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1. Introduction
Tissue engineering currently stands out among innovative
technologies, aimed at solving the problem of organ and
tissue donor shortages for transplantation to patients with
irreversible injuries. Tissue engineering is based on inter-
disciplinary technologies that allow construction and grow-
ing biofunctional materials both inside and outside the
patient body in order to replace or regenerate damaged
tissues or organs. This approach implies formation of a
three-dimensional tissue structure with the required phys-
ico-chemical properties, including the mechanical ones, in
place of a defect. Besides, this process is accompanied by
restoration of biological functions of the damaged organ,
which is the main difference from application of traditional
implants made of inert materials.
1
The most promising field in tissue engineering is based
on the creation of tissue-engineered constructs (TECs)
representing the integration of three components: polymeric
materials in the form of matrix-carriers, the so-called scaf-
folds, living cells, and biologically active molecules. A
tissue-engineered construct provides conditions that mimic
the environment in the replaced tissues of the body, which
determines penetration, colonization, attachment and pro-
liferation of either allogeneic or autologous cells of the
patient. Such an environment should reproduce the main
characteristics of the native extracellular matrix at the
cellular and subcellular level as closely as possible and
facilitate the synthesis of new tissue.
2
Scaffolds play an
https://doi.org/10.57634/RCR5068
Photopolymerization in 3D printing of tissue-engineered constructs
for regenerative medicine
Alla N. Generalova,
a, b
Polina A. Demina,
a, b
Roman A. Akasov,
a, b
Evgeny V. Khaydukov
a, b
a
Shemyakin ± Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russian Federation
b
Federal Scientific Research Center `Crystallography and Photonics', Russian Academy of Sciences,
Leninsky prosp. 59, 119333 Moscow, Russian Federation
iD iD iD iD
Contents
1. Introduction 1
2. Basic photoinduced reactions in 3D printing 3
2.1. Free radical chain reaction 3
2.2. Radical polycondensation based on thiol-ene addition 4
2.3. Redox radical reactions 5
2.4. Controlled radical polymerization 5
2.4.1. Controlled radical polymerization 6
with addition ± fragmentation chain transfer
2.4.2. Living controlled radical polymerization 6
with the addition ± fragmentation chain transfer
3. Main components of photocompositions 7
3.1. Ink composing materials 7
3.1.1. Natural polymers 7
3.1.2. Synthetic precursors of tissue engineering constructs 8
3.2. Initiators and radiation sources 8
3.2.1. Type I photoinitiators 9
3.2.2. Type II photoinitiators 9
3.2.3. Initiating systems activated by near-IR light 12
4. 3D printing for tissue-engineered constructs 13
4.1. Extrusiuon-based 3D printing 13
4.2. Lithography-based 3D printing 14
5. Biomedical applications 16
5.1. Cytotoxicity assessment 16
5.2. Obtaining tissue-specific constructs 17
5.3. 3D printing in situ 19
5.4. Drug delivery systems 20
6. Conclusion 21
7. List of abbreviations and designations 22
8. References 22
The progress in the field of tissue engineering is largely driven by the development of 3D laser printing technologies, which
allow precise creation of hydrogel scaffolds containing cells (the so-called tissue-engineered constructs), using photoinduced
radical reactions of polymerization and crosslinking. The review considers the main mechanisms and features of such
reactions, presents the most common materials for photocompositions, including natural and synthetic polymers and
precursors, and describes various mechanisms for photoinitiator activation. Advances in the field of photopolymerization
enable application of modern laser 3D printing techniques based on extrusion and stereolithography to design tissue-
engineered constructs in a wide range of sizes and shapes with a finely organized architecture. The integration of such
methods with the methods of bioengineering and cell technology is discussed, including for the creation of tissue-specific and
in vivo polymerized constructs.
The bibliography includes 225 references.
#2023 Uspekhi Khimii, ZIOC RAS, Russian Academy of Sciences
Received 3 October 2022
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
Russ. Chem. Rev., 2023, 92 (2) RCR5068
important role in stimulating tissue regeneration providing
the necessary physical, chemical, mechanical and biological
properties, including biocompatibility, biodegradation,
porosity, mechanical strength, elasticity. Moreover, their
surface should have high adhesion to cells in the absence of
cytotoxicity, immunogenicity, and pyrogenicity.
3, 4
The
properties of scaffolds are primarily determined by the
nature and chemical composition of the source material.
The most widely used materials are natural and synthetic
polymers that allow the formation of scaffolds with a
variety of properties. In particular, it is possible to obtain
polymer-based hydrogels, which have recently become of
great interest for their application as scaffolds. This is
because the structural and biochemical properties of such
hydrogels are similar to the properties of extracellular
matrix of most tissues, and their mechanical properties can
be adapted to the properties of soft tissues.
5
The hydrophilic
properties of hydrogels promote cell adhesion, and the
porous three-dimensional structure facilitates the diffusion
of cells and nutrients.
6
The hydrogel production is based on
the crosslinking reaction of hydrophilic monomers, oligom-
ers or macromonomers, which determines its structure,
shape, size and degradation rate. The crosslinking process
can occur both by covalent bond formation (radical or
cationic polymerization, reactions of addition, condensa-
tion or enzyme catalysis, reactions induced by gamma rays
or electron beams, etc.), and by non-covalent interactions
based on the action of electrostatic or hydrophobic forces,
Van der Waals forces, solvation, etc.
7
However, when
forming hydrogels, there is a problem of controlling their
internal architecture (e.g., porosity, pore size, their spatial
arrangement and relationships) and distribution of the
composition components within the hydrogel volume. In
addition, it is almost impossible to include living cells and
signal molecules at the stage of hydrogel formation due to
the use of solvents incompatible with cells, long time or
harsh conditions of gel forming reaction.
8, 9
One of the methods that eliminates these drawbacks is
the light-driven covalent crosslinking. This can be a photo-
polymerization reaction if hydrogels are formed from
monomers (oligomers), or photoinduced crosslinking
involving macromonomers (e.g., biomolecules).
{
As com-
pared to the above methods,
10
photopolymerization pro-
ceeds rather quickly (from a few milliseconds to minutes) at
low doses of initiating radicals and does not require high
temperature or extreme pH values.
11
The process can be
implemented in the presence of cells and bioactive mole-
cules, and without deposition of cells in the initial reaction
solution, which is called a photopolymerizable composition
(photocomposition). As a rule, the light-driven chemical
reactions are very efficient that is associated with the
formation of a minimum amount of byproducts. This is an
important condition for the production of biocompatible
scaffolds containing living cells.
In recent years, significant efforts of researchers have
been focused on the creation of photocompositions required
for the formation of three-dimensional (3D) functional
scaffolds, which are called ink, from monomers (oligomers
or macromonomers) with good biocompatibility, bioactiv-
ity and degradability.
12
Note that light allows non-contact
spatial and temporal control of the degree of macromole-
cule crosslinking, which determines the ability to make 3D
objects of different shapes that can be integrated into the
structure of native tissue.
The greatest success in the field of TEC fabrication by
photopolymerization was achieved by using additive tech-
nologies, namely 3D photoprinting, to produce copies of
three-dimensional computer models of products and proto-
types, based on their step-by-step formation (in the form of
layers) when adding material to the substrate.
13, 14
This
technology is based on two main approaches:
Ð photoprinting based on extrusion, when the cross-
linking reaction proceeds under the action of light as the ink
is supplied or deposited from the extrusion device;
Ð photoprinting based on lithography, which involves
the light-driven transition of the ink to a solid state.
The issue of obtaining 3D models in the process of
photopolymerization using 3D printing is covered in the
literature in sufficient detail (e.g., see reviews
2, 12, 15 ± 17
).
However, there is no systematized information on the
action of photoinitiators and the mechanism of the main
photoreactions occurring during the transformation of ink
into crosslinked hydrogels (scaffolds) under the action of
light. Usually such studies are limited to photoreactions
activated by UV and visible radiation and do not affect the
IR spectrum.
This review presents traditional and recently developed
reactions, as well as their mechanisms and peculiarities of
implementation, used to form the 3D structures when
irradiated with light of a wide spectrum Ð from ultraviolet
to infrared one. Such reactions include chain free-radical
A.N.Generalova. Professor,Doctor of Chemical Sciences, Chief
Researcher, Head of the Laboratory of Polymers for Biology, IBKH RAS;
Senior Researcher, Laboratory of Laser Biomedicine, FSRC
`Crystallography and Photonics' RAS.
E-mail: angeneralova@gmail.com
Research interests: synthetic and natural high-molecular compounds,
including their modification of surfaces of inorganic luminescent nano-
particles to solve the problems of bioimaging, diagnosis and therapy of
pathologically altered tissues; nanobiotechnology, including obtaining
polymeric structures in the process of reactions photoinduced by IR light
using anti-Stokes nano-phosphors.
P.A.Demina. Junior Researcher, Laboratory of Laser Biomedicine, FSRC
`Crystallography and Photonics' RAS; Junior Researcher Laboratory of
Polymers for Biology, IBKH RAS.
E-mail: polidemina1207@yandex.ru
Research interests: upconversion nanoparticles, functionalization of sur-
faces with biopolymers for in vitro and in vivo studies, preparation of
scaffolds by IR-induced 3D photopolymerization.
R.A.Akasov. PhD, Senior Researcher, Laboratory of Laser Biomedicine,
FSRC `Crystallography and Photonics' RAS; Researcher, Laboratory of
Biomedical Materials, IBKH RAS.
E-mail: roman.akasov@gmail.com
Research interests: experimental biological models for in vitro and in vivo
studies, cell technologies: 3D cultures, nanosystems and their application
in biomedicine; methods of laboratory diagnostics and therapy, and in vivo
scaffold implantation.
E.V. Khaydukov. Professor,Doctor of Physical and Mathematical Scien-
ces, Senior Researcher, Laboratory of Polymers for Biology, IBKH RAS;
Head of the Laboratory of Laser Biomedicine, FSRC `Crystallography
and Photonics' RAS.
E-mail: khaydukov@mail.ru
Research interests: development of upconversion nanoparticles for bio-
medical applications, in particular, in biosensorics, bioimaging, early
detection of tumor diseases, and phototherapy; optical coherence tomog-
raphy; photochemistry of riboflavin, upconversion processes.
Translation: S.B.Karlova
{In this review, to simplify presentation, the term `photopolymerization'
will be used in both cases.
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
2of26 Russ. Chem. Rev., 2023, 92 (2) RCR5068
polymerization, thiol-ene polycondensation, redox radical
polymerization, and controlled radical polymerization. The
most common ink materials and types of initiating systems
necessary for photopolymerization and fabricating scaffolds
of the required architecture are considered. The approaches
to produce scaffolds in a wide range of sizes, various shapes
with finely organized structure and surface morphology by
using modern laser technologies of 3D printing based on
extrusion and stereolithography are demonstrated. The
methods for scaffold creation in vitro and their implantation
are shown with an emphasis on the high-potential methods
of scaffold formation directly in the damaged area (in situ),
which ensures a high accuracy of tissue reconstruction. The
presented approaches allow the creation of structured TECs
with cell cultures both in the process of synthesis and when
cells are incorporated into the final products for wide
utilization in biomedical research.
2. Basic photoinduced reactions in 3D printing
Three components are necessary to obtain TECs in the
process of 3D photopolymerization: light, photopolymeriz-
able composition (ink), and cells. The most promising are
inks, whose irradiation with light leads to formation of
hydrogels. Hydrogel structures of liquid ink in the process
of 3D photopolymerization are traditionally obtained under
the action of UV and visible radiation using radical initiat-
ing systems. The latter activate chain reactions and partic-
ipate in double bond hydrotiolation, redox and controlled
reactions by the mechanism of `live' chains, as well as in the
presence of cationic initiation systems. However, the use of
cationic systems, despite the possibility of reaction proceed-
ing by the mechanism of living chains, which provides high
substrate conversion, is not suitable for working with living
systems. This is because cationic initiators generate strong
acids that negatively affect cell cultures.
18
In addition, the
presence of water slows down or even completely inhibits
the cationic polymerization reaction.
19
In addition, the
living chains mechanism can be implemented only in the
absence of oxygen, making impossible the creation of
scaffolds with cell cultures. For this reason, cationic systems
will not be considered in this review.
Of particular interest is photopolymerization under the
action of near IR (NIR) light, which affects tissues by low-
energy light quanta at great depths and provides a safe
environment for cell life in 3D technologies.
20
For this
purpose, initiation systems for two-photon polymerization
and systems based on energy transfer from the donor,
excited by NIR light, to the acceptor, which can generate
radicals that enter into the polymerization reaction, are
being developed.
15
2.1. Free radical chain reaction
Typically, the ink composition includes a radical-reactive
monomer (oligomer, macromonomer), an initiator or ini-
tiating system, and various additives such as drugs, poro-
gens, mechanical strength regulators, etc.
15
Acrylate and
methacrylate monomers and oligomers are most widely
used in 3D photopolymerization by the chain free-radical
mechanism in the TEC production. Free-radical initiators,
described in Section 3.2, are used to activate these
monomers.
In photoinduced chain polymerization of ink by the
radical mechanism, three stages can be distinguished: ini-
tiation, chain growth and chain growth termination
(Fig. 1). Each stage has its own kinetic features, which can
affect the micro- and macrocharacteristics of the resulting
hydrogels.
The polymerization process begins with generation of
radicals under the action of light with participation of a
photoinitiator or a photoinitiator system, which form
radicals that can activate the monomer (oligomer) due to
photolysis or electron (hydrogen atom) attachment. The
rate of radical formation from the initiator depends on the
incident light intensity, the photoinitiator activity and
concentration, the quantum yield and the number of
effective radicals generated per one act of photolysis.
Then these free radicals react with vinyl groups of mono-
mers (oligomers) to form new covalent bonds and reactive
radicals. The radicals enter into subsequent reactions with
vinyl groups, which leads to the growth of a polymer chain.
The growth process ends when the monomer (oligomer) is
exhausted and due to reactions of chain growth termina-
tion such as recombination of radicals, disproportionation
of radicals with formation of saturated and unsaturated
end groups, transfer reaction from the growing chain to
other components of the reaction medium Ð solvent,
monomer, etc.
12
Examples of basic monomers with a vinyl end group
capable of entering into light-driven chain radical polymer-
ization are shown in Fig. 1. The most common inks for
making 3D objects are (meth)acrylate monomers (oligo-
mers): polyethylene glycol diacrylate, triethylene glycol
dimethacrylate, copolymer of bisphenol A with glycidyl
methacrylate, trimethylolpropane triacrylate, bisphenol A
ethoxylate diacrylate.
15
The structure and density of crosslinking of the polymer
chains, formed by radical chain polymerization can be
controlled by changing the concentration of initiator, the
number of vinyl groups (e.g., by varying the monomer or
oligomer concentration) and the light intensity. As a rule,
mechanically strong scaffolds with long degradation times
in the case of biodegradable materials are formed at a high
degree of crosslinking.
