American Journal of Engineering and Applied Sciences

Review

Bio-Inspired Materials: Exhibited Characteristics and
Integration Degree in Bio-Printing Operations

Antreas Kantaros

Department of Industrial Design and Production Engineering, University of West Attica, Greece

Article history Abstract: In the last decade, additive manufacturing techniques, commonly
Received: 25-10-2022 known under the term "3d printing" have seen constantly increasing use in
Revised: 16-11-2022 various scientific fields. The nature of these fabrication techniques that
a operate under a layer-by-layer material deposition principle features several
de facto advantages, compared to traditional manufacturing techniques.
These advantages range from the precise attribution of pre-designed complex
shapes to the use of a variety of materials as raw materials in the process.
However, its major strong point is the ability to fabricate custom shapes
with interconnected lattices, and porous interiors that traditional
manufacturing techniques cannot properly attribute. This potential is
being largely exploited in the biomedical field in sectors like bio-printing,
where such structures are being used for direct implantation into the
human body. To meet the strict requirements that such procedures dictate,
the fabricated items need to be made out of biomaterials exhibiting
properties like biocompatibility, bioresorbability, biodegradability, and
appropriate mechanical properties. This review aims not only to list the
most important biomaterials used in these techniques but also to bring up
their pros and cons in meeting the aforementioned characteristics that are
vital in their use.

Email: akantaros @uniwa.gr

Keywords: 3D Printing, Bio-Printing, Biomaterials, Fused Deposition
Modeling (FDM), Stereolithography (SLA) Direct Ink Writing (DIW) Laser-
Guided Direct Writing (LGDW)

Introduction Bio-printing is the additive process of creating cell
patterns by successively depositing cells along with

Additive manufacturing, most commonly referred biomaterials that form a substrate thereby fabricating

to as "3D printing", can be described as the additive living human constructs with similar biological, chemical
process where a three-dimensional object is fabricated and mechanical properties for proper recuperation of
by laying successive material layers at controlled speed tissues, scaffolds and organs. Materials used in this
and layer thickness. These materials can be process are known as biomaterials and should exhibit
biomaterials, metals, ceramics, plastics, resins, biocompatibility, i.e., being compatible with the human
concrete, or other materials. Even though printing time, body, bioresorbability, i.e., being able to be naturally
processing speed and printing resolution have been absorbed by the human body, biodegradability 1.e., the
constantly improving over recent years, still, the lack capacity for biological degradation and appropriate

mechanical properties dependent on the implantation spot
(Bielenstein et al., 2022; Kowalewicz et al., 2021; Guo et al.,
2022; Kantaros et al., 2016).

In this context, the field where the aforementioned bio-
printing technique is growingly being adopted is tissue
engineering. Tissue engineering is an innovative,

of variety in 3D printable materials persists. Emerging
fields like 3D printing of biomaterials, 3D printing of
tissues, and high viability cell printing are highly
dependent upon printing ink’s compatibility and
flowability with the current printing techniques

available. This study Heponts the advances in 3D printing multidisciplinary field that incorporates the fundamental
materials for emerging biomedical fields and their principles of engineering and biology to invent structures
compatibility with currently available printing techniques. with elevated biological functions. Regarding clinical

Y, Science © 2022 Antreas Kantaros. This open-access article is distributed under a Creative Commons Attribution (CC-BY) 4.0
: : li ;
U3 Publications icense

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DOI: 10.3844/ajeassp.2022.255.263

applications, one of the most decisive targets of tissue
engineering is to surpass the numerous barriers imposed
by current treatments that are currently based mainly on
organ transplants and biomaterial use for implantation
(Guillotin and Guillemot, 2011). Organ and _ tissue
malfunctions are a major problem regarding human health
and well-being. Especially in cases where the human body
fails to self-heal using its mechanisms, targeted medical
intervention is crucial.

