Optogenetics and Its Transformative Role in Tissue Engineering and Regenerative Medicine

Abstract

Optogenetics, a revolutionary technique leveraging light stimulation for precise cellular control, holds immense potential in regenerative medicine. Offering unparalleled spatial and temporal accuracy, the entrance of optogenetics into tissue engineering and regenerative medicine (TERM) empowers researchers to modify genes precisely and reversibly, control signal pathways and antibodies, as well as alter biomaterial properties. Optogenetics provides unprecedented control in the manipulation of physiological regeneration, replication of intricate developmental processes, and tissue engineering in a laboratory setting. Although further investigation is required for the safe and feasible injection of optogenetic systems into human bodies, the use of optogenetics has already led to a great amount of research on several tissues and systems. It has found diverse applications in TERM of skin and connective tissue, endothelium, bone, cartilage, and muscle; with researchers leveraging optogenetics for precise control over the in vitro or in vivo production of these tissues, the investigation of critical proteins and pathways, the creation of light‐controlled wound coverings and even as a tool for directional tissue growth in living subjects. Optogenetics emerges as a transformative force in shaping the future of medical science, demonstrating a pivotal role in advancing regenerative medicine and paving the way for innovative therapeutic strategies.

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Nano Select
REVIEW
Optogenetics and Its Transformative Role in Tissue
Engineering and Regenerative Medicine
Emine Camcıoglu1Deniz Ghasemi Mohammadrezaloo1Bircan Dinc2
1Faculty of Medicine, Bahcesehir University, Istanbul, Turkey 2Department of Biophysics, Faculty of Medicine, Bahcesehir University, Istanbul, Turkey
Correspondence: Bircan Dinç (bircan.dinc@bau.edu.tr)
Received: 22 January 2024 Revised: 21 May 2024 Accepted: 23 May 2024
Keywords: biomaterials | cellular control | optogenetics | regenerative medicine | tissue engineering
ABSTRACT
Optogenetics, a revolutionary technique leveraging light stimulation for precise cellular control, holds immense potential in
regenerative medicine. Offering unparalleled spatial and temporal accuracy, the entrance of optogenetics into tissue engineering
and regenerative medicine (TERM) empowers researchers to modify genes precisely and reversibly, control signal pathways
and antibodies, as well as alter biomaterial properties. Optogenetics provides unprecedented control in the manipulation of
physiological regeneration, replication of intricate developmental processes, and tissue engineering in a laboratory setting.
Although further investigation is required for the safe and feasible injection of optogenetic systems into human bodies, the use of
optogenetics has already led to a great amount of research on several tissues and systems. It has found diverse applications in TERM
of skin and connective tissue, endothelium, bone, cartilage, and muscle; with researchers leveraging optogenetics for precise
control over the in vitro or in vivo production of these tissues, the investigation of critical proteins and pathways, the creation
of light-controlled wound coverings and even as a tool for directional tissue growth in living subjects. Optogenetics emerges as
a transformative force in shaping the future of medical science, demonstrating a pivotal role in advancing regenerative medicine
and paving the way for innovative therapeutic strategies.
1 Introduction
Several years have elapsed since optogenetics, a technology
involving the introduction of light-sensitive proteins into living
systems for manipulation or monitoring, transcended its origins
in neuroscience, becoming accessible to any ambitious scientist
seeking to control proteins using a straightforward beam of light.
Since then, there have been many great optogenetic undertakings
in several scientific fields—one being regenerative medicine.
After all, with its undeniable connection to knowledge gained
from embryologic and developmental science, the technology
of tissue engineering, and medicine as a whole, it shouldn’t be
surprising for researchers in regenerative medicine to desire a way
of clicking on and off the proteins.
To understand how regenerative medicine can benefit from
this tool which sprouted from bacterial proteins and grew into
prominence through neuroscience, it is imperative that we first
understand what regenerative medicine is. There are many
definitions of regenerative medicine, though most of them are too
lengthy. Perhaps the shortest and clearest is that of Mason and
Dunnill [1]:
Regenerative medicine replaces or regenerates human
cells, tissue or organs, to restore or establish normal
function.