9
However, the different lengths of
the growing kinetic chain determine heterogeneity of the
scaffold structure. This heterogeneity is associated with the
concentration gradient and diffusion limitations, since the
high rate of kinetic chain formation leads to auto-acceler-
ated chain growth and diffusion-controlled chain termina-
tion. In particular, steric hindrances that arise in the process
Initiation
IhnI
I+M M
Chain growth
M+M M
2
M
2
+M M
3
Chain termination
M
n
+M
m
P
n+m
(recombination)
M
n
+M
m
P
n
+P
m
(disproportionation)
M
n
+HX P
n
7H+X (chain transfer)
Monomers
OR
O
R-acrylate
OR
O
R-methacrylate
NR
O
R-acrylamide
HR = Me, Et,
Pr and, etc.
Figure 1. Scheme of radical chain polymerization and examples of
basic monomers. The following designations are used: I is initiator,
M is monomer, P is polymer, R is alkyl group.
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
Russ. Chem. Rev., 2023, 92 (2) RCR5068 3of26
of polymer network formation limit chain termination
reactions, and, correspondingly, the total concentration of
radicals increases. The rate of polymerization increases and
the auto-acceleration process starts, leading to formation of
a heterogeneous system in which intramolecular chain
transfer reactions can also take place. As a result, the
radicals responsible for branched and cyclic structures can
be formed along the chain,
21
i.e. the heterogeneity of the
scaffold structure increases.
The chain nature of polymerization determines the high
rate of hydrogel formation, but the dependence of kinetics
on many factors causes a number of limitations. First of all,
we should note the inhibitory effect of atmospheric oxygen,
required for TEC formation with cells. The negative effect
of oxygen on polymerization is manifested in deactivation
of the excited states of the initiator and formation of
inactive peroxide radicals, which affects the efficiency of
the entire process. This leads to incomplete crosslinking of
the material during 3D photopolymerization and reduces
printing accuracy, and also has an effect on the shape
retention over time,
22
which is crucial for complex structure
creation. In addition, it is necessary to use low intensity
radiation and minimize the number of generated radicals,
since both of these factors have a cytotoxic effect on cells.
23
The above limitations of photoinduced free-radical
chain reactions (relatively poor control of crosslinking
kinetics, inhibitory effect of oxygen, presence of unreacted
double bonds, formation of heterogeneous structures)
necessitates the development of new approaches to scaffold
production. One of the ways is the use of orthogonal click
reactions for photoinduced radical crosslinking.
2.2. Radical polycondensation based on thiol-ene addition
Orthogonal click reactions based on hydrotiolation of
double bonds (thiol-ene reactions) have attracted much
attention due to more precise control of light-driven radical
crosslinking as compared to the chain processes. Thiol
reactions with highly reactive carbon ± carbon double
bonds are well known.
24
They can proceed as polyconden-
sation (radical step polymerization), which will be discussed
below, or by the reaction of Michael addition.
25
Thiol
reactions provide polymer scaffolds with less heterogeneity
because they allow control over crosslink density, cell size
and mechanical properties by varying functionality, length
and concentration of a crosslinking agent. In addition,
thiol-ene reactions are insensitive to the presence of water
and oxygen and can proceed under mild conditions with
higher efficiency, selectivity and rate as compared to the
chain radical process.
26
Under the action of radicals formed at the stage of
initiation, the sulfide groups of thiol-containing molecules
are converted into reactive thiyl radicals (Fig. 2). Then,
these intermediate thiyl radicals form thioether bonds with
molecules containing vinyl groups. The growth of a polymer
chain occurs according to a stepwise mechanism, in which
the sequential addition of monomers (oligomers, macro-
monomers) leads to a gradual increase in molecular weight.
When using multifunctional crosslinking agents, a network
structure is formed. To obtain scaffolds, a reactive oligomer
and a multifunctional thiolated crosslinking agent are
usually utilized. As in the case of a chain reaction, the rate
of polycondensation and the properties of a final product,
depend significantly on a number of factors. In general, the
thiol-ene reaction proceeds very quickly (within a few
seconds) with crosslink formation and this allows the
control of the final product properties by controlling the
crosslinking degree.
15
The main vinyl monomers capable of participating in
the thiol-ene reaction are shown in Fig 2. The following
thiol-containing compounds are most widely used as the
second component for obtaining 3D objects: tris(3-mer-
captopropionate) trimethylolpropane (TMPMP), tetra-
kis(3-mercaptopropionate) pentaerythritol (PETMP),
tris[2-(3-mercaptopropionyloxy)ethyl]isocyanurate (TMI)
and tetrakis(2-mercaptobutanoate) pentaerythritol
(PE-1).
15
It is worth noting that some vinyl derivatives, along with
the thiol-ene reaction, can simultaneously participate in free
radical chain processes that result in scaffold formation by
two mechanisms. This case can be considered as mixed
polymerization. For example, (meth)acrylates form cross-
links with multifunctional thiols during polycondensation
and also enter into a radical chain reaction, i.e. copolymer-
ization can take place in one material. This format of
photoinduced crosslinking allows expansion of scaffold
properties during their production. However, the complex
kinetics of these radical processes can potentially cause
phase separation in the scaffold formed by different mech-
anisms.
27
The reactivity of vinyl groups is determined by their
electron density, stability of intermediate radicals, and
steric hindrances. End groups with high electron density
react more actively than groups in the main chain or groups
with lower electron density. The conversion of monomers
(oligomers, macromonomers) depends not so much on the
kinetics of various thiol-ene reactions but on the vinyl
group ability to polymerize in a given material and such
effects as auto-acceleration and diffusion-controlled chain
termination determined by the crosslinking degree. These
factors allow one to reach the same substrate conversion for
processes with different kinetics. In addition, thiol-ene
Chain termination
Chain growth
I
hn
I
Initiation
I+R
1
7SH R
1
7S
R
1
7S+R
2
R
1
SR
2
R
1
SR
2
+R
2
R
1
SR
2
R
2
(homopolymerization)
R
1
SR
2
+R
1
7SH R
1
SR
2
+R
1
7S
(chain transfer)
P
n
+P
m
(disproportionation)+M
m
M
n
P
n+m
(recombination)+M
m
M
n
Norbornen
Monomers
NR
O
H
R-acrylamide
R
alken
OR
simple vinyl
ether
O R
O
vinyl ester R-methacrylate
OR
O
R-acrylate
OR
O
Figure 2. Scheme of thiol-ene addition.
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
4of26 Russ. Chem. Rev., 2023, 92 (2) RCR5068
reactions provide more precise control over the depletion of
vinyl groups compared to the chain process since the cross-
linking degree is controlled by the concentration of the
crosslinking agent rather than the reactive groups. This
allows consistent, controlled crosslinking, as well as incor-
poration of biomolecules important for tissue engineering
into the scaffold network.
28
However, there are a number of restrictions for the wide
application of thiol-ene systems in 3D photopolymeriza-
tion. The scaffolds obtained in this way are characterized by
a short shelf life and an unpleasant odor, caused by
formation of byproducts with a disulfide bond, which can
be easily oxidized. Moreover, thiol-ene reactions, due to the
stepwise growth kinetics, lead to formation of homogeneous
hydrogel networks with flexible thioether bonds. As a
result, soft materials with a low elasticity modulus are
obtained.
29
Though, the mechanical properties of the result-
ing products can be improved by introducing a second
monomer, such as methacrylate,
15
or an oligomer, such as
oligourethane with norbornene end groups.
30
The storage
stability of the source thiols can be improved by using
stabilizers, preventing formation of disulfides,
31
or by
creating new monomers. For instance, Chen et al.
32
have
synthesized a highly stable double thiol-ene photocurable
composition containing tetrakis(3-mercaptobutanoate) pen-
taerythritol (PE-1) and triallyl-1,3,5-triazine-
2,4,6(1H,3H,5H)-trione (TTT), which showed high thermal
stability during storage.
In general, the thiol-ene photocrosslinking reaction is a
very promising approach to produce scaffolds with finely
tuned properties by 3D photopolymerization.
2.3. Redox radical reactions
Redox reactions are also used to produce scaffolds in the
course of photopolymerization. These reactions can involve
both monomers (oligomers) with vinyl groups, conventional
for radical chain polymerization (see Fig. 1),
33, 34
and poly-
mers containing groups, which can be activated during
redox reactions under the action of photosensitizers and
then participate in radical polymerization or crosslinking
reactions.
35
Dyes or additives, effectively absorbing light and passing
into excited states, can be used as photosensitizers, capable
of oxidizing (reducing) reactive groups. Photosensitizers
absorb light of longer wavelengths and they do it with
greater efficiency than organic small molecules. The energy
of the absorbed photon can be converted into a chemical
potential that affects organic substrates in various ways.
36
The photosensitizer must have a high absorption coefficient
at the wavelength of the exciting light, a high quantum yield,
and a sufficient stability to catalyze the photoreaction.
37
To
generate radicals, it is necessary to use an initiating system of
type II (see Section 3.2.2) that contains a photosensitizer and
a co-initiator. In this case, the excited photosensitizer mole-
cule reacts with the corresponding co-initiator, which exhib-
its the properties of an electron donor or acceptor or
hydrogen atom donor responsible for generation of corre-
sponding radicals or radical ions.
38
The fulfillment of these
conditions determines the ability of photosensitizers to
generate free radicals through electron transfer or hydrogen
atom abstraction from the substrate (Fig. 3). Further poly-
merization proceeds according to the scheme of the radical
process, in which the stages of chain growth and chain
termination are distinguished (see Fig. 1). The ink composi-
tion includes traditional radical monomers (oligomers, poly-
mers) containing vinyl groups, as well as, for example,
moieties of tyramine or tyrosine.
12
It is important to note that, in the presence of oxygen,
photosensitizers additionally undergo side reactions, which
lead to the formation of singlet oxygen, superoxides, and
then hydrogen peroxide.
37
These side reactions cause regen-
eration of the ground electronic state of photosensitizers
and consumption of the formed radicals. As a result, the
rate of photoreactions increases as well as the degree of
reactive group crosslinking.
Side reactions of the first type include energy transfer
from a photosensitizer in an excited (triplet) state to an
oxygen molecule in the ground state. As a result, singlet
oxygen, which can easily oxidize hydroxyl, sulfide, and
amino groups, is generated. After reactions associated with
electron transfer and hydrogen atom abstraction from the
substrate, the photosensitizer radicals can enter into secon-
dary reactions with triplet oxygen, accompanied by super-
oxide formation. In addition, primary radicals can interact
with each other and react with oxygen in the ground state,
resulting in formation of hydrogen peroxide.
It is known that the reaction of photoinduced cross-
linking hardly proceeds under the anaerobic conditions.
35
Reactive oxygen species in the form of radicals may not
participate in the process of radical crosslinking of mono-
mers (oligomers, macromonomers), but they are necessary
for activation of the photosensitizer and generation of
reactive radicals by the initiating system. Therefore, a very
important property of photosensitizers is the ability of easy
energy transfer to triplet oxygen. This feature of redox
polymerization determines the significant difference from
the radical chain process, when oxygen inhibits the photo-
induced crosslinking reaction. Examples of initiating sys-
tems are presented in Section 3.2.2.
When using photosensitizers in the process of 3D photo-
polymerization, it is necessary to take into account their
cytotoxic properties, as well as the ability to generate
reactive oxygen species upon irradiation. These side effects
can be minimized by reducing the concentration of photo-
sensitizers to a level that allows maintaining the kinetics of
the photocrosslinking reaction without a significant cyto-
toxic effect on cells.
2.4. Controlled radical polymerization
Controlled radical polymerization (CRP) is often used to
synthesize polymers with specific compositions, topologies,
and architectures. It is based on the replacement of irrever-
sible bimolecular chain termination with a reversible reac-
Initiation
IhnI*
Chain growth
or
RH
R
co-initiator (D/A)
R+M MA is acceptor,
D is donor
H abstraction I7H+R
I
/
7
+R
++/ 7
R
M+M M
2
M
2
+M M
3
+
I*
(A/D)electron
transfer
Figure 3. Scheme of redox radical photopolymerization (chain
termination reactions see in Fig. 1).
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
Russ. Chem. Rev., 2023, 92 (2) RCR5068 5of26
tion of radical growth under the action of agents that
transfer the chains into an inactive state. As a result,
macromolecular radicals participate in successive activa-
tion7desactivation cycles. Activation at the end of the
polymer chain leads to the appearance of a labile end
group capable of cleavage under certain conditions to
form a radical that can continue the growth of the polymer
chain. The process of repeated cycles of radical activation ±
desactivation ensures the stepwise growth of all chains
during polymerization.
39
In particular, the CRP methods are being developed that
use light to detach the end group, which transfers into a
growing radical, and this allows spatiotemporal control
over the process of chain growth.
40, 41
Currently, active
application of such CRP methods for scaffold production
by 3D photopolymerization starts.
2.4.1. Controlled radical polymerization with
addition ± fragmentation chain transfer
Acrylate-based monomers (oligomers) are most widely used
as a starting material for 3D photopolymerization resulting
in a rigid hydrogel network due to the high rate of polymer
chain growth. However, the lack of control over this
reaction leads to formation of inhomogeneous materials
with uncontrolled chain crosslinking and, accordingly, brit-
tle materials, which significantly narrows the scope of their
application. The key direction in the development of 3D
photopolymerization is the creation of new ink that forms
homogeneous networks in the process of radical stepwise
growth similar to the thiol-ene reaction, but devoid of its
drawbacks (see Section 2.2).
42
Such ink is suitable for
fabrication of 3D structures with high impact toughness
and low shrinkage stress.
43
One of the approaches is based on radical polymer-
ization with the addition ± fragmentation chain transfer
(AFCT). During this polymerization, the growing radical
is attached to the AFCT agent, followed by deactivation
(fragmentation) of the resulting adduct radical, which leads
to the formation of a polymer chain with a vinyl end group
and a radical capable of initiating a new chain (Fig. 4).
44
This method allows the control of the polymer molecular
weight and introduction of functional end groups. The
process is very similar to the reversible addition ±fragmen-
tation transfer polymerization (RAFT),
45
but the growing
radical reacts with the AFCT agent much slower than in a
controlled living RAFT system (see Fig. 4).
44
Chain transfer agents in the AFCT process, such as allyl
sulfides,
46
allylsulfones,
42
vinyl sulfone esters,
42
are the
promising additives for photopolymerizable ink controlling
the formation of a uniform network, which improves the
thermal and mechanical properties of the material.
However, the use of AFCT polymerization reduces the
rate of radical reaction, especially at high concentrations of
agents. This can increase significantly the time required for
layer photocuring and, accordingly, duration of 3D printing
based on the creation of multilayer structures.
2.4.2. Living controlled radical polymerization
with the addition ± fragmentation chain transfer
In the RAFT-polymerization, free-radical polymerization
can be implemented using the mechanism of living chains, in
which re-initiation, introduction of new monomers into the
formed polymer network and visible- or UV-light-induced
functionalization are possible.
47
This polymerization pro-
ceeds with the help of agents containing groups with an
unsaturated bond at the end of the chain (mainly C
=
S),
such as dithioether, dithiocarbonate, dithiocarbamate, etc.,
which enter into the exchange reaction due to addition and
fragmentation.
To date, only a few examples of RAFT agent utilization
for 3D photopolymerization are presented in the literature.