Even though tissue engineering may be newly
introduced, the initial concept of tissue substitution was
first expressed in the 16th century. Gasparo Tagliacozzi
(1546-1599), Professor of Surgery and Anatomy at the
University of Bologna, can be considered as the
initial/first documented relevant case when he managed to
construct a nose replacement by a forearm flap in “De
Custorum Chirurigia per Insitionem” announced in 1597
(O'brien, 2011). The primal clinical application of human
cells in this field involved skin tissue development by
utilizing fibroblasts, keratinocytes, or a scaffold (that
would act as a tissue substrate). In another published
work, the regeneration procedure of rabbit articular
surfaces by utilizing allograft chondrocytes along with
collagen gel is also reported (Wakitani ef al., 2002). A
published literature work by Langer and Vacanti (1993)
named "Tissue Engineering" is considered a_ great
contribution towards advancing tissue engineering research
on a worldwide scale (Ikada, 2006).

The first decade of the current century is linked with
the first successful cases of developing the first solely lab-
grown organs by utilizing 3D_ bioprinters. The
contribution of Anthony Atala is considered fundamental
in this field, where cell therapies, tissue engineering
constructs, and organs for many diverse areas of the body
have been developed (Anthony, 2022). In addition, he is
attributed with the development of 3D _bioprinters
(Murphy and Atala, 2014) and, in 2006, he and his team
developed the first lab-fabricated organ (a human bladder)
for implantation to humans (Atala et al., 2006). Dr. Paolo de
Copp1’s work is also fundamental in this sector, where
a tissue-engineered tracheal replacement for a child
being developed by him and his team was reported
(Elliott et al., 2012).

Lattice tissue engineering structures fabricated by bio-
printers and compatible biomaterials must exhibit a
variety of desired characteristics in the sense of providing
a proper substrate for 3D tissue development as the final
target. More commonly referred to as "scaffolds", they act
as bioresorbable constructs in the spot of the defect with
the role of cell-encapsulated tissue constructs containing
cells/hydrogels (Dong et al., 2017; Nicodemus and
Bryant, 2008). They can be categorized as "acellular
scaffolds" (scaffolds with no cells, like hip and knee
implants, etc.) or "cellular scaffolds" (scaffolds with cells,
like skin constructs). Bio-printed scaffolds should exhibit

256

the desired mechanical behavior in terms of providing
mechanical integrity in the healing and degradation
periods. Controlled porosity is also desired, where an
interconnected pore network is considered crucial for
blood and nutrient flow. The aforementioned dictates the
ability for scaffold fabrication in well-defined geometrical
shapes (Kantaros and Piromalis, 2021). Bio-printing
techniques exhibit high potential in this field, by being
able to provide dimensional stability, reproducibility, and the
fabrication of pre-designed interconnected porosity networks
at distinct sizes (Moroni et al., 2006; Amirkhani et al., 2012).
Several published works describe cases of bio-printed
scaffold structures (Moroni et al., 2006; Leong et al., 2003;
Gauvin et al., 2012; Kantaros, 2022). Stages of the bio-
printing process are depicted in Fig. 1.

3D Bioprinting Techniques
Stereolithography (SLA)

SLA printing is a 3D printing technique that uses a
laser or a DLP projector to cure photopolymer resin
layer-by-layer. SLA 3D printers use a UV laser or a
DLP projector to cure a specific layer of photosensitive
resin in a tank. The light source cures or hardens the
resin, forming a very thin sliced solid layer. This slice
bonds to the previously formed layer or the build plate. The
build plate then moves away from a value that equals the pre-
determined layer thickness. This is how the process of object
formation is repeated until the complete object is created.

SLA printing produces higher resolution than FDM
printing because it uses a light source to solidify the
material, resulting in small-sized prints. The horizontal
resolution of an SLA scanner depends on the size of the
light source spot and can range from 30 to 140 microns.
The vertical resolution (or Z-direction resolution) varies
from 25 to 200 microns (Jo and Song, 2021). To create a
good print, you need to set the layer height and the support
placement correctly. Figure 2 depicts an SLA 3D Printer
apparatus schematic.