For the aim of restoring or establishing normal function in
the human body, various techniques are commonly applied in
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly
cited.
© 2024 The Author(s). Nano Select published by Wiley-VCHGmbH.
Nano Select, 2024; 5:e202400013
https://doi.org/10.1002/nano.202400013
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FIGURE 1 The main therapies of TERM are shown. Tissue engineering with its three pillars, stem cells produced from embryonic or adult origin,
scaffolds with varying porousness and growth factors are on the circle. They come together with regenerative medicine treatmentssuch as nanomedicine,
immunomodulation, and gene therapy to form the complex field of TERM. Created using BioRender.com.
tissue engineering and regenerative medicine (TERM). Tissue
engineering itself is built on three major aspects; scaffolds, cells,
and growth factors, and it comes together with regenerative tech-
niques such as immunomodulation, gene therapy, nanomedicine,
and more to form the basis of this field [2](Figure1).
Scaffolds are formed of polymeric biomaterials and act as
replacements for the extracellular matrix, providing structure and
attachment to the cells either endogenous or exogenous that are
growing into the material [3]. These “endogenous or exogenous
cells” are “endogenous” for in vivo genetically modified stem cells
that already inhabit the relevant adult tissue and “exogenous” for
ex vivo allogeneic stem cells acquired from an outside source. In
three categories, stem cells are adult tissue-resident stem cells
that maintain the mature tissues, especially ones with rapid
proliferation such as blood, epidermis, and intestine, embryonic
stem cells (ES) which are manufactured from preimplantation
embryos in culture, and induced pluripotent stem cells (iPSC)
which are stem cells de-differentiated from genetically engi-
neered adult cells [4]. In conjunction with cells and scaffolds,
growth factors as well as regenerative techniques are used to
trigger and maintain critical processes such as cell differentia-
tion, proliferation, and neovascularization in the newly grown
tissue [2].
The convergence of these elements gives rise to revolution-
ary techniques, including biological nanomachines and 3D
bioprinting, offering a promising outlook for the future of
TERM [5]. 3D-bioprinting technology, in particular, is already
at the level needed for various biomaterials and cell types
to be printed in structures close to the clinically required
size and shape. Cartilage, bone and skin are examples of
three-dimensional bioprintable tissues that are currently in
research [6].
However, while 3D-bioprinting of living tissue is a powerful tool,
it is faced with some challenges that are common to TERM
in general, such as the necessary physiological heterogeneity
of tissues, or the development of stem cells into the required
type and number of functional cells [6]. For example, Guillot
et al. [7] describe the difficulties of expanding, differentiating,
and choosing the right differentiation stage of an ES-derived
cell to be clinically transplanted. According to the authors, an
ES-derived cell transplanted while it’s too immature may not
differentiate into the required cell type in vivo, as the adult body
does not mimic the environment (niche) required for the fetal
development that leads to said functional cell, and it also carries
a higher tumorigenic risk. Conversely, an ES-derived cell that is
too mature at the time of transplantation might not be capable of
the necessary adaptation that will allow it to survive and function
within the body (Table 1).
The solution to functional tissue creation, Lane et al. [4] propose,
lies in manipulating the tissue to form the necessary cell niche.
By using the major components of a niche—cell-cell interactions,
secreted factors, ECM, mechanical factors like stress or stiffness,
or signaling molecules both environmental and intracellular—
Lane et al. claim that it is possible to produce the correct behavior
in stem cells and thus mimic the endogenous tissue formation
that occurs naturally during embryogenesis. The accurate simula-
tion of a niche is not easy. There are more than a few components
that affect the behavior of stem cells. Spatial components such
as ECM, mechanical factors, or structural requirements can be
provided by 3D-bioprinting which offers accurate cell, growth
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TABLE 1 Summary of the significant advancements of optogenetics in TERM for different systems. Contains brief descriptions of the studies
mentioned in Chapters 4.1, 4.2, 4.3, 4.4, 4.5, and 4.6.