In particular, hydrogels based on acrylates and acrylamides,
such as N-isopropylacrylamide, n-butyl acrylate, and poly-
ethylene glycol methyl ester acrylate, can be obtained by the
mechanism of light-driven living radical polymerization in
the presence of sodium trithiocarbonate derivatives as a
RAFT agent and 10-phenylphenothiazine as an organic
photochemical redox initiator. Using this system, we can
effectively switch the state of radicals in the growing chain
from addition to fragmentation and back under the action
of visible and UV light. During the polymerization, the
molecular weight of polymers increases linearly without a
gel effect. The proposed mechanism of this process is shown
in Fig. 4. The electron transfer from the photoexcited
photocatalyst (I*) activates the molecule of sodium trithio-
carbonate with the formation of P
n
.radical, which partic-
ipates either in the growth reaction or in chain transfer by
the RAFT mechanism. Sodium trithiocarbonate (TTC rad-
ical in the case of UV light; TTC anion and an ionic
complex with I
+
.in the case of visible light) can deactivate
the growing polymer chain to form a polymer containing a
TTC fragment at the end of the chain, and the photo-
initiator in the ground state. Under the action of light these
components are able to re-enter the catalytic cycle (rever-
sible activation).
Photocatalyzed RAFT polymerization enables embed-
ding of a new monomer into the product by the mechanism
of living chains. Depending on the nature of monomers and
the number of crosslinks, secondary gels with a different
network structure and, accordingly, with different chemical
and mechanical properties can be obtained on the basis of
the initial gel.
48
However,therateofCRPbythemecha-
I
hn
I
Initiation
Reversible chain transfer (growth) reaction
P
n
+
Mk
p
S SR
Z
TTC-drivative
S SP
n
Z
R
S SP
n
Z
+R
Reinitiation
RM
Chain growth
P
m
+
Mk
p
S S
P
n
Z
S SP
n
Z
P
m
S SP
m
Z
+P
n
Mk
p
Chain termination
P
m
,P
n
R, termination
P
m
P
n
I,
Figure 4. Scheme of living controlled radical polymerization with
addition-fragmentation chain transfer (RAFT). TTC is trithiocar-
bonate, Z is activating group.
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
6of26 Russ. Chem. Rev., 2023, 92 (2) RCR5068
nism of living chains is lower than that of radical chain
polymerization, which may limit its use in 3D photopoly-
merization if it is necessary to assemble the product rapidly.
3. Main components of photocompositions
The necessary components of photopolymerizable compo-
sitions (ink) are as follows: a material, which can form
hydrogel structures; a photoinitiating system that generates
radicals under the action of UV, visible or IR radiation, and
cells that can be included in the structure both at the stage
of ink photopolymerization and after the sample prepara-
tion. The formation of TECs in the form of scaffolds with
cells and their application in biomedical research are dis-
cussed in Section 5. Components that improve rheological
and mechanical properties, as well as porosity, degradation,
visualization of hydrogels, etc., can be introduced into the
photocomposition.
3.1. Ink composing materials
Scaffolds in the form of hydrogels are water-swollen poly-
mer networks, consisting of natural and (or) synthetic
materials, usually biodegradable. Great prospects for the
use of hydrogels in tissue engineering are associated with the
possibility of their rational design to mimic the functions
and structures of damaged or lost tissues. The highly
hydrated environment of hydrogels reproduces the aqueous
environment of tissues and allows the introduction of cells,
bioactive molecules and drugs into them.
In addition, the physicochemical properties of hydrogels
can be easily adjusted for specific tasks. For example, by
adjusting the crosslinking degree, we can obtain gels with
well-defined network parameters, which allow the diffusion
of components to ensure the supply of nutrients or oxygen
to the cells. Controlling the crosslink density, which also
affects the mechanical properties of hydrogels, enables the
formation of hydrogel networks whose viscoelastic proper-
ties and stiffness are comparable to those of native tissues.
An important property of hydrogels is the possibility of
their biofunctionalization, for example, with bioactive mol-
ecules, various labels or drugs to improve adhesion, migra-
tion, visualization, proliferation and differentiation of
encapsulated cells.
49
The unique properties of hydrogels listed above deter-
mine the prospects of their application as scaffolds for
repair of affected tissues. Materials on the basis of which
hydrogels can be obtained in 3D photopolymerization are
classified according to their origin: natural, synthetic and
hybrid ones. Examples of such materials are presented
below.
3.1.1. Natural polymers
Natural compounds are widely used as the main component
of ink, since they are usually characterized by high bio-
compatibility, biodegradability and bioactivity. One of
these compounds is gelatin, which is formed during collagen
denaturation. Gelatin contains adhesive peptide sequences
(e.g., RGD peptide) that allow embedded cells to attach and
proliferate in gelatin hydrogels. In addition, gelatin con-
tains peptide sequences, sensitive to endogenous enzymes
(matrix metalloproteinases), which contributes to its effec-
tive biodegradation. To carry out photoinduced crosslink-
ing by the mechanism of radical chain polymerization,
methacryloyl groups are introduced into gelatin (by ester-
ification with methacrylic anhydride). Gelatin can also be
modified with reactive groups that enter into radical thiol-
ene reactions. For example, Bertlein et al.
50
introduced allyl
groups using the reaction of glycidyl ether with gelatin at
65 8Cinanalkalinemedium.VanHooricket al.
51
synthe-
sized gelatin derivatives with norbornene by reacting the
primary amino groups of protein with 5-norbornene-2-carb-
oxylic acid upon activation with carbodiimide. Both mod-
ifications provide polymer capable of reacting with multi-
functional thiol crosslinkers to form a hydrogel. To incor-
porate desired biomolecules into the gel or to perform
chemical crosslinking, gelatin can be functionalized with
thiol groups.
52
Hyaluronic acid (HA), which is an endogenous poly-
saccharide consisting of repeating unbranched units of
glucuronic acid and N-acetylglucosamine, causes significant
interest. Hyaluronic acid is found in many connective
tissues and is involved in a number of biological processes,
such as wound healing, maintaining tissue homeostasis,
etc.
53
The prospects for using HA to create scaffolds in 3D
photopolymerization are associated with the possibility of
its degradation in the body under the action of hyaluroni-
dase and oxidizing agents, as well as with its easy modifica-
tion due to the presence of carboxyl and hydroxyl groups in
the structure. For instance, methacrylate units were intro-
duced into HA by a polymer-analogous reaction with
methacrylic anhydride
54
or glycidyl methacrylate in the
presence of triethylamine.
55
Hyaluronic acid, like gelatin,
can be modified with norbornene by the reaction of 5-nor-
bornene-2-carboxylic acid with hydroxyl groups
56
or by
the reaction of 2-methylamino-5-norbornene with carboxyl
groups.
57
However, the modification of the carboxyl group
in HA was shown
58
to decrease the acid ability to interact
with cellular binding sites, such as CD44. For this reason,
the methods have been developed to maintain the concen-
tration of carboxyl groups, for example, by interacting the
HA hydroxyl groups with cysteines.
59
Such HA derivatives
afford the preparation of hydrogels with a wide range of
degradation times and with the most diverse architecture,
which significantly expands the range of TECs created.
Chondroitin sulfate is proteoglycan found in connective
tissue such as cartilage and synovial fluid. This biomolecule
can be degraded by the action of enzymes. An important
property of chondroitin sulfate is the possibility of its
modification by introducing vinyl groups, for example, by
reaction with glycidyl methacrylate in the presence of
dimethylaminopyridine, proceeding through the formation
of a salt with tetrabutylammonium.
60
Other materials of natural origin can also participate in
3D photopolymerization: dextran, silk fibroin, collagen,
decellularized extracellular matrix, alginate, k-carrageenan,
and chitosan. Such biopolymers can be modified by poly-
mer-analogous reactions or by interaction with functional
compounds capable of participating in the photoinduced
crosslinking. For example, to introduce vinyl groups into
dextran, it is reacted with methacrylic anhydride,
61
while for
the modification of silk fibroin, it is reacted with glycidyl
methacrylate in the presence of lithium bromide at 60 8C.
62
Methacrylate units are introduced into chitosan through
interaction of hydroxyl groups with activated carbonyldii-
midazole.
63
The advantage of natural materials in 3D photopolyme-
rization concerns the formation of hydrogels, characterized
by high efficiency in cell encapsulation, biological activity,
ability to degrade. They possess minimal immunogenicity,
and contain functional groups responsible for the introduc-
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
Russ. Chem. Rev., 2023, 92 (2) RCR5068 7of26
tion of photopolymerizable units. The wide use of natural
polymers is limited by unpredictable molecular mass distri-
bution and variation of properties for each batch, which
impedes the control of mechanical parameters and the rate
of biodegradation of the formed scaffolds.
3.1.2. Synthetic precursors of tissue engineering constructs
Synthetic materials, in contrast to natural compounds,
allow the formation of hydrogels with well-reproducible
properties and ability to control network formation, gelat-
ion kinetics, degradation rate, and mechanical properties.
Hydrogels based on synthetic materials can be obtained by
3D photopolymerization from acrylate- and methacrylate-
based monomers and oligomers by the mechanism of
radical chain polymerization and redox polymerization
(see Sections 2.1, 2.3), as well as from thiol-containing
monomers and norbornene, N-vinylamides, vinyl and
propyl esters of organic acids, maleimide, etc.bythe
mechanism of radical thiol-ene reactions (see Section 2.2).
Among synthetic polymers employed for hydrogel for-
mation, polyethylene glycol (PEG) is the most widely used.
This polymer is characterized by high hydrophilicity and
low protein adsorption, which determines the long circula-
tion time of structures based on this polymer in the
circulatory system in vivo.
64
An important property of
PEG is the possibility to incorporate vinyl moieties by
utilizating simple methods. For example, the reaction of
PEG with acrylic or methacrylic acid chlorides in the
presence of triethylamine allows introduction of end acryl-
ate or methacrylate units, which can enter into 3D photo-
induced crosslinking by the mechanism of radical chain
polymerization.
65
The hydrolytic degradation of PEG-
based hydrogels is ensured by the introduction of a-hydroxy
acids between the vinyl groups and the main PEG chain.
66
Polyethylene glycol can also be modified by end thiol
groups or norbornene fragments to carry out thiol-ene
crosslinking.
27
Polyvinyl alcohol (PVA) is actively used for hydrogel
preparation due to PVA hydrophilicity and the presence of
hydroxyl groups, which can be easily modified. Fragments
with vinyl groups are introduced in PVA, similar to natural
polymers, via the esterification with methacrylic anhy-
dride.
67
Modification of PVA with tyramine units, which is
carried out by interaction of carboxylated PVA derivatives
with tyramine under carbodiimide activation promotes the
formation of hydrogels in the redox radical 3D-photopoly-
merization.
68
Polylactide (PLA), as well as copolymers of lactide with
glycolide (PLGA) and polycaprolactone (PCL) are exam-
ples of polymers that provide mechanically strong, biocom-
patible and biodegradable scaffolds. Different ratios of
PLA and PLGA determine the hydrophilicity and regulate
the rate of hydrolytic degradation, since PLA containing a
methyl group decomposes slower than PLGA.
69
To increase
the hydrophilicity and to control the mechanical properties
of the material, it is copolymerized with PEG
70
or poly-
ethylene glycol dimethacrylate.
71
Introduction of units with
double bonds into such polymers, for example, by inter-
action with methacrylic anhydride, results in the formation
of porous materials of a gyroid structure, which can
function as soft tissue scaffolds.
72
Synthetic polymers are also used to increase the strength
of photocurable materials by improving homogeneity and
controlling the crosslinking degree.
73
For example, flexible-
chain polymers Ð polysiloxanes Ð have shown great
potential for achieving this goal. The addition of polysilox-
anes, which, along with chain flexibility, have the ability to
reduce surface tension, leads to a change in adhesion,
morphology, surface energy, thermal stability, mechanical
properties, and hydrophilicity of scaffolds. Advincula and
co-workers
74
proposed a method for performing 3D photo-
copolymerization of oligomers of methacryloxypropylme-
thylsiloxane and methacrylate. The scaffold consisting of
this composition was characterized by high tensile strength,
good elasticity, and high compressive strength.
Despite a number of advantages of synthetic material, a
significant disadvantage of such scaffolds obtained from
them is low adhesion and weak cell proliferation on the
surface and in the bulk. For this reason, the modification of
the polymer surface is developed to improve its adhesive
properties by incorporating adhesive proteins or peptides
(e.g., RGD peptide)
75
or molecules with heparin-binding
sites.
49
In general, synthetic polymers do not have the biological
activity of natural materials; therefore, hybrid ink, which
includes both natural and synthetic polymers,
67
is often
used (see Section 5.2).
3.2. Initiators and radiation sources
Initiating systems based on one-, two-, or multicomponent
photoinitiators play a key role in photopolymerization,
which can proceed both via the radical and cationic mech-
anisms (the latter is not considered in this review).
Photoinitiating systems not only determine the reaction
mechanism, but also affect its patterns, the rate, and the
final properties of the polymer, such as hardness and
viscosity. The main parameters of photoinitiator selection
are the maximum absorption wavelength and the molar
extinction coefficient. The efficiency of a photoinitiator
relates directly to its structure, which determines the
absorption range and quantum efficiency of photochemical
and photophysical processes involving reactants in excited
states.
76
Regardless of the type and mechanism of initiation,
a photoinitiator should have the following properties:
Ð absorption wavelength falls within the emission band
of the source;
Ð high quantum efficiency;
Ð good solubility in photocomposition;
Ð lack of cytotoxicity;
Ð high thermal stability;
Ð storage stability.
In addition, the course of photopolymerization is
affected by the structure and physicochemical properties of
the initial monomers (oligomers, macromonomers), the
presence of oxygen, stabilizers or other additives in the
photocomposition; thickness of the irradiated layer, type
and intensity of the radiation source. In the case of in vivo
photopolymerization, it is important to use low toxicity
initiators, especially when exposed to light. Free radicals
formed during initiation can react with the main compo-
nents of living cells Ð cell membranes, proteins, and nucleic
acids Ð and cause damage.
77
Applying photopolymeriza-
tion to the creation of three-dimensional TECs requires
understanding of the initiation reaction mechanisms, which
can maintain cell viability without loss of the method
resolution.
Photopolymerization is initiated by light irradiation of a
photoinitiator or photoinitiating system that converts pho-
ton energy into generation of reactive radicals.
78
The light
source can be xenon lamps, mercury lamps, LEDs, or lasers.
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
8of26 Russ. Chem. Rev., 2023, 92 (2) RCR5068
The source photon wavelength can be in the UV
(190 ± 400 nm), visible (400± 700 nm) or even in the near-
IR (700 ± 1000 nm) range of the spectrum. This Section
presents the main types of photoinitiating systems that are
used in 3D photopolymerization; specific examples of
radiation sources that can activate these systems are given
in Section 5.
Photoinitiators activated by UV and visible light can be
of two types. In the case of type I initiators, free radicals are
formed during homolytic photodissociation of the initial
molecule. Type II initiators include mainly the systems
consisting of a photoinitiator- co-initiator pair. In such
systems, the process of electron transfer or hydrogen atom
abstraction, leading to generation of corresponding radicals
or radical- ions, occurs under the action of light. Photo-
polymerization can also proceed under NIR light, but in
this case, the two-photon initiating systems or systems
based on upconversion nanoparticles, in which resonant
energy transfer occurs, are applied.
