a chemical crosslink-
ing mechanism [151]. Recent advances have been focused on the development of new
Polymers 2021, 13, 1209 16 of 26

crosslinking agents to improve the printability and accuracy of chitosan systems and, at the
same time, add biological properties to the system to promote cell adhesion, migration, or
differentiation. For example, the group of Prof. San Roman developed an ionic crosslinker
based on phytic acid (G1 Phy) for 3D printing [152]. This new crosslinker allowed the 3D
printing of low concentrate chitosan/GelMA to obtain scaffolds with excellent mechanical
and biological properties.
Another approximation to crosslink chitosan based bioinks for their use in laser-
assisted bioprinting was developed by He et al. The authors provided a photocurable
bioink based on the copolymerization of chitosan and acrylamide (AM) [153]. The capacity
to use this bioink in laser-assisted bioprinting allows the preparation of complex 3D
hydrogel scaffolds with high strength and good biocompatibility [154].
A different approach was presented by Puertas et al. in which the carboxymethyl
derivative of chitosan was crosslinked with partially oxidized hyaluronic acid via Schiff
base formation [155]. This study presented a novel bioprinting methodology based on a
dual-syringe system with a static mixing tool that allowed the in situ crosslinking of the
reactive hydrogel-based ink in the presence of living cells. This new approach allowed the
use of low viscosity solutions while obtaining 3D printed scaffolds with good mechanical
stability and proliferation of encapsulated cells.

4.1.3. Other Natural Polymer Based Bioinks

There are a great variety of natural polymers that are being used as bioinks. In the
previous topics, we showed different examples using GelMA in combination with alginate
or chitosan. Other natural polymers such as hyaluronic acid, fibrinogen, agarose, collagen,
or silk have been used as bioinks. Due to their bad mechanical properties or low cell affinity,
these natural polymers need to be modified, crosslinked, or blended to obtain adequate
properties for their use as scaffolds. For example, Skardal et al. developed a bioink based on
GelMA and methacrylated hyaluronic acid. This bioink allows the direct incorporation of
cells due to the good biological properties [156]. This group also developed another bioink
based on the combination of fibrin and collagen with stem cells [157]. This bioink was used
to print a full-thickness skin as a carrier of stem cells for the treatment of wound healing.

4.1.4. Sacrificial Bioinks

Sacrificial bioinks are used to provide the necessary mechanical properties during
the bioprinting step. After the scaffold is printed, the sacrificial bioink will be removed to
create open spaces to allow cell adhesion and migration. Normally, water-soluble synthetic
polymers are used as sacrificial bioinks due to their low adhesion to natural polymers.
Synthetic polymers such as pluronic, PVA, or PEG are commonly used as sacrificial
bioinks in natural polymers scaffolds like in the research of Zou et al. Here, the authors
used PVA as a sacrificial bioink to prepare a porous scaffold of alginate agarose. First,
a support scaffold of PVA was printed, and then the empty space was filled with an
alginate/agarose/HUVECs bioink. Once the scaffold was finished, PVA was removed,
with cell media forming a porous structure (Figure 7).
Important progress has been made in recent years in the use of sacrificial polymers.
Jian et al. developed a bioprinting method using two different inks for meniscal reconstruc-
tion [154]. The system consists of two nozzles; one of them prints PCL by high-temperature
melt deposition, forming the principal construct that provided the physical properties,
and the second nozzle uses a mix of GelMa, ECM, and chondrocytes, and it is deposited
in the free space between PCLs. Once the scaffold is finished, PCL only provides the
necessary mechanical properties to the scaffold in the first days and is degraded, forming a
microchannel in the scaffold that allows the transport of nutrients to the cells.
Polymers 2021, 13, 1209 17 of 26

Figure 7. Flow diagram for the biofabrication of the large-size, hollow, and micro-fluid channel
networks valentine-shaped heart. Reprinted from [158], with permission from Elsevier.