Ink materials compatible with the SLA process should
exhibit properties like biocompatibility, stability under
exposure to UV light, low viscosity, and optical
transparency (Rasheed ef al., 2021).

|
e-

Fig. 1: Distinct stages of the bio-printing process

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x-y scanning motor

Light source

Platform moving assembly

Z Printed part

se Resin tank
Photosensitive

resin

Fig. 2: Stereolithography 3D Printing schematic

Bio-Inks Compatible with SLA Process
Poly (D, L-Lactide) (PDLLA)

PDLLA is a versatile polymer with many applications
in the medical field, including as a scaffold material for
tissue engineering, as a controlled delivery system for
drugs, and as synthetic nerve conduits made out of
PDLLA, B-TCP, and collagen for regeneration of
peripheral nerves (Hofmann et al., 2012; Lin et al., 2006;
Lin et al., 2017).

Literature works describe the fabrication of composite
scaffolds from HA biocement embedded in PDLLA
oligomers using SLA 3D printing technology using ethyl
2,4,6-trimethylbenzoylphenylphosphinate as a
photoinitiator and N-Methyl-2-Pyrrolidone (NMP) as a
diluent. With increasing percentages of ceramic in the
resin, the viscosity of the resin increases and so a non-
reactive diluent, such as NMP, is necessary to help
maintain the desired viscosity for SLA. It has been found
that as the concentration of HA powders increases, the
elasticity of the material increases. (Cai et al., 2019).

Poly (Propylene Fumarate) (PPF)

PPF is used in SLA due to exhibiting pho-to-cross-
linkability. It is also biodegradable and possesses elevated
mechanical properties. In the majority of cases, it is being
used in SLA by creating a solution consisting of PPF as
the base polymer and Diethyl Fumarate (DEF) as a
solvent. The solvents were used in an attempt to avoid
premature crosslinking of the polymer. Along with the
above-mentioned solution, a photoinitiator is required in
SLA. In this case, bisacryl phosphrine oxide is used. A
proper balance between PPF and DEF is essential. It has
been observed that with a ratio higher than 0.5 of PPF to
DEF mechanical strength decreases significantly. On the
other hand, adding DEF decreases the viscosity of the
solution and hence improves printability. The recent

257

introduction of a ring-opening polymerization method
allows the precise determination of PPF molecular mass,
viscosity, and molecular mass distribution. Decreased
molecular mass distribution assists in the time-certain
resorption of the fabricated structures. Newly introduced
post-polymerization and post-processing functionalization
methods have increased the number of biomedical
applications that use PPF material.

Recently, literature works suggest that the influence of
PPF molecular mass in scaffolds fabricated via the SLA
technique proved critical regarding the degradation rate
and bone regeneration in vivo. PPF with lower mass
exhibited finer behavior in healing rates while no
inflammation and host cell acceptability were reported
(Kondiah et al., 2020; Mamaghani et al., 2018).

PEGDA and GelMA Inks

Literature works report the use of SLA-based 3D bio-
printing for a novel cell-laden cartilage tissue
construction. The resin used was comprised of 10%
Gelatin Methacrylate (GelMA) as base material, various
percentages of Polyethene Glycol Diacrylate (PEGDA),
biocompatible photo-initiator and trans-forming growth
Factor-Beta 1 (TGF-B1) embedded nanospheres fabricated
via a core-shell electrospraying technique. It was found that
adding PEGDA to GelMA hydrogel greatly improved
printability while compressive modulus also elevated
proportionally with PEGDA whilst the swelling ratio
decreased. Cells grown on 5/10% (PEGDA/GelMA)
hydrogel present the highest cell viability and
proliferation rate. The TGF-B1 embedded in nano-
spheres can keep a sustained release for up to 21 days
and improve the chondrogenic differentiation of
encapsulated MSCs. Therefore, such materials feature
high potential in cartilage regeneration processes
(Martinez-Garcia et al., 2022; Jiang et al., 2022).

Fused Deposition Modeling (FDM)

In this technique, the material is led to the extrusion
nozzle as a liquid of predefined viscosity or it is melted by
the heated nozzle to form a layer on the build platform. The
required ink is fabricated in the form of a solid filament
which is then heated to a semi-molten state in the stage of
extrusion. A temperature-controlled nozzle then oozes out
the filament material. The forced-out extruded material is
deposited onto a platform in a layer-by-layer deposition
principle. After completing one layer, the platform gets
lowered further and then the next layer gets deposited. The
main parameters decisively contributing to the final
properties of the material are layer thickness or height,
printing speed, infill percentage, nozzle temperature,
retraction, shell thickness, and support potential presence
(Kantaros and Karalekas, 2013; Antreas and Piromalis,
2021; Kantaros et al., 2022; Kantaros and Karalekas 2013;
Tsaramuirsis et al., 2022; Kantaros et al., 2013).