3.2.1. Type I photoinitiators
When absorbing light, photoinitiator molecules of type I
transfer into an excited singlet or triplet state, which is
accompanied by the homolytic bond cleavage. This photo-
chemical decomposition generates free radicals, mainly by
the Norrish type 1 reaction, which can initiate radical
polymerization or crosslinking.
79
Any weak bond can be
cleaved, but initiators that break up at the C7C bond
adjacent to the carbonyl group are most commonly used
(a-splitting) (Fig. 5). To activate such a decay, UV radia-
tion is required (Table 1). The first photocurable materials
were obtained using benzophenone as an initiator excited
with UV-vis light (253 nm).
80
The main limitations of the
first photoinitiators were associated with their hydropho-
bicity and, accordingly, poor solubility in an aqueous
medium, as well as the necessity to use high-energy UV
radiation, which can damage cells. The development of
methods for the synthesis of water-soluble photoinitiators
guided the growing interest in biomedical applications of
photopolymerization, which is reflected in the increase in
the number of scientific publications in this field.
7
One of the main ways to increase the solubility of
traditional radical photoinitiators is their chemical modifi-
cation by introducing appropriate groups. They include
nonionic groups of ethers, polyesters, hydroxyethers;
91
ionic groups of quaternary ammonium salts, sulfonates,
carboxylic acids, and thiosulfates.
92
For example, the
hydroxyethoxy group imparts hydrophilic properties to the
initiator Irgacure 2959, which has been widely used in the
last two decades for producing hydrogel TECs, and con-
tributes to an increase in solubility in water [0.7 wt.
(vol.)%]. Another advantage of using this compound is the
absence of a cytotoxic photoreaction byproduct such as
benzaldehyde.
81, 93
Photoinitiator Irgacure 2959 absorbs in the UV spec-
trum (200 ± 370 nm) and dissociates with the formation of
benzoyl and ketyl radicals. Unsaturated double bonds of
monomers (oligomers, macromonomers) predominantly
react with benzoyl radicals, which initiate radical chain
polymerization or thiol-ene polycondensation.
94
However,
light in the short-wavelength UV spectrum (<300 nm) is
phototoxic and mutagenic, which is unacceptable for
experiments with cells. Therefore, to excite Irgacure 2959,
a source with a wavelength of 365 nm, close to the visible
light spectrum, is usually used, which reduces the efficiency
of the reaction, since the molar extinction coefficient of
Irgacure 2959 at this wavelength is rather low (only
4molL
71
cm
71
).
95
This makes it necessary to increase
the irradiation intensity, exposure time and photoinitiator
concentration during photoreaction.
At present, along with the modification of known
initiators aimed at an increase in their solubility in water,
the task is to expand the absorption range to create more
efficient initiating systems.
96
Photoinitiators have been
obtained, which include monoacylphosphine oxides and
bis(acyl)phosphine oxides, absorbing in the wavelength
range of 380 ±450 nm, and almost insoluble in water. One
of the common photoinitiators of this series Ð
diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (Daro-
cure TPO) Ð has an absorption band in the range of
350 ± 380 nm. However, even the energy of visible light
(wavelength of up to 420 nm) is sufficient for the formation
of radicals from such an initiator capable of inducing
radical polymerization (see Table 1). In addition, Darocure
TPO is uncolored and can be used to produce optically
transparent 3D objects with high mechanical strength,
which compares favorably with Irgacure 2959 mentioned
above.
84
To obtain water-soluble initiators, TPO derivatives
were synthesized, among which lithium phenyl(2,4,6-trime-
thylbenzoyl)phosphinate (LAP) was widespread. This ini-
tiator is highly soluble in water (8.5 wt.%) and has a high
molar extinction coefficient (e=218Lmole
71
cm
71
)ata
wavelength of 365 nm. The high efficiency of photoinitia-
tion contributes to rapid formation of scaffolds on its
basis.
97
This compound also weakly absorbs in the blue
spectrum (l=405 nm,e=25 L mole
71
cm
71
). Thus, LAP
is used to obtain 3D objects upon excitation with both UV
and visible light in the process of radical chain polymer-
ization and polycondensation.
11
3.2.2. Type II photoinitiators
In the case of type II photoinitiators, as a rule, a more
complex mechanism of initiation is implemented. The
energy of photons in the visible spectrum, which is often
used to activate such photoinitiators, is usually less than the
dissociation energy of individual bonds in organic com-
pounds. This significantly limits the choice of highly effec-
tive initiators operating in the visible range, and it is
necessary to create two- or multi-component initiating
systems containing an initiator and co-initiator. The activa-
tion of type II initiators proceeds slower and less efficiently
in comparison with initiators of type I, which is associated
with the presence of competitive reactions involving the
monomer, co-initiator, and atmospheric oxygen.
To date, multicomponent photoinitiation systems
based on electron transfer and hydrogen atom abstraction,
as well as systems that include both processes, have been
developed to obtain the corresponding radicals or radical
ions that initiate photopolymerization.
17
Electron transfer
occurs upon interaction of an excited electron donor or
C
O
R
2
R
3
R
1
hn
O
+C R
2
R
3
R
1
Figure 5. Type I initiator disintegration.
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
Russ. Chem. Rev., 2023, 92 (2) RCR5068 9of26
acceptor (in the triplet state) with the second component
(electron acceptor or donor, respectively) in the ground
state (Fig. 6). In some cases, it is possible to quench the
triplet state of the photoinitiator by the monomer, but the
main way of radical formation proceeds through interac-
tion with co-initiators.
Most often, amines are used as co-initiators due to
their reducing properties. It is believed that the mecha-
nism of such a reaction is based on interaction of amines
with the photoinitiator in the excited state due to electron
transfer with the formation of an intermediate ion pair (or
exciplex) in the form of the photoinitiator radical anion
Table 1. Examples of photoinitiators for 3D photopolymerization.
Title Structural formula Wavelength,
a
nm Photopolymerization mechanisms Ref.
Type I initiators
Benzophenone 253 Radical chain, thiol-ene reactions 17, 80
2-Hydroxy-1-[4-(2-hydr- 274 (365) Radical chain, thiole-ene reactions 81
oxyethoxy)phenyl]-
2-methylpropan-1-one
(Irgacure 2959)
2-Benzyl -2-(dimethylamino)- 365 (420) Radical chain, thiol-ene reactions 82
1-(4-morpholinophenyl)- 800 Two-photon polymerization 83
butan-1-one (Irgacure 369)
(Diphenylphosphoryl)- 350 (420) Radical chain 84
(mesityl)methanone
(Darocure TPO)
Lithium phenyl (2,4,6-tri- 375 Radical chain, thiol-ene reactions 11
methylbenzoyl)phosphinate
(LAP)
Type II initiators
Camphorquinone 475 Radical chain, thiol-ene reactions 85
Eosin Y 525 Radical chain, thiol-ene reactions 86
Two-photon polymerization 15
Riboflavin 450, 475 Radical chain 82, 87,
88
Zinc meso-tetraphenyl 420 Controlled radical polymerization 89
porphyrin (ZnTPP)
Two-photon initiators
2,5-Bis[4-(diethylamino)- 750 ± 800 Two-photon polymerization 90
benzylidene]cyclopentan-
1-one (BDEA)
a
The absorption maximum is indicated; the working wavelength, which is used to activate photoinitiators, is given in parentheses.
O
O
HO
O
HO
O
N
OEt
N
O
P
O
P
OOLi
O
O
O
Br
HO
Br
O
Br
OH
Br
O
O
N
NN
NH
O
O
OH
OH
OH
HO
NN
NN
Ph
Ph
Ph
Ph
Zn
Et
2
N
O
NEt
2
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
10 of 26 Russ. Chem. Rev., 2023, 92 (2) RCR5068
and the amine radical cation.
98
Then, proton transfer
occurs with the formation of an active radical from an
amine-based co-initiator and a photoinitiator ketyl radi-
cal, as it is shown in the example in Fig. 6. The ketyl
radical is inactive with respect to the double bond and is
deactivated by recombination with another radical or by a
disproportionation reaction. As a result, the process of
radical chain polymerization is initiated by co-initiator
(amine) radicals.
Other types of co-initiators are also described in the
literature. For example, thiols, which are involved in photo-
induced electron transfer followed by proton transfer and
the formation of free S-centered radicals capable of attach-
ing to double bonds and initiating radical polymerization
(see Fig. 6), have found wide application (see Fig. 6).
99
S-radicals are very active, insensitive to the inhibitory
action of oxygen, and can attach to both vinyl and allyl
groups. An important role of thiols is associated with their
participation in photoinduced radical thiol-ene polyconden-
sation reactions used to form hydrogels.
Phosphorus-containing compounds can also act as co-
initiators in photopolymerization, acting as an electron or
hydrogen atom donor. In particular, Laleve
Âeet al.
100
showed that an excited photoinitiator can cause the abstrac-
tion of a hydrogen atom from a phosphorus-containing
compound with a labile hydrogen7phosphorus bond (see
Fig. 6). Such a path of the reaction without an electron
transfer stage was proved via the spectral method by the
absence of an intermediate ion pair.
One of the most widely used type II photoinitiators is
bornane-2,3-dione, called camphorquinone (see Table 1).
This compound is usually part of an initiating system with
tertiary amine such as ethyl 4-(dimethylamino)benzoate
when excited with visible light. Camphorquinone has been
successfully used to prepare hydrogels with encapsulated
viable cells,
85
but its low solubility and reactivity in water,
as well as the yellow color of polymerization products, limit
its application in most 3D printing technologies.
Dyes are of great interest as photoinitiators for visible
light-driven polymerization. Such compounds are usually
highly soluble in water and can participate in electron
transfer reactions from an excited state molecule to a
suitable co-initiator capable of triggering the photopolyme-
rization process as a result of activation (see monograph,
101
p. 48). Dyes in the excited state act either as reducing or
oxidizing agents, and their reaction with co-initiators pro-
ceeds through redox stages due to electron transfer. Most
often, eosin Y, methylene blue, rose bengal, erythrosin are
used in photopolymerization.
15
For instance, eosin Y,
which was first used to initiate photopolymerization, has
e=60803 L mole
71
cm
71
at the wavelength of 539 nm and
it rapidly transforms into the triplet state when irradiated.
When interacting with an amine-based co-initiator, for
example, with triethanolamine (TEA), a hydrogen atom is
abstracted and the deprotonated TEA radical initiates the
chain and thiol-ene radical reactions. However, these proc-
esses are rather slow and require additional accelerators
such as N-vinylpyrrolidone or N-vinylcaprolactam. The
main disadvantage of eosin Y is the need to use a large
number of components in the initiating system, which
hampers optimization of the process.
86
The possibility of
using eosin Y without participation of a co-initiator was
demonstrated
102
using a successful example of a photo-
induced crosslinking reaction of hyaluronic acid containing
tyramine units by the redox mechanism with formation of
dityramine bridges (see Section 2.3).
Some works have demonstrated the great potential of
riboflavin (vitamin B2) as a type II initiator. This water-
soluble biocompatible compound does not exhibit a cyto-
toxic effect and has a broad absorption spectrum with four
maxima at 223, 267, 373, and 444 nm, which makes it
particularly attractive as an alternative to synthetic initia-
tors.
82, 87
To initiate radical polymerization by the redox
mechanism, riboflavin requires the presence of a co-initiator
as an electron donor. Various riboflavin-based initiating
systems have been developed to prepare methacrylate
hydrogels involving triethanolamine
87, 103
and
L
-arginine.
88
The degree of formation of riboflavin radicals and, con-
sequently, the reaction rate depend strongly on pH of a
photocomposition. In addition, it has been proven that after
irradiation with UV or visible light in the presence of
oxygen, riboflavin produces such reactive oxygen species
as hydroxyl radical, peroxide radical anion, singlet oxygen,
etc. This effect can significantly accelerate initiation of
photopolymerization (see Section 2.3).
35
The photochemical properties of metal complexes, in
particular, strong absorption in the visible range, relatively
Electron transfer
O
3
*
hn
O
3
*
7
N
H
+
HOH
OH
Ketyl radical
OHHO
recom-
bination
O
3
*
R7SH
hn
O
37
HS7R
N
H
*
+
N
+
proton transfer
MP
chain
growth
proton transfer
S7R
+MP
chain
growth
OH
Hydrogen atom abstraction
P O
Hhn
I7H+R
2
7P=
OI+R
2
dispropor-
tionation
Figure 6. Schemes of initiation with type II photoinitiator (ben-
zophenone) via electron transfer (with amine and thiol as co-
initiator) or hydrogen atom abstraction.
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
Russ. Chem. Rev., 2023, 92 (2) RCR5068 11 of 26
long-living excited states, and suitable redox potentials
determine the possibility of participating these compounds
in redox processes (see Table 1). First of all, they can be
used in controlled radical chain transfer polymerization by
the addition-fragmentation mechanism (AFTP, RAFT)
(see Section 2.4). For example, such processes actively
employ an initiating system based on complexes of
tris(2,2
0
-bipyridine) dichlororuthenium(
II
)(Ru)hexa-
hydrate (e=14600 L mole
71
cm
71
at 450 nm). Irradia-
tion with visible light excites the ground state of Ru
2+
ion,
whichisoxidizedtoRu
3+
by transferring electrons to a co-
initiator such as sodium persulfate. After accepting elec-
trons, persulfate dissociates into sulfate anions and sulfate
radicals capable of triggering radical chain polymerization
or thiol-ene polycondensation.
104
The zinc meso-tetraphe-
nylporphyrin (ZnTPP) photosensitizer, used in photody-
namic therapy of cancerous tumors, also seems interesting
(see Table 1).
89
An effective system for initiating radical
chain polymerization of hydroxyethyl methacrylate upon
irradiation with light at a wavelength of 420 nm was
obtained through combination with diphenyliodonium
complexes.
105
Despite the great potential, the use of these
systems is limited due to the presence of a metal in their
composition, which may have a toxic effect, and low
storage stability.
3.2.3. Initiating systems activated by near-IR light
In comparison with UV and visible light, NIR radiation
does not lead to strong photodamage of the material, has
low scattering in tissues and a large penetration depth. One
of the possible ways of 3D photopolymerization under the
NIR light action is based on the use of two-photon
polymerization (2PP). This process is similar to traditional
single photon polymerization based on absorption of a
single photon. However, in the case of 2PP, high-intensity
laser radiation is used to trigger nonlinear absorption of
two photons from the NIR spectrum (*800 nm) which
leads to the molecule transition through a virtual energy
level into an excited state.
106
The energy difference between
the ground and excited states is equal to the sum of energies
of two photons (Fig. 7 a,b). Since in this case the energy of
molecule transition to an excited state is in a quadratic
dependence on the incident light intensity, 2PP allows
creation of finely tuned objects at the micro- and nanolevels
with a resolution of <100 nm in the depth of photo-
composition at a relatively high printing speed.
108
As a
rule, femtosecond laser radiation is used to implement the
2PP process. It should be noted that the main technological
development for biomedical applications of the 2PP method
was owing to the efforts of a scientific team led by
Prof. B.N.Chichkov, first at the Laser Center Hannover
(Germany), and then at Leibniz University Hannover
(Germany).
The use of low-energy photons, which are safe for cells,
determines the possibility of 3D structure formation in the
presence of living cells as well.