4.1.5. Evolution of the Bioinks

The continuous research into new materials and processes promotes the fast evolution
of the bioinks. In recent years, potential candidates have been emerging quickly. Decellu-
larized ECM, self-assembling peptides, cellular aggregates, or nanobiocomposites have
emerged in recent years [159–162].
From the point of view of the polymer field, the incorporation of nanomaterials into
a bioink in order to improve the stiffness, shear-thinning, degradation, or stability is
interesting. One of the most important nanocomposites is nanocellulose. Nanocellulose is
derived principally from bacteria [163], and its use, combined with other bioinks, integrates
the common properties of the cellulose: high stiffness, modulus, hydrophilicity, and thermal
stability. For example, Han et al. studied the effect of nanocellulose on alginate/gelatin
bioinks [164]. Their result showed that the incorporation of a small amount of nanocellulose
improved the printability, stability, and fidelity of the structure, but high amounts of
nanocellulose promoted a negative impact on the elongation and compression yield.

4.2. 3D Printing for Biomedical Applications

Current research on 3D printing in biomedical applications can be classified into
the following areas: (i) printing of bioactive and biodegradable scaffolds and (ii) directly
printing tissues and organs.

4.2.1. Bioactive and Biodegradable Scaffolds

One of the main research fields in tissue engineering is the development of advanced
scaffolds for tissue regeneration. In this case, the bioink will be a polymer system alone or
a mixture incorporating cells, where the polymer systems play the role of the ECM. Com-
pared with traditional scaffold-fabrication methods (salt-leaching, cryogels, or gas-foaming)
that prepared simple-shapes supports with an inhomogeneity porosity, 3D printing can
prepare complex structures, with the adequate shape to fill the defect and with an effec-
tive control of the porosity and microstructure. These systems require the presence of an
interconnected porous network to allow cell growth and migration and flow transport of
nutrients [120].
Bioactive scaffolds obtained by 3D printing can be divided into two families: scaffolds
printed without cells (cell-free scaffolds) and scaffolds directly printed with cells (cell-
loaded scaffolds).
Cell-free scaffolds are normally prepared from high water content polymer systems that
present a high biocompatibility and a controlled biodegradation. The facility to prepared
complex structures makes this kind of scaffold suitable for the reconstruction of complex
tissues like osteochondral tissue. Osteochondral tissue is composed of different layers with
Polymers 2021, 13, 1209 18 of 26

different structures and compositions [25]. 3D printing is capable of producing scaffolds

that simulate the structure of this tissue. For example, Gao et al. designed a multilayer
system using GelMA with or without hydroxyapatite to obtain a scaffold that simulates
the ECM in the osteochondral tissue [165]. The design of a proper bioink is a crucial
point for the correct regeneration of the tissue. In this sense, Ma et al. developed a novel
polymeric/ceramic bioink for customizing craniomaxillofacial bone reconstruction [166].
The bioink was based on a hyperelastic PEGylated urethane composited with microscale
β-TCP. This bioink presented a high biocompatibility and osteoinductivity due to its
biomimetic composition. In addition, it presented adequate mechanical properties for
surgical manipulation and it had the capacity of osteoregeneration.
Cell-loaded scaffolds were developed due to the problems of seeding cells directly into
3D printed scaffolds (inhomogeneous distribution and inefficient cell adhesion). The main
objective of this technique is to simulate the structure of the ECM in vitro to allow cell
growth and differentiation. This technique allows the preparation of semi-functional tissues
like in the case of the group of Prof. Jorcano. This group developed a functional skin by
3D bioprinting using extrusion bioprinting to deposit different layers composed of fibrin
and fibroblast or keratinocytes to form the dermis and the epidermis layers [167]. With
this technique, the authors obtained a functional skin that can be used for implantation in
burned tissues.

4.2.2. Directly Printing Tissue and Organs

The preparation of cell-loaded scaffolds does not assure the final functionality of
the scaffold due to the dispersion of the cells and growth factors. For this reason, recent
advances in 3D bioprinting are focused on the evolution of a direct-printing technology:
tissue structures with physiological functions, containing seed cells, growth factors, and
nutritional components [119]. One of the main goals is the pre-vascularization of the
scaffolds, because the absence of a vasculature is one of the leading causes of failure
for current 3D bioprinted scaffolds [168]. Recent advances have obtained functional
vascularized tissues that showed better biointegration. Kim et al. developed a perfusable
vascularized human skin formed by an epidermis, dermis, and hypodermis [169]. The
system involves the preparation of a support of PCL and gelatin followed by the impression
of the different skin layers: (1) The hypodermis layer was composed of fibrinogen and
adipose-derived ECM with human adipocytes. (2) The vasculature was printed using a
gelatin/glycerol/thrombin bioink with human umbilical vein endothelial cells. (3) The
dermal layer was composed of fibrinogen, dermal-derived ECM, and human dermal
fibroblast. (4) The final epidermis layer was composed of human keratinocytes. The result
showed a fully functional skin with a microenvironment close to a real skin that can be
used to test skin drugs or similar (Figure 8).