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Filament is led
to the extruder

Filament

The extruded material is laid down
on the model where it is needed.

The print head and/or bed is moved
to the correct X/Y/Z position for placing
the material

Fig. 3: Fused Deposition Modeling (FDM) schematic

Figure 3 depicts a Fused Deposition Modeling (FDM)
3D Printer apparatus schematic.

The majority of FDM printers are compatible with a
wide range of inks. However, the viscosity should be
greater than 6 x 10’ MPa/s, they should be molten at
temperatures between 200 and 250°C and a fast rate of
solidification is needed to melt them. In addition, the ratio
of elastic modulus to melt viscosity should be less than
5x 10°s1.

Bio-Inks Compatible with FDM Process
Poly (Caprolactone)

PCL has properties that make it well-suited for
melt-based extrusion processes. The low acquisition
cost and shear-thinning properties of this material make
it well-suited for use in medical devices and its thermal
stability makes it a desirable choice for medical
applications. Published literature works suggest that
PCL can be used to fabricate a tissue-engineered
scaffold to be used as a restoration substrate of breast
tissue after a partial mastectomy operation (Jwa et al.,
2022). Other reported literature cases indicate the use
of PCL material combined with sodium Mesoglycan
(MSG) which exhibited high rates in targeted wound
healing (Liparoti et al., 2022) and the design and bio-
printing of a novel wound-dressing material by
incorporating Juglone (5-hydroxy-1,4-
naphthoquinone) to a 25% Polycaprolactone (PCL)
scaffold (Ayran et al., 2022).

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Poly (Lactic Acid)

Polylactic Acid (PLA) is one of the most commonly
used polymers for FDM due to advantages such as
biocompatibility, biodegradability, and low cost. The
melting point temperature of this material makes it
suitable for forming filaments and it can be extruded at a
temperature of 180-250°C (Kantaros et al., 2021). One of
the challenges with PLA is the release of acidic by-
products when it degrades. There was a significant
decrease in the physiological acidity level due to the
release of lactic acid. To reduce the likelihood of acidic
release, PLA and ceramics are combined to create a
composite material. The composite material also tends to
increase the strength of compressive forces, making it a
good candidate for tissue engineering processes.

Polyether Ether Ketone (PEEK)

Peek is a semi-crystalline thermoplastic polymer that
has a melting temperature between 330-340°C and a
service temperature of 260°C (Ikada, 2006). Due to its
high melting point, it was initially excluded from its use
in FDM processes. Recent advances in FDM printer
technology have allowed the use of PEEK in FDM
printers. Published literature suggests that the main
factors affecting the 3D printing of PEEK in FDM
processes are the melting point temperature, the extrusion
speed, and the extrusion force. (Dorovskikh ef al., 2022).

Poly-Vinyl Alcohol (PVA)

Poly-Viny] Alcohol (PVA) is a synthetic polymer created
by vinyl alcohol and acetate monomers. The presence of the
latter provides biocompatibility, biodegradability, and bio-
inertia. PVA is soluble in lukewarm water and can be used in
FDM techniques in filament form. The tensile properties of
this material are highly comparable to those of human
articular cartilage, thus, providing a suitable substrate for
bone cell ingrowth (Chua et al., 2004). Its hydrophilicity and
chemical stability allow extreme pH and temperature
exposure and its semi-crystalline form ensure proper oxygen
and nutrients flow to the cell. PVA is extensively used in
various load-bearing implant cases like cranio-facial defects
and bone tissue regeneration treatments (Oka et al., 2000).