109
Initiators of two-photon
polymerization should have low absorption at the working
wavelength and also contain conjugated systems of p-elec-
trons and groups with strong donor-acceptor properties in
the structure (see Fig. 7c). After photon absorption, an
electron in the initiator is probably transferred from the
donor-acceptor group to the p-system. The transfer of an
electron from the initiator to the monomer generates
exciplex (excited state) and leads to the formation of
radicals that initiate polymerization.
110
Two-photon polymerization uses type I initiators capa-
ble of homolytic decomposition upon irradiation, such as
LAP. However, such compounds are characterized by
relatively low p-conjugation and weak two-photon absorp-
tion that determines insufficient efficacy in the processes
with excitation by IR-radiation.
111
Chichkov et al.
83
reported the use of Irgacure 369 as an initiator for two-
photon polymerization. However, the small absorption
cross-section in the IR spectrum and the absorption max-
imum at a wavelength of 369 nm were the reason for
insufficient efficiency of this photoinitiator irradiated by a
laser source with wavelengths of 750 ± 800 nm.
112
Some well-known type II initiators, such as eosin Y,
erythrosin, and rose bengal, in combination with amines,
have been successfully used as two-photon polymerization
initiators.
15
However, these systems have the long process
time and the high dose of irradiation, therefore, the task
was to create initiators that ensure fast photopolymeriza-
tion at low intensity of laser radiation. Specially synthesized
water-soluble derivatives of chromophores, for example,
1,4-bis(4-{N,N-bis[6-(trimethylammonio)hexyl]amino}styryl)-
2,5-dimethoxybenzene (WSPI) tetraiodide
113
and (2,5-
bis[4-(diethylamino)benzylidene]cyclopentan-1-on (BDEA)
showed high efficiency in two-photon polymerization (see
Table 1).
90
Chesnokov and co-workers
114
demonstrated a
relatively high polymerization rate at low NIR excitation
power for imidazole-containing compounds with phenan-
throline and phenanthrene fragments, used directly as two-
a
Excited
state
Ground
state
Fluorescence
Fluorescence
UV
UV IR
IR
hn
1
hn
2
hn
1
One-photon Two-photon
Ink
b
DpD2hn[D p*D] [D pD]
7+
M
[M ]
7
[D pD]
+
c
Laser beam
405 nm
Focal plane
975 nm
Figure 7. Energy diagrams of initiator excitation: schematic (a)
and fluorescence (b) images of beam focusing in ink for one-photon
(left) and two-photon polymerizations (right),
107
as well as the
mechanism of two-photon polymerization initiation (c).
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
12 of 26 Russ. Chem. Rev., 2023, 92 (2) RCR5068
photon polymerization initiators and in combination with
amines.
Along with the great advantages of NIR radiation and
the fabrication of high-resolution products, one of the
significant limitations of 2PP is expensive equipment,
namely femtosecond laser systems, which somewhat hinders
the development of this technology.
Hybrid organo-inorganic structures are considered to be
promising initiating systems excited by IR radiation. The
mechanism of such structure action is based on the resonant
energy transfer between the components. Successful exam-
ples of using upconversion nanoparticles (UCNPs) as inor-
ganic components are presented in the literature. In these
nanoparticles, low-energy NIR radiation (975 nm) is con-
verted into UV (360 nm), visible (450, 475 and 650 nm) and
NIR (800 nm) light with higher energy through real energy
states of trivalent ions of rare earth elements.
115
UCNPs can
be excited by CW radiation from cheap semiconductor
lasers with a relatively low intensity (several orders of
magnitude lower than in the case of a two-photon process).
Together with UCNPs, traditional photoinitiating systems
can be exploited to create 3D structures. This process occurs
only if the UCNP emission spectrum overlaps with the
absorption spectrum of the photoinitiator.
For example, UCNPs doped with Tm
3+
ions were used
as a source of UV light when irradiated by a laser with a
wavelength of 980 nm. Overlapping of the UCNP emission
band (360 nm) with the absorption band of the photo-
initiator Irgacure-819 was the main condition for the
initiator activation and the generation of radicals during
polymerization. This hybrid initiator system allowed for-
mation of 3D structures from an optically transparent
polyethylene glycol diacrylate oligomer.
116
In addition, the
structures of UCNPs with the Irgacur 369 were employed
for crosslinking reaction of methacrylated oligocarbonate,
as well as the structures of UCNPs with the Darocure TPO
photoinitiator were employed for photocuring of the com-
mercially available E-shell 300 resin.
117
4. 3D printing for tissue-engineered constructs
Additive technologies, known as 3D printing, have been
used since the 1980s to produce specific complex objects
without using mechanical processing or special molds.
118
The application field of such processes is constantly expand-
ing, covering a variety of areas, including biology and
medicine.
119
In particular, 3D printing technology, which
includes both rational design and production of TECs,
plays a leading role in tissue engineering.
120
A special
position is occupied by 3D printing, based on the polymer-
ization or light-driven ink crosslinking. During irradiation,
curing occurs, which allows easy and quick separation of
the object from the original liquid composition. In this
review, when describing technologies, the term photocuring
will be used, which implies the crosslinking or polymer-
ization. Photoinduced processes are characterized by a high
spatiotemporal resolution, they proceed with a minimum
amount of byproducts, and allow creation of the required
three-dimensional structures from monomers (oligomers,
macromonomers), cells, and biologically active molecules.
Great opportunities of 3D printing are associated with the
formation of multifunctional 3D objects of various archi-
tectures with controlled chemical, optical, and mechanical
properties.
12
Usually the process of 3D printing includes three stages:
1) design of 3D-structures by means of computer simu-
lation;
2) presentation of 3D-structures in the form of layers;
3) sequential printing of layers resulting in the 3D
structure formation.
As it was noted above, two main approaches are used to
obtain 3D TEC models (Fig. 8):
Ð printing by extrusion, based on a photoreaction
activated by light before, after or immediately upon photo-
composition exit from the extruder;
Ð printing based on lithography, when a photoreaction
occurs in a layer of a photocomposition irradiated with
light.
These methods provide structurally organized func-
tional TECs for various biomedical purposes.
12
4.1. Extrusiuon-based 3D printing
The principle of extrusion printing consists in pressing ink
through the nozzle and forming the layers that make up the
product on the platform using a computer created design.
The deposition of each subsequent layer of ink occurs by
moving either the extruder above the platform or the plat-
form below the extruder. As a result, three-dimensional
objects are created by continuous layer-by-layer application
of material. In the case of 3D printing, ink after the extruder
is irradiated with light to form hydrogels. The object can be
illuminated in different positions between the nozzle and the
platform, starting from the nozzle outlet and ending with
irradiation directly on the platform (see Fig. 8 a). A typical
extrusion 3D photoprinter consists of a dosing head, which
can move along two axes and a platform where photocured
before after during
Dosing
head
Movement
Ink
Ink
Substrate Object
Dead zone O
2
Ink: cured
SLA DLP CLIP
uncured
z
z
zz
y
y
Scanning
laser system
x
x
Photocuring
Extrusion
Lithography
a
b
DLP-projector
Figure 8. Schematic representation of extrusion-based 3D print-
ing technologies involving inkcuring before, after and during
extrusion (a), and lithography (b). SLA is stereolithography, DLP
is digital light projection, CLIP is continuous liquid interface
production. The figure is created by the authors.
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
Russ. Chem. Rev., 2023, 92 (2) RCR5068 13 of 26
ink is deposited.
121
The ink is supplied mechanically (by
piston) or pneumatically (by air pressure) through the
nozzle with small holes (dies) (see Fig. 8 a).
122
Successful extrusion 3D printing is grounded on the ink
requirements, specific for the technology, which determine
the possibility of given composition extrusion and its high-
precision deposition to reproduce the desired 3D structure.
First of all, the rheological properties of ink should allow it
to pass through the printhead, usually having a nozzle
diameter of 100 ± 800 mm, which is optimal for dosing the
material. Constriction of the nozzle and corresponding high
shear stresses that occur in the nozzle are the reasons for the
preferred use of ink with low viscosity for layer-by-layer
deposition. However, finished scaffolds must maintain
integrity, shape and structure under the action of external
forces. Ink consiting of high-viscose materials or with high-
polymer content can be used to produce structures with
high resolution and precision, and the balance of these
properties must be maintained in such a way that the
viability and function of the cells included in the material
are not reduced.
123
Various approaches to scaffold forma-
tion are developed that meet the structural requirements
along with presevation of the biological activity of cells. In
particular, these approaches include layer-by-layer deposi-
tion and total irradiations of the entire structure,
124
ink
crosslinking prior to extrusion,
125
the use of additives that
give the products the properties of a non-Newtonian liquid
(with low viscosity in the dosing head and with high
viscosity when leaving the nozzle),
126
ink deposition into
supporting moulds,
127
etc.
One of the promising approaches to extrusion 3D
printing is photocuring of the composition during extrusion
(in situ)(seeFig.8a). In contrast to processes involving
crosslinking reactions before or after extrusion, in the case
of in situ, the formation of threads occurs after ink irradi-
ation in a photopermeable capillary immediately after leav-
ing the nozzle. This allows the use of a non-viscous ink
without any additives or additional processing steps. More-
over, a homogeneous photocomposition is maintained,
which determines the constant pressure in the dosing device
and production of polymer structures of a given geometry at
a high level of viability of encapsulated cells.
128, 129
The technology of multi-jet 3D printing (MJP), which
uses a printhead with an array of nozzles (from 96 to 448
pieces) is of great interest. This technology allows deposi-
tion of materials of various colors, hardness, strength, etc.
on the substrate with a high resolution, providing a print
layer thickness of 16 mm. However, MJP printers are
expensive, as is the ink, which must have a low viscosity to
be used in this technology.
130
Based on extrusion, including multi-jet printing, the
concept of 4D printing has been developed. This concept
was first presented in 2013 using the example of creating
products of complex configuration due to the shape mem-
ory effect.
131
The main feature of 4D printing is the ability
to produce 3D objects that can change their shape, proper-
ties, or function over time in response to the action of such
external stimuli such as temperature, light, water, etc.
132
In
addition, this technology allows significant time and mate-
rial saving when forming thin-walled or network struc-
tures,
133, 134
and also printing single- and multicomponent
hydrogels.
135
A change in the shape of constructs is based
either on the use of smart materials or on the creation of a
localized self-deformation inside the printed object during
or after printing.
136
4D printing also provides maturation of
cell population in time when they are co-printed with
hydrogels.
137
The main limitations of extrusion-based 4D
printing are slow printing speed and relatively low resolu-
tion.
Currently, extrusion-based 3D printing is actively used
to create TECs in a wide range of sizes: from small cellular
hydrogel objects to complex anatomical structures, which
cannot be achieved by other methods.
138
However, scaling
affects the printing speed and leads to a relatively low
resolution during production (*100 ± 200 mm).
139, 140
The
last disadvantage can be eliminated by structural changes in
the printer, for example, by reducing the nozzle diameter,
although in some cases this may be accompanied by a lower
rate of cell survival and clogging of hole in the nozzle.
141
Together with possibility of obtaining TECs of various
sizes, the advantages of this method are the simplicity and
availability of inexpensive commercial equipment, a wide
range of suitable ink, including those with high viscosity,
the possibility of incorporating cells at a significant concen-
tration comparable to those for natural tissues, as well as
minimal ink losses in the process of production.
12
To date,
various types of tissues have been produced by extrusion 3D
printing: bone,
142
cartilage,
143
cardiac,
144
and nervous tis-
sues.
145
4.2. Lithography-based 3D printing
Currently, there are a large number of 3D printing tech-
nologies that are based on the process of lithography:
formation of two-dimensional objects on the ink surface.
One of the first developed methods, actively and widely
used both in biology and medicine, and in various indus-
tries, is stereolithography (SLA). This method was
patented
146
in 1984 by Charles Hull, a co-founder of 3D
Systems, a leader in 3D printing. In Russia, key competen-
cies in the field of stereolithography were developed at the
Institute of Problems of Laser and Information Technolo-
gies of the Russian Academy of Sciences under the guidance
of Academician V.Ya.Panchenko.
The general principle of SLA is to scan the ink surface
with a laser beam from a source (usually at a wavelength of
355 nm) located above (below) the tank. As a result of
irradiation, the composition is photocured on the platform
in the form of a layer, which is a cross-section of a 3D
contruct. Then the platform is lowered (raised) by the
thickness of the next layer and surface scanning with a
laserbeamisrepeated(seeFig.8b). The process continues
until a cured 3D construct corresponding to a computer
model is obtained.
147
This technology allows resolution
from 50 to 250 mm. The formation of each layer is con-
trolled by the laser beam movement, which can occur over a
large surface area and print constructs of a significant size
(upto50650 cm) with little or no loss in performance
precision.
Based on SLA, a masked stereolithography printing
technology was developed; it consists in applying thin layers
of ink according to a physical pattern or mask of the
corresponding contour, followed by material irradiation
with UV light. Irradiation causes material curing and
removal of non-irradiated liquid composition from the
work area. Recently, this method has been replaced by
film transfer imaging (FTI) technology, which, thanks to
introduction of digital projectors, has become almost anal-
ogous to digital image projection (DLP),
148
presented
below. Stereolithography is devoid of some disadvantages
of extrusion-based printing. For example, during the photo-
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
14 of 26 Russ. Chem. Rev., 2023, 92 (2) RCR5068
curing process, no pressure is applied to the liquid compo-
sition leading to cell death. However, significant restrictions
of this method are the insufficient choice of cell-laden
ink,
149
and the fact that commercial SLA systems are
mainly intended for the production of solid polymeric
materials that do not meet the requirements of bio-
medicine.
150
Among modern 3D printing technologies based on
lithography, the DLP method and the liquid crystal
display method (LCD) should be distinguished. The main
difference between all lithographic technologies, including
SLA, is the light source and visualization system with
almost the same type of control system and layer-by-layer
printing of 3D structures. In DLP technology, a layer of ink
is cured with light from a DLP digital projector using a
DLP matrix (usually from the tank base). This allows
simultaneous irradiation of the entire polymer layer, creat-
ing objects according to the mask formed by the DLP
matrix, in contrast to SLA technology, where point scan-
ning occurs. This approach provides high resolution
(10 ± 50 m) at a higher printing speed compared to similar
SLA parameters.
151
In addition, the DLP method uses
visible light (405 nm) instead of UV radiation, incompatible
with semiconductor materials of a DLP projector, which is
especially important in the case of photocuring of
compositions containing ce lls (see Fig. 8 b).
152
The use of a
DLP matrix as a dynamic mask generator allowed the
development of the projection micro-stereolithography
method (PmSL), which was used to obtain complex 3D
structures 0.6 mm in size under UV irradiation with a high
resolution.
153
However, simultaneous illumination of the entire layer
is possible only for a limited area. At present, the size of the
resulting layers varies from 100660 to 1906120 mm.
Therefore, DLP 3D printing is mainly used to print small
objects with high precision.
The LCD printing technology is similar to DLP, but the
mask is formed by the LCD matrix, which transmits the
LED light only to the areas that need to be cured. The
advantages of LCD printing are low cost and good reso-
lution, but the light intensity is very low since only 10% of
light can come from the LCD screen.