Figure 8. Schematic diagram exhibiting the 3D cell printing process for fabrication of a 3D full-thickness skin model.

4.3. Future Perspectives in 3D Printing

Considerable advances have been achieved in the use of polymers for 3D bioprinting.
However, this field is still in the early stages of development. One of the principal key
Polymers 2021, 13, 1209 19 of 26

factors is the development of a bioink adequate to our system. The bioink needs to
have a good printability and geometry accuracy, adequate mechanical properties, and
good biocompatibility and needs to be biodegradable. Many bioinks have already been
formulated and used, but researchers continue to develop new compositions, looking to
obtain better properties and new biofunctionalities. These characteristics allow the 3D
bioprinting of fully functional tissues and organs.
Recently, 4D bioprinting technology has emerged as a powerful platform to obtain
stimulus responsive bioprinting. This new methodology is in its first stages. Only a proof
of concept with smart polymers has been developed. In the next years, this technology will
evolve into more sophisticated systems and will be used in the advanced biomedical field.

5. Summary and Future Direction

In this review, we have entered into the relationship between natural polymers and
new biofabrication techniques. The use of 3D printing, microfluidics, and electrospin-
ning techniques has been widely investigated for the biofabrication of naturally-derived
polymer scaffolds with encapsulated cells. Important challenges must be addressed for
the successful biofabrication of these cell-laden natural scaffolds. On the one hand, the
poor mechanical strength of natural polymers makes the processing and material manipu-
lation difficult. In this sense, different modifications and blends have been investigated
to improve the mechanical properties of the processed scaffold. On the other hand, the
application of these techniques must ensure that the viability and functionality of the
encapsulated cells are not negatively affected during the processing. Controlling both
material parameters (e.g., solvent, viscosity, or polymer concentration) and processing
parameters (e.g., pressure, voltage, or feed rate) is essential to avoid stress damage of cells
during the fabrication, and therefore to provide high cell viability, metabolic activity, and
proliferation of the encapsulated cells. Therefore, it is one of the main challenges that need
to be addressed. In the last years, different modifications, blends, and adaptations of the
biofabrication process have been investigated, but we are still in the first stages of the
development of these technologies. There are indeed still many technological issues and
limitations that need to be solved. One of the most important concerns is the clinical imple-
mentation of the cell-laden scaffolds fabricated using these techniques. Some preliminary
in vivo studies in animal models have been performed. However, more research studies
regarding immunological response and vascularization (which are essential for effective
tissue implantation) are still necessary to assess the real applicability of the materials for
clinical applications. Despite all these limitations and challenges, many efforts are being
made to develop more complex techniques to simulate the complexity of native tissues
and overcome the processing limitations, so we cannot exclude the possibility that in a few
years biofabrication techniques will evolve and allow obtaining fully functional organs
and tissues.

Author Contributions: Conceptualization, A.M.-B., M.P.-B., L.G.-F.; investigation, A.M.-B., M.P.-B.,

L.G.-F.; resources, A.M.-B., M.P.-B., L.G.-F.; writing—original draft preparation, A.M.-B., M.P.-B.,
L.G.-F.; writing—review and editing, A.M.-B., M.P.-B., L.G.-F. All authors have read and agreed to
the published version of the manuscript.
Funding: This research was funded by the Ministry of Science, Innovation, and Universities (Spain)
(MAT2017-84277-R); Apadrina la Ciencia-Ford Foundations (Fellowship of A.M.-B)); and Leibniz
ScienceCampus Living Therapeutic Materials (Saarbrücken, Germany).
Institutional Review Board Statement: Not Applicable.
Informed Consent Statement: Not Applicable.
Data Availability Statement: Not Applicable.
Acknowledgments: Luis García Fernández is a member of the SusPlast platform (Interdisciplinary
Platform for Sustainable Plastics towards a Circular Economy) from the Spanish National Research
Council (CSIC).
Polymers 2021, 13, 1209 20 of 26

Conflicts of Interest: The authors declare no conflict of interest.

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