Direct Ink Writing (DIW)

DIW is an extrusion-based 3D printing exhibiting
similarities with the FDM method that uses a nozzle to
extrude materials onto a build platform in a layer-by-layer
manner. By utilizing this technique, controlled deposition
of raw materials in a highly viscous liquid state is
achievable which allows them to retain their shape upon
the deposition stage. DIW can be considered more
versatile than FDM as it can use a large variety of
materials ranging from ceramics, hydrogels, plastic, food,
and even living cells. The prime parameters which decide
the final properties of the product are nozzle size,

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viscosity and density of the material, printing speed, and
thickness kept between the layers. In DIW, similarly to
FDM, the use of support structures is vital in cases of
complex geometrical shapes featuring overhangs and
steep deposition angles. However, the use of dissolvable
materials as supports helps towards overcoming this issue
due to their ability to be easily removed upon the
completion of the printing procedure. The post-
fabrication processing stages also assist in the elevation of
the printed item’s mechanical properties (such as
elastic modulus) by using UV-curing apparatus
(Shuai et al., 2013a-b). Figure 4 depicts a Direct ink
writing schematic

Ink materials using the DIW method should exhibit a
fast rate of gelation and maintain proper structural
integrity upon the completion of the printing process.

Laser-Guided Direct Writing (LGDW)

LGDW is a laser-assisted direct writing technique that
is capable of depositing cells with micrometer accuracy.
The technique of cell deposition using a weakly focused
laser beam can be used on a variety of surfaces and
matrices. Laser-guided bio-printing is a process in
which a laser beam is used to direct cells onto a
receiving substrate (Bunea et al., 2021).

Bio-Inks Suitable for DIW and LGDW
Hydrogel Inks

Hydrogels are complex three-dimensional networks of
hydrophilic polymers that can absorb a large amount of
water. The key advantage of those materials is their high
biocompatibility and biodegradability rates because of
their ability to supply the proper conditions that favor the
encapsulation of viable cells further protecting the cells
without hindering cell-cell interaction. Hydrogel inks
should be able to flow fluently under working pressure
conditions by exhibiting controlled viscosity. Also, they
ought to provide sufficient structural integrity upon the
completion of the printing, furthermore as a fast rate of
gelation which will be controlled by exploiting shear
thinning (Tamo ef al., 2022). The literature desired
approach to design a hydrogel ink is to fabricate a polymer
solution that forms a network upon the completion of the
printing process. The network thus formed could be
physically or chemically crossed-linked using external
stimuli like temperature, light, or ion concentration
(Rioux ef al., 2022). The majority of natural polymers
such as gelatin, cellulose, collagen, fibrinogen, alginate,
and agar and synthetic polymers such as polyacrylamide,
polyurethane, Polyethene Glycol (PEG) are being utilized
in hydrogel fabrication in 3D __ bio-printing
(Ramezani et al., 2022; Teixeira et al., 2022).

The rate of proliferation towards the targeted tissue
decreases as the polymer concentration and cross-linking

259

density increase. However, due to their increased
viscosity, higher polymer concentrations are ideal for
extrusion-based DIW. Mechanical properties increase as
polymer concentration increases. Shear stress rises with
viscous inks, high pressure, and small diameter nozzles
and can cause cell death during extrusion. Shear stresses
greater than 60 MPa have been found to cause 35% or
more cell death (Duan, 2017). Thus, when using the DIW
technique, shear stress is the most important parameter
influencing resolution in hydrogel bioprinting.

Gelatin-Methacryloyl (GelMA)

GelMA is a semi-synthetic hydrogel consisting of
gelatin derivatized with methacrylamide and
methacrylate groups. Sauty et al. (2022). experimented
that GelMA hydrogels can be synthesized with a
specific Degree of Functionalization (DoF) and
adjusted to the intended application as a three-
Dimensional (3D) cell culture platform and GelMa was
also shown to support cartilage tissue formation using
chondrocytes and MSCs (Sauty et al., 2022). Piao et al.
(2021) found out that while dispensing cell-laden
GelMa from a glass capillary significantly higher
pressure was required; however, cell vitality and
proliferation weren't significantly affected.

Bio-printing neural tissues via DIW dictate some
specific ink material requirements. More specifically, the
elastic modulus (1.e., stiffness of the matrix of composite
ink used) affects neural cell growth, vitality, and cell
signaling. It has been found that brain tissues are
compatible with a stiffness of 0.5 MPa approximately
which is much less as compared to bone or cartilage
tissues. Therefore, soft hydrogels need to exhibit low
interfacial tension which allows cells to move across
the tissue implant line. This can be achieved by a
combination of two or more printing inks where one
will possess the required biological property and the
other with the task to regulate stiffness percentage
(Mohd et al., 2022).