16
Despite the great promise of the presented 3D lithog-
raphy methods, there are problems that require the
improvement of modern technologies. For example, the
printing quality is reduced by the stair-stepping effect,
since only flat layers with sharp edges can be produced.
154
A solution to this problem has been presented in the
literature in the form of a technology called computed
axial lithography (CAL). In this technology, radiation
affects the entire volume of the printed product at once, in
contrast to SLA technology, in which the effect occurs
pointwise, or DLP and LCD methods, in which a two-
dimensional layer is illuminated. Thanks to this feature, the
CAL technology leads to a multiple acceleration of the
printing process.
155
Lithography-based 3D printing is also limited by the
relatively low printing speed, caused by the low rate of
photopolymerization and process discreteness (printing
does not occur when the platform is moved). The new
technology of continuous liquid interface production
(CLIP) (see Fig. 8 b), based on continuous printing in a
liquid medium, was developed in 2015 by Carbon 3D
Corp.
156
This technology allows printing of more uniform
structures 25 ± 100 times faster than the conventional 3D
printing methods. The innovation of the method lies in the
presence of a dead zone: a thin layer of uncured ink between
the formed construct and the tank bottom. This zone is
located above the base of the tank made of a specially
designed membrane that transmits radiation from the
source and oxygen, supplied from below. Projection irradi-
ation by the DLP technology activates photocuring, and
oxygen inhibits radical reactions, stably maintaining the
presence of a liquid ink layer. This dead zone, whose size
can be adjusted by the flow of oxygen, ensures the continu-
ity of printing, i.e. at the moment the photocured layer is
deposited on the 3D structure, the next layer of ink is
irradiated.
150
Despite the advantages of this method, its
efficiency can be fully achieved only for ink with low
viscosity, which determines the rapid supply of material to
the printing area, and when creating hollow structures that
do not require a large amount of material.
Holographic 3D printing, or interference lithography,
which uses two or more light sources to create an interfer-
ence pattern is of great interest. By superimposing light
waves, a pattern with a periodic structure is formed due to a
local change in the light intensity.
157
This technology is well
known in the field of creating nanostructured substrates,
microframes, 3D photonic crystals, and microsieves.
158
Holographic 3D printing is a fast and accurate method for
obtaining photocurable structures and, despite the limited
number of templates, it is suitable for producing porous
tissue contructs.
157
One of the advanced technologies of stereolithography,
distinguished by ultra-fast making of structures with sub-
micron 3D resolution (*100 nm), is based on the 2PP
process (see Section 3.2.3). In this technology, the initiating
system responsible for polymerization is activated by a
focused beam of a femtosecond NIR laser (*800 nm) due
to nonlinear absorption.
159
The main advantages of 2PP as compared to traditional
single-photon polymerization include the possibility of
producing three-dimensional images in the ink volume.
This is due to the fact that IR light cannot initiate single-
photon polymerization, while in the case of 2PP, initiation
is possible, but only in the region of laser beam waist
(voxel).
160
Thus, the laser beam can pass through the ink
without polymerizing the material on its way and induce
polymerization only in the focal volume. The strong non-
linear dependence of two-photon absorption on intensity
allows localization of the region of photoreactions in ink
near the voxel and unprecedented resolution of the method.
The possibility of producing arbitrary three-dimensional
structures is implemented by three-dimensional movement
of the voxel over the photocomposition volume (see page 10
in Ref. 161).
The simplest 2PP-based setup consists of a laser source,
focusing optics, a moving platform, and a 3D printing
control system. Nanoscale resolution can be achieved by
controlling the power density of the laser pulse and the
printing speed. Traditional lithographic materials are usu-
ally used as photopolymerizable components in 2PP ink,
but printing the products with nanoscale precision
(<100 nm) is a relatively time-consuming process, which
can hardly be implemented through other 3D printing
technologies.
107
Despite the numerous advantages of the 2PP-based
technology, optical systems for such polymerization are
currently still expensive. Products can be made only at
high intensity of laser radiation (mainly on the order of
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
Russ. Chem. Rev., 2023, 92 (2) RCR5068 15 of 26
TW cm
72
) and from a single material, which excludes
production of multifunctional structures.
120
In addition,
the height of the printed construct is limited by micro-
objective working distance used to focus the laser pulses in
the ink.
162
The development of lithography-based 3D printing is a
particularly promising avenue in the creation of artificial
bone grafts, since this technology has great prospects for
making products with a complex structure and high reso-
lution.
163
It should be noted that this technology provides
the possibility to obtain not only porous structures, filled
with cells or bioactive components, but also constructs
simulating, for example, hollow vessels,
164
which are diffi-
cult to make using extrusion-based printing.
5. Biomedical applications
Successful application of photoinduced 3D printing tech-
nology in regenerative medicine for obtaining tissue-engi-
neered constructs requires consideration of the
requirements for cytotoxicity and immunogenicity of mate-
rials, as well as the correspondence of mechanical and
chemical properties of the tissue in which scaffolds of this
type are supposed to be used. Since the concept of photo-
induced 3D printing implies the maximum convergence of
photopolymerization process and cellular technologies, the
most interesting are investigations with polymerization in
the tissues of the body (in situ photopolymerization). In this
case, the cells are introduced into the scaffold at the time of
preparing photocomposition for a tissue bioequivalent and
their subsequent autologous transplantation as part of such
bioequivalents.
5.1. Cytotoxicity assessment
Cytotoxicity of materials and methods used is one of the
most important factors that must be taken into account for
the successful application of 3D printing technology. And if
the scaffold material can be chosen in advance by selecting
the most biocompatible materials for this purpose (see
Section 3.1), then the toxic effect of the technology itself is
much more difficult to assess (see p. 250 in monograph
165
).
As it can be seen from the data given in Table 1, a number
of photoinitiators are excited by UV light, which itself can
induce photooxidative stress in the cell. The use of UV
radiation is not a fundamental limitation for photopolyme-
rization in living systems: for example, earlier LAP and
Irgacure 2959 initiators were used to photopolymerize
primary human chondrocytes in scaffolds based on meth-
acrylated gelatin irradiated with 20-W LEDs (l=405 and
365 nm, respectively).
166
Cells retained their viability for
28 days, but the authors noted a decrease in cell viability by
this time in all samples. An important problem in the use of
initiators in any available spectrum range is the formation
of radicals necessary to start photopolymerization proc-
esses. The generated radicals can seriously damage the
structure of cellular biomacromolecules, and this principle
underlies photodynamic therapy.
167
Sometimes, to protect
cells, antioxidants, such as ascorbic acid
168
or hydrogen
sulfide, are added to the photocomposition immediately
before polymerization.
169
This approach allows an increase
in the survival rate of encapsulated cells, but, in turn, it
slows down the polymerization process.
168
In most cases,
researchers make up with a certain number of cells that die
during photopolymerization by choosing such cultures and
photoinitiators to minimize this effect. In particular, chon-
drocytes,
170
fibroblasts,
171
mesenchymal stem cells,
172
and
keratinocytes
173
(i.e. all the cell types most in demand in
tissue engineering) tolerate photopolymerization well.
It should be noted that the inclusion of cells in the
photocomposition can also affect the process of photo-
crosslinking and lead to formation of inhomogeneities
within the gel. It was shown that encapsulation of chon-
drocytes in a PEG-based composition followed by a cross-
linking reaction during irradiation with a UV lamp
[l= 352 nm, excitation intensity (I
ex
)=5 mW cm
72
]
caused a decrease in the bulk and local density of hydrogel,
and thiol-mediated interactions between dithiol crosslinkers
and free thiol groups on the cell surface played an important
role.
174
This effect was more pronounced with increasing
cell density during encapsulation. Encapsulation of chon-
drocytes in fluorescently labeled hydrogels resulted in for-
mation of hydrogel with a density gradient around the cell,
which was eliminated by treating the cells with estradiol
antioxidant prior to encapsulation. During hydrolytic deg-
radation of PEG hydrogel in the presence of cells, spatial
density variations were also observed.
174
The photopolymerization technology can also be used to
encapsulate multipotent cells, which are extremely sensitive
to environmental conditions. Thus, the composition on the
basis of methacrylated glycol-chitosan and type I collagen
had excellent biocompatibility at the example of reprog-
rammed multipotent cells. At that, the spread of cells and
the formation of structures similar to muscle fibers occurred
without undesirable osteogenic or chondrogenic differentia-
tion, which was confirmed by gene expression analysis.
175
Despite the fact that photoinitiators (Irgacure 2959,
riboflavin) excited by UV- and blue-visible light were used
in almost all the works mentioned above; cell survival was
>90%. The transition to photoinitiators activated by
visible light in the green and red spectra allows both an
increase in cell survival due to lower irradiation energy and
light penetration to the depth of the photocomposition
(biological tissue), which is beneficial for the photopolyme-
rization process as a whole. Eosin Y, which has good
solubility in water and low toxicity, often acts as such an
initiator.
176
Structures obtained with its help under LED
irradiation (l= 525 nm, I
ex
=5±100 mW cm
72
) can sup-
port the functioning of mesenchymal stem cells while main-
taining their ability to proliferate and differentiate.
177
However, it should be taken into account that an increase
in the concentration of eosin Y in the photocomposition
negatively affects cell viability and mechanical properties of
hydrogel, its swelling coefficient, and porosity; i.e. cell
adhesion. Thus, an increase in the concentration of eosin
Y from 0.01 to 0.04 mole L
71
in a methacrylated gelatin-
based photocomposition (GelMA) using the DLP technol-
ogy (laser with l= 405 nm and I
ex
=48.6 mW cm
72
)
resulted in a decrease in the viability of mouse fibroblast
cells NIH-3T3 from 91.5 to 75.6%, while the Young s
modulus of compression increased from 4.40 to 14 kPa,
and cell adhesion was maximum at eosin Y concentration in
the composition equal to 0.04 mole L
71
(see Ref. 178).
A further development of this concept was the use of
two-photon initiating systems (see Section 3.2.3), which,
under the action of pulsed femtosecond radiation at a
wavelength of 800 nm (80 fs at 75 MHz, power of
330 mW), allow printing of tissue bioequivalents with high
cell viability.
179
However, in terms of abundance, two-
photon systems are still inferior to photoinitiators in the
UV and visible spectra. It should be noted that the require-
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
16 of 26 Russ. Chem. Rev., 2023, 92 (2) RCR5068
ment for low phototoxicity of initiators becomes much more
stringent when turning to in situ photopolymerization
technology, i.e. directly in a living organism, as there is a
risk of damage and mutation of cells of a person own
tissues. Therefore, to implement this approach, the use of
NIR radiation, deeply penetrating and neutral for cell,
seems to be the most promising due to the technology of
two-photon polymerization or participation of upconver-
sion nanoparticles that transform the NIR radiation into
visible and blue light.
180
The search for new potential photoinitiators can pro-
ceed not only by applying irradiation from the transparency
window of biological tissues, but also through the use of
various nanocompositions that can perform several func-
tions at once: photoinitiation and visualization or photo-
initiation and drug delivery. For example, carbon
nanotubes containing the anti-inflammatory drug formono-
netin, which is intended for the treatment of spinal cord
injuries, have been proposed as such a nanocomplex. Nano-
composites were introduced into GelMA and irradiated
with UV light (l=365 nm,I
ex
= 6.7 mW cm
72
) to activate
photopolymerization, while a short irradiation time (up to a
minute) allowed the process to proceed without reducing
cell viability (survival rate of > 80%).
181
It should be noted that cytotoxicity is the most impor-
tant and easily detectable biocompatibility factor, but far
from being the only one. Thus, adhesion of cells to a
polymer is also important for the successful use of 3D
printing technology, but the goals of increasing cell adhe-
sion can be different. For example, in the case of a
nanocomposite hydrogel containing GelMA, nanohydro-
xyapatite (nHAP), quaternized chitosan (QCS), and nano-
particles of functionalized polyhedral oligomeric
silsesquioxane (POSS), the goal was to improve cell adhe-
sion to stimulate the regeneration of skull bones.
182
Irga-
cure 2959 and POSS nanoparticles irradiated by UV light
(l=365 nm, I
ex
=50 mW cm
72
) served as a photoinitiat-
ing system and, together with hydroxyapatite and QCS,
increased the mineralizing ability and cell adhesion. The
prepared nanocomposite hydrogels had high mechanical
properties and high cytocompatibility, including the ability
to osteodifferentiate.
To achieve the opposite effect, Wang et al.
183
prepared
ink based on PLA with monoethyl fumarate, N-vinylpyrro-
lidone, and Irgacure 2959 as a photoinitiator. UV irradi-
ation at an Ultralum crosslinking cabinet (l=365 nm,
I
ex
=50 mW cm
72
) led to formation of scaffolds with low
cell adhesion, illustrated in mouse fibroblast culture L929.
The resulting non-cytotoxic scaffolds can be used as anti-
adhesion barrier materials (for example, stents and any
tubular structures).
5.2. Obtaining tissue-specific constructs
An important advantage of using photoinduced 3D poly-
merization technology in regenerative medicine is the pos-
sibility of obtaining complex structures with a given
geometry and biomechanical properties. For example, a
strategy for 3D bioprinting of a trachea using photocros-
slinkable tissue-specific ink (Fig. 9) was recently pro-
posed.
184
One of the main difficulties in creating a trachea
bioequivalent is its heterogeneity: alternant cartilaginous
rings and vascularized rings of fibrous tissue; it is also
important to form the tracheal epithelium, which is neces-
sary for implementation of the mechanical and physiolog-
ical functions of the trachea. Since the trachea is a tubular
organ, the requirements for the mechanical properties of
ab
Trachea
Scaffold of cartilage rings
Scaffold between cartilage rings
Scaffold between cartilage rings
Traheal TEC
GelMA
CSMA
ACMMA
HAMA
8-PEG-NHS
ADMMA
1mm
1mm
1mm
Figure 9. Schematic representation of tracheal TEC obtained by ink photopolymerization (to make scaffolds of cartilage rings and fibrous
tissue) and amidation reaction (for interfacial binding) (a), and 3D images of cell distribution in tracheal TEC in three projections, made
using fluorescence microscopy: chondrocytes labeled with green fluorescent protein (GFP) are stained green; fibroblasts labeled with red
fluorescent protein (RFP) are stained red (b). GelMA is methacrylated gelatin, CSMA is methacrylated chondroitin sulfate, ACMMA is
methacryloyl-modified cell-free cartilage matrix, HAMA is methacrylated hyaluronic acid, 8-PEG-NHS is polyethylene glycol succinic acid
ester, ADMMA is methacryloyl-modified cell-free matrix of dermis. Adapted from paper.
184
ThefigureispublishedundertheCreative
Commons Attribution 4.0 International license (CC BY 4.0).
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
Russ. Chem. Rev., 2023, 92 (2) RCR5068 17 of 26
material are also high. The authors of the study
184
pro-
posed alternant layer-by-layer printing with two types of
ink: chondrocyte- and fibroblast-loaded ones. Photocros-
slinkable ink of the first type consisted of methacryloyl-
modified gelatin, chondroitin sulfate, and cartilage acellular
matrix and was intended to create analogs of cartilage rings.