System
Study
type Significance Cell/animal type
Photoreceptor
molecule(s)
Study
reference(s)
Skin and
connective
tissue
In vivo Enhancing diabetic chronic wound
healing using optogenetically engineered
exosomes
Mice CRY2/CIBN [160]
Skin and
connective
tissue
In vivo Controlling wound contraction through
optogenetic manipulation of intracellular
pathways
Fibroblast-like
kidney cells
VVD,
CRY2/CIBN
[32, 79]
Skin and
connective
tissue
In vitro Developing light-controlled state- or
property- changing hydrogels for wound
dressing
—UVR8,
phytochromes,
LOV-domain
[51, 66, 89]
Skin and
connective
tissue
In vivo Controlling transcription of genetic
engineering tools & Deleting gene to alter
hair growth
Mice CRY2/CIBN [10]
Vessels In vivo
(embryo)
Inducing neovascularization and
angiogenesis in embryo using increased
VEGF transcription
Chick embryo
chorio-allantoic-
membrane
PhyB/PIF6 [65]
Vessels In vitro Understanding angiogenesis-related
interaction of kindlin-2 and αVβ3 integrin
in endothelial cells
Endothelial cells LOV-domain [52]
Vessels In vitro Inducing increased αVβ3 activation and
endothelial cell migration through
optogenetic manipulation of talin
Endothelial cells CRY2/CIB1 [140]
Vessels In vitro Investigating the potential of optogenetic
calcium influx to stimulate angiogenesis
Endothelial cells hBACCS2 [141]
Vessels In vitro Studying the effects of optogenetically
induced Akt1 signaling on endothelial
cells and its downstream substrates
Endothelial cells CRY2/CIB1 [142]
Vessels In vitro Controlling vascular endothelial barrier
strength through reversible and
temporally precise manipulation of Rho
GTPase activity using optogenetics
Endothelial cells LOV-domain [143]
Bone In vitro Manipulating BMP signaling pathway for
bone regeneration
Chondrogenic
cells
LOV-domain [47]
Bone In vitro,
In vivo
Controlling Lhx8 transcription to regulate
cell proliferation and osteogenic
differentiation during bone regeneration
Mice LOV-domain [144]
Bone In vitro,
In vivo
Controlling Lhx8 and BMP2 expression to
optimize bone regeneration
Mice LOV-domain [145]
Cartilage In vitro Achieving zonal structure of cartilage
tissue through optogenetic control of
TGF-b signaling pathway
Human MSCs CRY2/CIBN [146]
Muscle In vivo Stimulating muscle contraction in
genetically modified mice
Mice ChR2 [147]
Muscle In vivo Evaluating muscle loading effects on
Achilles tendon’s insertion using
optogenetics
Mice ChR2 [148]
Muscle In vitro Controlling 3D muscular structures for
simulating exercise and movement
Skeletal muscle
myoblasts
ChR2 [148–150]
Muscle In vitro Studying the connection between muscle
contraction and nearby angiogenesis
Myoblasts &
endothelial cells
ChR2 [41]
(Continues)
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TABLE 1 (Continued)
System
Study
type Significance Cell/animal type
Photoreceptor
molecule(s)
Study
reference(s)
Muscle In vivo Treating muscular atrophy after nerve
injuries or diseases using optogenetics
Mice ChR2 [116, 151, 152]
Peripheral
nerves
In vitro Studying the optogenetic stimulation of
peripheral nerves in vitro to enhance
neurite outgrowth and Schwann cell
migration
Dorsal Root
Ganglions (DRGs)
ChR2 [17]
Peripheral
nerves
In vitro Creating calcium ion influx to induce
Schwann cell proliferation, differentiation
and myelination
Schwann cells CatCh [153]
Peripheral
nerves
In vivo Enhancing nerve regeneration after injury
through optogenetic stimulation
Mice ChR2 [154]
Peripheral
nerves
In vivo Examining the efficacy of a
bioluminescent-optogenetic system for
enhancing nerve regeneration in vivo
Mice ChR2 [155, 156]
Peripheral
nerves
In vivo Manipulating cAMP levels to study
peripheral nerve regeneration
Zebrafish bPAC [157]
Peripheral
nerves
In vitro,
In vivo
Guiding axon growth using a
photoactivatable form of DCC
Chick DRG, C.