Recent research suggests several approaches to
combining nucleic acid delivery and bio-printing. One of
them is developing gene-activated bioink. In this case, the
nucleic acid of interest and its delivery mechanism could be
incorporated in a single step by encapsulating it in a
bio-printable material, resulting in a gene-activated
bio-ink (Wu ef al., 2019).

Inkjet Bioprinting

Inkjet printing is a non-contact, controlled 3D printing
process that allows for the dispersion of droplets with
volumes ranging from 1-100 picoliters that include cell
viability. Droplets are extruded from a nozzle in one of
two ways: Drop by Drop (DOD) or Continuous Inkjet (CD
(CIJ). DOD is the more suited of the two for tissue
engineering. Furthermore, DOD can be classified into
three types according to the depositing techniques.

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Extrusion nozzle

Moving platform

Fig. 4: Direct ink writing schematic

Inkjet 3D bio-printer

Thermal _—_ Electromagnetic

: : Piezoelectric Piezoelectric crystal

Build platform

Bio-ink __Bio-ink reservoir _

_Heater element _ element

Fig. 5: Different Inkjet 3D bio-printer schematic

Thermal (using heat to expand and deposit the material
before the nozzle), electromagnetic and mechanical are
considered some examples. Individual drops with
diameters ranging from 25 to 50 um are generated
according to predetermined requirements in DOD inkjet
3D bio-printing. In CIJ 3D _ bio-printing continuous
streams of individual droplets possessing a volume of
100 um in diameter are ejected. It is found that as far
as tissue engineering is concerned, electromagnetic and
thermal inkjet printing has not been broadly adopted
since these processes tend to affect the cell wall and its
vitality after sonication at 15-25 Hz. That is the reason
why thermal inkjet is more widely used for better cell
vitality (Xiao et al., 2022; Yang et al., 2022;
Aversa et al., 2021a-b; Aversa et al., 2022; Yang et al.,
2022). Multiple inkjets print heads consisting of

260

several individual nozzles are being utilized to fasten the
process. In the case of Thermal Inkjet Printing, ink is super-
heated and bubbles are created which expand further until the
ink is released through the nozzle. The heating temperature
can reach up to 300°C for a few microseconds, thus, not
affecting the viability of biologically printed DNA, cells,
tissues, and other organs. Cell viability, in this case, has been
found out approximately 85% (Tofan et al., 2022). Figure 5
depicts different inkjet3D bio-printer schematics.

In this technique, ink requirements dictate properties like
desired viscosity and surface tension as viscosity affects
clogging, and surface tension has an immense contribution
to the shape of the drop not only after emerging from the
nozzle but also on the substrate. The viscosity of the ink
should be below 10 centipoises while surface tension should
ideally range between 28-350 mn m! (Yang et al., 2022).

Conclusion

3D Printing technology has greatly evolved in the past
decade, with several different techniques being introduced in
various fields and sectors. Available equipment now features
reduced fabrication times as well as newly introduced
materials, exhibiting a variety of properties. The introduction
of 3D bio-printers utilizing bio-materials compatible with the
human body offers an unprecedented ability to fabricate
highly controlled porous interconnected structures that act as
biological substrates for human cells to proliferate and lead
to grown tissues. These structures must exhibit a variety of
properties such as biocompatibility, bioresorbability as well
as appropriate mechanical behavior. In this context,
advanced biomaterials, like bio-inks acting as raw materials
for 3D bio-printers, now offer the ability to produce high
viability cell, tissue, and even direct DNA fabrication. The
careful tuning of process parameters in the 3D bio-printer
settings as well as the continuous introduction of new
biomaterials possesses an unrivaled way to realize the full
potential of this technology.

Funding Information
The authors have not received any financial support or
funding to report.

Ethics

This article is original and contains unpublished
material. The corresponding author confirms that all of the
other authors have read and approved the manuscript and
no ethical issues involved.

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