The ink of the second type included methacrylate-modified
hyaluronic acid, 8-arm-polyethylene glycol-succinic acid
ester, and methacryloyl-modified derm acellular matrix,
and it was suitable for reproducing a vascularized annulus
fibrosus (see Fig. 9 a).
184
Polymerization under UV-LED
irradiation (l=365 nm, I
ex
=20 mW cm
72
) occurring in
both types of ink, and the amidation reaction for ring
integration provided fast crosslinking with the formation
of hydrogel networks, improved mechanical properties of
material, high cell adhesion (see Fig. 9b), as well as sat-
isfactory specific tissue regeneration.
A similar task was associated with restoration of the
intervertebral disc, a heterogeneous structure that consists
of nucleus pulposus, annulus fibrosus, and two cartilage
plates connecting adjacent vertebrae. To restore the inter-
vertebral disc, hydrogel of methacrylated gelatin and hya-
luronic acid, which allowed fixing the gelatin-like structure
and triggering differentiation of mesenchymal stem cells
characteristic of the nucleus pulposus, was obtained
under the action of UV irradiation (l=365 nm,
I
ex
= 7.0 mW cm
72
).
185
It is important to note that photo-
induced 3D polymerization for both cases presented above
enabled flexible control of both the scaffold composition
and structure and its mechanical properties, successfully
combining the photopolymerization process with cellular
technologies.
Currently, CLIP 3D printing technologies provide an
opportunity to create structures with a complex architecture
and required mechanical properties, including those infilled
with cells and utilizing growth factors in photocomposi-
tions.
186
Imaging agents can be additionally introduced into
the formed scaffolds for noninvasive monitoring of prod-
ucts after implantation. Ding et al.
187
made bioresorbable
stents based on poly(1,12-dodeca-methylenecitrate) metha-
crylate containing a radiopaque compound, iodixanol,
using a micro-CLIP printer (l= 365 nm). The latter
allowed stent detection in X-rays for at least 4 weeks after
their formation and implantation. In addition, it has been
found that the mechanical properties of stents can be
controlled by changing the concentration of iodixanol.
Another difficulty is associated with the restoration of
functional tissues, primarily the muscle ones.
188
It is shown
that low-density GelMA hydrogels can be rapidly (<1 min)
formed under the action of visible light
(I
ex
=203 mW cm
72
) and successfully used to encapsulate
pluripotent human stem cells while maintaining their high
viability. The initial stiffness of the resulting constructs is
220 Pa, even so they support cell growth and dynamic
remodeling of the microenvironment, and also promote
highly efficient (>70%) cell differentiation into cardiac
tissue to obtain spontaneously contracting tissue-engineered
constructs on the 8th day of differentiation.
189
Deshmukh et al.
190
proposed another method for creat-
ing muscle structures based on a combination of acousto-
fluidics and photopolymerization. For example, muscle
progenitor cells (myoblasts) were injected into gelMA-
based hydrogels (5.0 wt.% GelMA and 0.1 wt.% LAP)
and subjected to acoustic action to orient the cells into
patterns corresponding to muscle fibers. Then, the cross-
linking reaction was carried out under irradiation with a
UV-LED (l=405 nm, I
ex
=10 mW cm
72
) focused with
the help of a microscope objective. As a result, parallel
strands, imitating the structure of skeletal muscles, were
obtained and increased formation of myotubes and their
spontaneous twitching were observed there. The authors
believe that such an approach will allow the design of
skeletal muscles, as well as tendons, ligaments, vascular
networks, and their combinations thereof in the future.
An alternative option for creating patterns in hydrogels
can be the introduction of scaffolds, for example, in the
form of electrospun polycaprolactone fibers, into GelMA
hydrogels.
191
To do this, polycaprolactone fibers and cells
were added to the ink and polymerized. The mechanical
properties of hydrogels containing such fibers were signifi-
cantly higher than those of pure hydrogels. Encapsulated
cells there grew faster as compared to conventional hydro-
gels. The authors attribute this effect to the texture of fibers,
similar to the extracellular matrix, which can increase the
biological activity of the material.
Photopolymerization also allows the creation of neces-
sary patterns without introduction of additional compo-
nents. A photopolymerizable biocompatible a-elastomer
poly (glycerol sebacate) acrylate has been proposed for the
development of an in vitro model of muscle regeneration
and proliferation.
192
The mechanical properties of the con-
struct were controlled by varying the light intensity during
the DLP printing process (l= 385 nm) to match the specific
tension of the skeletal muscles. The formation of large-
diameter channels and coating of extracellular matrix with
proteins enhanced cell proliferation, and in vivo implanta-
tion of such a construct into a muscle tissue defect showed
the promise of this technology. Li et al.
193
used the DLP
technology (l= 405 nm, I
ex
=30 mW cm
72
) to form the
so-called multichannel nerve guidance conduits from
GelMA with PC-12 cells, promoting the directed growth of
axons to facilitate nerve regeneration, which is one of
several clinical treatments for nerve conduction damage.
Printing of small tubular structures similar in size to
blood vessels or glandular ducts is of great interest.
194
Recently, it was proposed to form such structures by
extruding a hydrogel of methacrylate-modified hyaluronic
acid, containing cells, directly into an aqueous solution of a
photoinitiator (riboflavin + triethanolamine), which is
excited by a semiconductor UV laser (l= 450 nm , power
of 900 mW) during scanning.
173
Diffusion of free radicals
from the solution into the extruded structure initiated
hydrogel crosslinking from the construct surface to the
center. Thus, it was possible to form a crosslinked wall,
whose thickness was determined by penetration of free
radicals into the hydrogel volume.
Other approaches include utilization of dynamic DLP
technology with an Omnicure S2000 UV curing system
(l= 365 nm), which allows production of three-dimen-
sional branching vessel-like constructs with encapsulated
cells, whose viability was maintained for at least 48 h after
polymerization. However, this method requires significant
engineering efforts and accurate calculation of the light
dose at different depths of the hydrogel.
195
Another example of an important and technically chal-
lenging biomedical application of 3D printing is retinal
reconstruction. Recently, the two-photon photolithography
of acrylated polycaprolactone loaded with retinal progenitor
cells from induced human pluripotent cells has been pro-
posed for this treatment.
196
Using the Nanoscribe Photonic
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
18 of 26 Russ. Chem. Rev., 2023, 92 (2) RCR5068
Professional GT two-photon lithography system
(l= 780 nm) with direct 3D laser drawing allowed the
obtaining of structures with a high resolution (up to 1 mm),
which is very important in the case of retinal reconstruction.
Such scaffolds supported the survival of retinal progenitor
cells in vitro. Subretinal implantation of the resulting scaf-
folds in a porcine retinitis pigmentosa model did not cause
inflammation, infection, and local or systemic scaffold
toxicity after one month.
196
In some cases, attention to photoinduced 3D polymer-
ization is caused by the possibility of using tools designed to
solve other problems in this field. So, polymerization lamps
have proven themselves well in dentistry, for example, the
VALO wireless dental lamp (Ultradent Products) with
l= 395 ± 480 nm, which was proposed for regenerative
dentistry using the GelMA composition with the LAP
photoinitiator and encapsulated odontoblast-like cells
OD21. During photopolymerization, >80% of cells
retained their viability, which, however, decreased with
increasing light exposure time and increasing LAP concen-
tration.
197
5.3. 3D printing in situ
Bioprinting in situ, i.e. directly at the site of damaged tissue
repair is a logical development of the technology of photo-
induced 3D printing of tissue-engineered constructs, since it
can provide minimal invasiveness and high accuracy of
tissue reconstruction, including tissues with a complex
structure. However, there are a number of difficulties, such
as maintaining the ink composition under in vivo condi-
tions, controlling the rheological and mechanical properties
of the material, and time-limited printing. Some require-
ments for the resulting structures are internally contra-
dictory: the synthesized constructs must, on the one hand,
be strong enough to retain their shape for a long time, and
on the other hand, they must be soft enough and capable of
degradation so that the encapsulated cells can proliferate
and perform therapeutic functions.
198
Compositions based on PEG and Irgacure 2959 photo-
initiator with introduced mesenchymal stem cells (MSCs)
were injected into a cartilaginous defect of the rabbit knee
joint under sterile conditions (362 mm in size) and photo-
cured under UV light irradiation (l=365 nm, I
ex
=5
mW cm
72
).
199
In all cases, the hydrogel was easily formed
in the defect without loosening or displacing from the
chondral defect. During the experiments, chondrogenesis
occurring in MSC cells and the ability of hydrogel to
controlled uptake were confirmed, while the animals in the
experimental group had the highest recovery rates.
One of the most significant restrictions in the transition
to bioprinting directly in the body is the low light perme-
ability of tissues, especially in the UV and blue spectra. The
simplest solution to this problem is to use hydrogels to treat
wounds and skin defects. Thus, a hydrogel based on
hyaluronic acid with grafted groups of methacrylic anhy-
dride and N-(2-aminoethyl)-4-[4-(hydroxy-methyl)-2-
methoxy-5-nitrophenoxy]butanamide with introduced
lyophilized amniotic medium was developed in 2022 to
treat the diabetic wounds.
200
Irradiation with UV light
using a LED (l=365 nm, I
ex
=5 mW cm
72
,LAPasa
photoinitiator) resulted in hydrogel photocuring in situ
within 3 s. At that, o-nitrosobenzaldehyde groups which
can form a covalent bond with the amino groups of the
tissue surface, were formed, and this ensured strong hydro-
gel adhesion to the tissue. It was shown that it significantly
accelerated the healing of diabetic wounds by regulating
macrophage polarization and promoting angiogenesis.
Irradiation through a fiber optic wire can be proposed as
another approach to solving the problem of delivering light
to the area of in situ photopolymerization. The success of
this approach was demonstrated by injecting a photopoly-
merizable hydrogel containing mesenchymal stem cells into
the nucleus pulposus of a rabbit intervertebral disc. The
composition used included GelMA, LAP and MSC. After
injecting this composition, a fiber optic wire was inserted
into the disc space and irradiated with light at a wavelength
of 405 nm at a power of 100 mW for 5 min. The results of
magnetic resonance imaging (MRI) and histological analy-
sis confirmed the success of this minimally invasive
approach, although it was not possible to avoid the for-
mation of osteophytes and defects. It is interesting that
MSCs after irradiation showed increased anabolic activity,
which may indicate a photobiological effect of light with
l= 405 nm.
201
Utilizatiuon of IR and NIR light is optimal for
biomedical applications, since it does not cause photo-
damage and, getting into the transparency window of bio-
logical tissue, it can deeper penetrate into it. This deter-
mines the intensive development of research related to the
use of the IR spectrum for printing under in situ conditions.
One of the first studies demonstrating the possibility of in
situ photoinduced printing was the reconstruction of bone
defects, in particular, the calvarium bones, using nano-
hydroxyapatite (n-HA) and a nanosecond pulsed laser
emitting at a wavelength of 1064 nm.
202
It was shown that
the ink material based on n-HA is biocompatible with
osteoblast cells and does not cause inflammation in vivo,
and the formed disks can start the restoration of a critical
damage of the mouse calvaria bone (a hole of 4 mm in
diameter) within a month. However, the insufficiently good
mechanical properties of n-HA prevented healing in all
cases, and later a more complex composition was proposed
to solve this problem, including, in addition to n-HA,
mesenchymal stromal cells and collagen.
203
Revasculariza-
tion of the implanted area was implemented via laser
printing with the help of the same laser. Obtaining the
circulatory system of a given structure was achieved by
applying red fluorescent protein(RFP)-labeled endothelial
cells into a mouse calvaria bone defect, filled with collagen
containing mesenchymal stem cells and vascular endothelial
growth factor.
204
A 3D printing technology using upconversion nano-
particles (UCNPs) in the presence of LAP was proposed in
2020.
180
In this case, UCNPs converted NIR laser radiation
(l= 975 nm) into light with a wavelength of 365 nm, which
initiated polymerization in a DLP printer and formation of
tissue-engineered constructs, including rather large ones,
such as a human ear.
A similar approach was also proposed in work
205
for
the restoration of soft tissues. The authors injected low-
viscosity ink into the injury site through a thin needle, and
polymerization was activated by UV-radiation of UCNPs,
excited by NIR-light of a continuous-wave laser
(l= 975 nm, power of 9 W) using the DLP technology.
Since the process was carried out using a focused beam, it
was possible to carry out gradient photopolymerization,
which allowed the control of both the mechanical and
adhesive properties of the hydrogel by regulating the
power of NIR irradiation and the concentration of
UCNPs.
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
Russ. Chem. Rev., 2023, 92 (2) RCR5068 19 of 26
In the study,
206
three-dimensional photosensitive poly-
mer hydrogels loaded with cells were printed using reactions
of orthogonal two-photon cycloaddition and crosslinking of
polymers under laser irradiation (l= 700 ± 900 nm, power
of 0. 7 ± 2 mW) with the help of a two-photon microscope
(Scientifica 2-Photon microscope). This in vivo 3D printing
of stem cells derived from donor muscles resulted in the de
novo myofibril formation in mice. It is important that the in
situ photoprinting technology allows formation of both
microstructures (several tens of microns in size), for exam-
ple, in the case of two-photon printing, and macrostruc-
tures. The latter are used, for example, to replace a full-
layer cartilage defect in a model of a large animal (sheep) by
printing discs with a diameter of 8 mm based on GelMA
and a VA-086 photoinitiator, excited by an Omnicrue
LX400 UV-LED (l= 365 nm, I
ex
=130 mW cm
72
). This
technique does not lead to postsurgical complications and
ensures rapid cartilage regeneration.
207
It should be noted that the technology of in situ photo-
printing can be automated and robotized, which is impor-
tant for further introduction of this method into clinical
practice.
198
For example, a portable bioprinter that uses ink
based on an aqueous two-phase emulsion containing
GelMA and PEG was created.
208
The presence of two
immiscible phases determined the possibility of pore for-
mation, which promotes the transfer of liquid and oxygen,
as well as cell proliferation. In addition, these TECs, while
maintaining biocompatibility, were characterized by high
elasticity and withstood multiple mechanical compressions.
The 3D printing process was carried out in the presence of a
LAP photoinitiator irradiated by five UV-LEDs
(l=365 nm, U=4.2 V, I= 20 mA). The presented bio-
printer has been proposed to make wound dressings.
Another bioprinter was developed as a robot with six
degrees of freedom and the ability of quick calibration to
improve printing accuracy.
209
It allowed repairing an osteo-
chondral defect (465 mm) in a rabbit joint within about
60 s with a printing error of <30 m. The ink consisted of
methacrylated a-hyaluronic acid and a crosslinking agent
(branched PEG with terminal acrylate groups); a DLP
printer with a UV laser from Prism (China) was used. It
should be noted that for known bioprinters, despite the
obvious problems with the depth of light penetration, it is
typical to utilize UV initiators due to their availability and
reliability.
It is important that the in situ printing technology allows
creation of not only relatively inert biological structures
(bones, cartilage, derm), but also muscles, for which func-
tionality is important, in particular, the ability to contract.
Thus, a handheld printer and ink that used a commercial
Laponite
1
nanogel for controlled release of vascular endo-
thelial growth factor (VEGF) and a GelMA-based hydrogel
as a supporting scaffold were developed. As a result of
curing under the action of a UV laser (l=365 nm), a
scaffold with high cell adhesion was obtained
(Fig. 10 a,b).