elegans
CRY2 [158]
Peripheral
nerves
In vitro,
In vivo
Manipulating calcium signaling and
Raf/MEK/ERK and AKT pathways to
guide axon growth and regeneration in
peripheral nerves
Fruit fly
(Drosophila)
larvae
CRY2/CIBN [145]
factor, and biomaterial positioning, but while 3D-bioprinting can
indeed mimic the three spatial dimensions of a niche, it cannot
assist in the control of the vital fourth dimension: time [8].
2Time as the Fourth Dimension of TERM
Regeneration, growth, and complex tissue functionality are all
processes dictated by fundamental cellular actions such as pro-
liferation, migration, differentiation, and death. These cellular
actions, in turn, are dictated by a variety of signals. Special
molecules (ligands) interact with cells in meaningful chemical
codes and form cascades, triggering further signals which even-
tually lead to a change in target genes or proteins—these are
called cell signaling pathways [9]. The cell signaling pathways can
be categorized as intracellular pathways, which are stimulated
in a cell by internal changes or factors, extracellular pathways,
stimulated in a cell by an external factor or in-between matrix
molecules, and intercellular pathways, stimulated during cell-
cell interactions like cell contact, paracrine signaling, exosome
formation [9]. All of these various signals come together in an
array of complex fluctuating environmental cues that form the
specific niche which will determine the activity of the cells within
its range [4].
The stem cell niche that promotes proliferation and forbids
differentiation into adult stem cells which Lane et al. [4]have
described is only a single example of the diverse and minutely
fluctuating niches formed in the natural lifecycle of living beings.
Embryogenesis, in particular, is a precise multistep developmen-
tal cascade of millions of niches triggering each other in precise
turns. This meticulous process cannot be achieved solely on
spatial signals, requiring the additional information brought by
temporal—also called dynamic—signaling [10].
In the case of simple signaling, the only information that can be
gleaned from a single ligand would be its presence or absence
at a single point in space, a so-called “on-off” signal. In the
far more complex dynamic signaling, on the other hand, a
single type of ligand can code a variety of information through
not only its presence but also through its delay, fold change,
duration, frequency, and more [11]. For example, in embryogenic
development, the sonic hedgehog protein (Shh) being secreted
from a concentration center grows less concentrated as it spreads
down the growing neural tube, leading to the development of
distinct neuron types down the ligand gradient [12].
Temporally coded information assists in diversifying the role
of each cellular pathway and allows for a small number of
specific and highly evolutionarily conserved pathways to have an
almost exclusive and vital role in an enormous portion of human
development [10]. An example of the complexity of information
coded by dynamic signaling is cardiac positioning: cardiac cells
begin developing in the intersection of two linear gradients,
dorsoventral Bone Morphogenic Protein (BMP, a part of the
TGF-βfamily) and anteroposterior Wnt, where BMP is high and
Wnt is low [13]. Another relevant example of the importance of
temporal dynamics is demonstrated by the formation of a stripe
pattern on the skin. A stimulatory factor activating its inhibitor to
affect its local concentration is called an Incoherent Feedforward
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Loop [5]. An incoherent feedforward loop with a short-range
activator and its long-range inhibitor, according to the Gierer-
Meinhardt equation, will form a signaling network that works
over time and will lead to a final ligand concentration in a stripe
pattern [14]. Neither 3D-bioprinting nor nanomachinery can
minutely mimic these temporal dynamics that are so integral to
the natural development of functional human tissue. Considering
the importance of oscillating, intersecting, delaying, and looping
signals in cellular processes, precise dynamic control is necessary
for the future of TERM. This is where optogenetics enters the
regenerative field with revolutionary potential.