210
It was shown that during direct UV-induced printing in
vivo (see Fig. 10 c), the proposed composite was attached to
skeletal muscles. At that, 7% GelMA in the composition
provided a stiffness slightly lower than the stiffness of the
rodent skeletal muscles, which allowed the scaffold to
deform without destruction. In addition, this scaffold had
a sustained release of VEGF, which promoted functional
muscle recovery in a mouse model of quadriceps injury,
reduced fibrosis, and improved anabolic response as com-
pared to untreated mice (see Fig. 10 d).
210
The GelMA-
based composites demonstrated strong binding at the tis-
sue7hydrogel interface due to hydrogen bonds and friction
arising during photocrosslinking of composite materials,
including heterogeneous ones (microgel + liquid precur-
sor).
211
As an interesting example of in situ photopolymeriza-
tion, we can note an approach, when the required scaffold
obtained in vitro through 3D printing or stereolithography
is placed in a tissue defect, a photopolymerizable gel is
additionally introduced into the defect, and polymerization
is used to fix the scaffold in the tissue. A similar approach
has been demonstrated in ex vivo repair of focal cartilage
defects (with a diameter of 3 mm and a depth of *2mm)in
pigs. At that, the construct obtained using a 3D printer was
placed in the area of the cartilage defect, and a photo-
composition was additionally introduced there: a mixture of
eight-chain PEG-norbornene (with a molecular weight of
10 kDa) and PEG-dithiol (1 kDa) in the ratio of 1 : 1, as
well as 0.05 wt.% of the photoinitiator Irgacure 2959. Then,
polymerization was carried out for 8 min using a DLP
printer (Autodesk Ember projection SLA-printer,
l= 352 nm, I
ex
=5 mW cm
72
).
212
Such a hybrid technol-
ogy may be useful when translating classic scaffolds into the
clinical medicine.
5.4. Drug delivery systems
3D printing is widely used to solve the problems of a
personalized approach in medicine, in particular, to adapt
pharmaceutical treatment, aimed at increasing its effective-
ness, to each patient. Depending on the genetic predisposi-
tion, lifestyle, environmental conditions for each person, it
is necessary to prepare personalized dosage forms. With this
approach, 3D printing is a unique tool that allows one to
quite easily create delivery matrices with an individual
optimal dose and composition of drugs, as well as a
personalized release profile.
213
One of the first reports on the use of photopolymeriz-
able hydrogels for drug delivery, in particular albumin,
dates back to the early 1990s. Hubbel and co-workers
214
synthesized PEG block copolymers with oligo(
D
,
L
-lactic)
acid or oligoglycolic acid containing end acrylate groups.
Biodegradable hydrogels on their basis, obtained in the
process of photoinduced crosslinking under the action of a
UV lamp (BlakRay Model 3 ± 100A, l= 365 nm,
I
ex
=8 mW cm
72
) in direct contact with tissues, demon-
strated high adhesion to tissues and continuous effective
release of albumin for 2 months. The choice of a suitable
wavelength, power and dose of radiation eliminated the loss
of protein stability and the biocompatibility decrease asso-
ciated with the formation of radicals.
85, 215
Due to their unique biological properties, hydrogels
based on natural polysaccharides are of great interest as
drug delivery matrices (see Section 3.1). For example,
Zhang et al.
216
obtained biocompatible chitosan-based
thermo- and pH-sensitive hydrogels for using them as
carriers of antitumor and anti-inflammatory drugs. Under
the action of a UV lamp (Osram Ultra-Vitalux 300-W), the
authors formed in situ hydrogels from a graft copolymer of
carboxymethylchitosan with N-isopropylacrylamide and
glycidyl methacrylate in the presence of Irgacure 2959
initiator, which contained an anticancer drug (5-fluorour-
acil) and anti-inflammatory agent (diclofenac sodium). The
release of these drugs was controlled by changing the degree
of inoculation, pH and temperature of the medium, provid-
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
20 of 26 Russ. Chem. Rev., 2023, 92 (2) RCR5068
ing drug protection at physiological temperature, low pH of
the medium (e.g., in a stomach) and demonstrating release
under conditions with a higher pH (e.g., in an intestine).
Thus, the release of diclofenac sodium increased from 27%
at pH 2.1 to 89% at pH 7.4
When delivering hydrophobic drugs, it is challenging to
control the dose and duration of release. The introduction
of such drugs into hydrophilic polymer matrices due to
physical interactions does not guarantee their stable release
over a long period of time. Cisneros et al.
217
demonstrated a
delivery method for simvastatin, a hydrophobic drug that
promotes bone growth and healing and prevents cartilage
degradation by counteracting tumor necrosis factor, inhib-
iting bone morphogenetic protein, and improving osteo-
genic stimulation. Simvastatin was incorporated into poly-
meric micelles of triblock copolymers of polylactide and
polyethylene glycol (PLA-PEG-PLA) and mixed with
N-methacryloylchitosan and polyethylene glycol diacrylate
(PEGDA); then, a crosslinking reaction under irradiation
with a 405 nm LED (power of 48 W) in the presence of LAP
was carried out. It was shown that the effect of the
controlled release of simvastatin on osteogenic stimulation,
as well as on the mechanical and biological properties of the
hydrogel as an implant, depends significantly on the poly-
mer compositions. Drug release continued for 17 weeks of
follow-up, with cytocompatibility of hydrogels confirmed in
all cases.
The creation of scaffolds that mimic the native extrac-
ellular matrix and are capable of releasing growth factors is
one of the important tasks of tissue engineering.
Modaresifar et al.
218
obtained a composite hydrogel based
on methacrylated gelatin and chitosan nanoparticles con-
taining angiogenic growth factor (bFGF) using photo-
polymerization under a UV lamp (l=365 nm,
I
ex
=10 mW cm
72
). It was shown that this hydrogel pro-
motes cell proliferation due to its biocompatible structure
and provides a stable bFGF release profile, which is of great
importance for angiogenesis processes.
The advantages of 3D printing technology, which allows
adjustment of geometric dimensions of scaffolds and dosage
of drugs, are reflected in the development of approaches to
creation of constructs for specific patients. Such approaches
are being used, for example, to create anatomically adapt-
able patches for personalized transdermal drug administra-
tion.
219
Goyanes et al.
220
used SLA printing to create a
nose-shaped mask containing salicylic acid as a treatment
for acne, an inflammatory skin disease. A commercial
product, polycaprolactone with salicylic acid, was incorpo-
rated into a PEGDA ± PEG ± LAP photoinitiator composi-
tion that was cured by irradiation with a 405-nm laser. SLA
printing demonstrated high resolution and achievement of
the required drug loading without loss of drug activity, as
well as the ability of construct fine-tuning in accordance
with the anatomical features of the patient. These studies
have shown the advantage of the SLA technology over
traditional thermoplastic extrusion.
220
One of the minimally invasive methods of drug delivery,
which has been actively introduced into medical practice
over the past 20 years, is based on the use of micro-
needles.
221, 222
Biosoluble microneedles started to be used
in transdermal drug delivery to improve penetration of low
and high molecular weight drugs through the skin barrier.
Such instruments may be in the form of a lancet or a
miniature hypodermic needle with a length of less than
500 mm, which can reduce injury and pain at the injection
site. Hollow microneedles are capable of delivering drugs
both by diffusion and by the exit through the needle open-
ing under the pressure, which allow adjustment of the
release profile over an extended period of time. For exam-
ple, Pere et al.
223
made polymer patches with microneedles
in the form of pyramids or cones using 3D printing
technology. These patches were produced using a Form 2
SLA printer (Formlabs) from a commercial Dental SG
biocompatible polymer with inkjet printed insulin-xylitol
coatings. A rapid release of insulin (*30 min) with main-
taining the protein properties was demonstrated.
Caudill et al.
224
showed the capabilities of CLIP-tech-
nology (Carbon's S1 CLIP printer, l=385 nm, I
ex
=1and
4mWcm
72
) to create PEG-based microneedles, which
were coated with model proteins (bovine serum albumin,
ovalbumin and lysozyme) using the same technology. The
high rate of in vitro and in vivo release, preservation of the
enzyme activity, and maintenance of the release profile for
72 h indicate the promise of such a spatially controlled
coating.
An innovative approach to microneedle fabrication
using femtosecond laser two-photon polymerization (2PP)
was presented by Ovsyanikov et al.
225
Three-dimensional
microneedle devices with different aspect ratios were cre-
ated from modified ceramic hybrid materials (Ormocer
1
)
under the action of femtosecond laser pulses (duration of 60
fs, frequency of 94 MHz, l= 780 nm, power of <450 mW).
It was found that microneedles do not break when penetrat-
ing into adipose tissue and the viability of human epidermal
keratinocytes is preserved on their surface. These results
demonstrate significant promise for the use of 2PP, which in
turn allows the use of a variety of polymers, low-cost
ceramics and other light-sensitive materials, as well as easy
scale-up of the process for industrial application. In addi-
tion, the use of 2PP for microneedle fabrication is a fast and
simple one-step process, which distinguishes it from tradi-
tional multi-step methods for making such specimen.
6. Conclusion
Based on photoinduced polymerization and crosslinking
reactions, 3D printing methods such as SLA, DLP and
CLIP allow quick and accurate fabrication of polymer
structures of various architectures without the use of
molds or mechanical processing. Significant progress in
this area is associated with the development of flexible and
adjustable radical photoprocesses that facilitate the devel-
opment of approaches to the creation of special ink taking
into account the features of photoprinting. However,
despite the active exploitation of photopolymerization and
crosslinking reactions, there remain the problems that limit
the further progressive development of 3D printing.
First of all, it is necessary to increase the variety of ink
with specific biological, rheological, physicochemical and
mechanical properties. This direction includes the search for
new precursors and photoinitiators, optimization and study
of new conditions for conducting photoreactions, as well as
the study of interaction between a photocomposition and
cells. In addition, an important direction is the creation of
ink capable of curing under longer wavelength irradiation,
which provides mild conditions for radical photoreactions
at a greater depth of light penetration.
Further development of approaches to perform photo-
induced radical processes, such as orthogonal click reac-
tions, redox and controlled processes, in situ crosslinking,
etc., will expand the range of methods for the formation of
hydrogels from ink with low viscosity. This is an effective
A.N.Generalova, P.A.Demina, R.A.Akasov, E.V.Khaydukov
Russ. Chem. Rev., 2023, 92 (2) RCR5068 21 of 26
tool for more efficient functioning and gradient inclusion of
cells of various lines by changing scaffold rigidity. The
absence of a high viscosity requirement also ensures the
chemical variety of ink, including the possibility of creating
multicomponent samples with controlled irradiation of a
photocomposition by light. Moreover, in the future, it is
necessary to study the mechanisms of reactions in more
detail and develop methods for controlling ink photoreac-
tions in order to obtain large centimeter-sized structurally
organized constructs containing cells within a few seconds.
Such advanced knowledge in the field of photoinduced
reactions will determine the development of new 3D print-
ing technologies, which promote creation of constructs with
specific patient anatomy for clinical application.
One of the main problems of 3D printing of large TECs
is the delivery of nutrients and the exchange of metabolites.
Currently, to solve this complex problem, active research on
photoreaction chemistry is being carried out to create
prevascularized scaffolds that ensure the delivery of
nutrients and the development of a blood circulatory
system. This can be achieved through additional spatiotem-
poral control of reactions, as well as through the light-
driven degradation of material throughout the construct
volume. Controlling the ratio of growing and degrading
blocks in a construct determines the architectonics of the
channel system, in which vascular growth can occur.
At present, the concept of 4D printing, based on the
study of material properties in time, is being actively
developed; it is positioned as a method for studying funda-
mental biological problems. For example, this concept can
be used to solve the important problem related to careful
control and adjustment of tissue repair when using 3D
tissue-engineered constructs, printed from compositions
that meet biomedical requirements in terms of both mate-
rial, functionality, and mechanical properties. In addition,
TEC can act as a model for various diseases, which has
great potential for studying the fundamental biological
processes associated with the disease progression and find-
ing new drugs for its treatment.
In conclusion, it is worth noting that further progress in
tissue engineering is determined by the development of new
approaches to light-driven production of multicomponent
constructs containing cells and drugs, as well as large
vascularized structures. In general, photoinduced 3D print-
ingofTECsisdevelopingataveryfastpace,andthereare
many challenges for further in-depth research of an inter-
disciplinary nature, which will open up new prospects in
numerous biomedical applications of this technology.
The review was financially supported by the Ministry of
Science and Higher Education of the Russian Federation as
part of the State Assignment of the Federal Research Center
`Crystallography and Photonics' of the Russian Academy of
Sciences (topic `Photocuring of polymer compositions').
R.A.Akasov (Russian Science Foundation Project
No. 21-79-10384) and P.A.Demina (Russian Science Foun-
dation Project No. 18-79-10198-P) express their gratitude
for the financial support to the Russian Science Founda-
tion, E.V.Khaydukov is grateful to Alexander von Hum-
boldt Stiftung (Germany).
7. List of abbreviations and designations
AFCT Ð Addition-Fragmentation Chain Transfer,
CAL Ð Computed Axial Lithography,
CLIP Ð Continuous Liquid Interface Production,
CRP Ð Controlled Radical Polymerization,
DLP Ð Digital Light Projection,
I
ex
Ð excitation intensity,
FTI Ð Film Transfer Imaging,
GelMA Ð Methacrylated Gelatin,
GFP Ð Green Fluorescent Protein,
HA Ð Hyaluronic Acid,
n-HA Ð Nanohydroxyapatite,
LAP Ð Lithium Phenyl(2,4,6-Trimethylbenzoyl)Phos-
phinate,
LCD Ð Liquid Crystal Display,
MJP Ð Multi-Jet 3D Printing,
MSC Ð Mesenchymal Stem Cells,
2PP Ð Two-Photon Polymerization,
PCL Ð Polycaprolactone,
PE-1 Ð Tetrakis(3-Mercaptobutanoate) Pentaerythri-
tol,
PEG Ð Polyethylene Glycol,
PEGDA Ð Polyethylene Glycol Diacrylate,
PETMP Ð Tetrakis(3-Mercaptopropionate) Pentaery-
thritol,
PLA Ð Polylactide,
PLGAÐCopolymerofLactidewithGlycolide,
POSS Ð Silsesquioxan,
PmSL Ð Projection micro-Stereolithography,
PVA Ð Polyvinyl Alcohol,
QCS Ð Quaternized Chitosan,
RAFT Ð Reversible Addition-Fragmentation Transfer
Polymerization,
RFP Ð Red Fluorescent Protein,
SLA Ð Stereolitography,
TEA Ð Triethanolamine,
TEC Ð Tissue-Engineering Constructs,
TMPMP Ð Tris(3-Mercaptopropionate) Trimethylol-
propane,
TMI Ð Tris[2-(3-Mercaptopropionyloxy)Ethyl]Isocya-
nurate,
TTC Ð Sodium Trithiocarbonate,
TTT Ð Triallyl-1,3,5-Triazine-2,4,6(1H,3H,5H)-Trione,
VEGF Ð Vascular Endothelial Growth Factor,
UCNP Ð Upconversion Nanoparticles.
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