Optogenetics is a non-invasive method for the precise temporal
manipulation of cell signals, environments, and interactions.
The maximum spatial precision achieved by optogenetics has
been demonstrated to be in micrometers, while its temporal
accuracy can get as precise as a single millisecond, providing an
unprecedented tool for precision in both academic and clinical
settings [15]. It is also a valuable tool due to being a clinically
applicable method that has up to now reached FDA approval in
clinical trials for ophthalmic treatment [16], promising a bright
outlook for optogenetic research in other areas as well.
Opsins can be used to stimulate excitable cells at will, while non-
opsin optogenetic molecules with more varied applications are
an ever-growing family of light-activated cellular remote controls.
With light stimulus, any number of cell activities or properties can
be precisely changed, allowing one to control the time at which
cells differentiate, proliferate, migrate, and more [5, 17]. Using an
optogenetic system to dynamically control Erk signaling, Johnson
and Toettcher [18] have demonstrated this exact versatility of
optogenetics in embryological research. In their research, they’ve
used light stimulation to induce Erk signals at different durations
in Drosophila embryos and discovered that activation in a 30-
minute pulse led to the formation of intermediate neuroblasts,
while a 1-hour activation instead triggered tissue contractility.
As exemplified by the work of Johnson and Toettcher [18], the
innumerable temporal patterns required for the replication of
fetal development beget a tool with the spatiotemporal precision
that optogenetic systems provide. These make optogenetics a
unique and invaluable tool for TERM research. The more optoge-
netic tools are developed, and the more they are applied to TERM,
the better this concept will be demonstrated.
3 Optogenetic Systems in TERM
Optogenetics is the technology of transgenetically expressed
proteins being used to accurately and noninvasively manipulate
biological structures using light stimulus [19]. Optogenetic sys-
tems are photosensitive proteins whose conformation or manner
of interaction with other molecules are changed by exposure to
light at a specific frequency and the effector molecules that are
fused to them, the whole of which is capable of changing the
biological state or function of a living organism when exposed to
the correct frequency of light [20].
There are many advantages to optogenetic systems compared to
others such as temperature-based or small molecule-based sys-
tems. These advantages can be summarized as; high specificity,
quick response and reversibility, lack of cross-interference in cells
that aren’t naturally photoreceptive, low toxicity to cells, and high
spatiotemporal precision [21].
3.1 Design of Optogenetic Systems
An optogenetic system can require the following three elements; a
photoreceptor to alter the activity of the effector accordingly with
light stimulation, an effector to induce the required response of
the target cell, and a guide to attach the system to the selected
place on the genome or to move it to the target localization in
the cell [5]. Of these, a pairing of a photoreceptor and an effector
alone (usually in light-control of intracellular signaling) or only a
photoreceptor on its own (usually in light-control of extracellular
biomaterial properties) can also be utilized.
The most important aspect of an optogenetic system is the
photoreceptor. These come in many types, from channel proteins
to protein cores [such as LOV domain—message to future emine]
to tetramers. There are five ways a photoreceptor can be utilized
in a system (Figure 2):
1. Ion flux, where an ion channel is opened or closed in response
to light stimulation, altering the intracellular electrochemical
composition. Opsin photoreceptors are all used in ion flux
systems [22].
2. Homodimerization or homooligomerization, where light
stimulation makes photoreceptors dimerize or multimerize
to bring together effectors that are more active in multimers.
Photoreceptors can also be attached to matrix proteins,
changing the mechanical properties of the proteins as they
dimerize in light. Photodissociable receptors work in the
exact opposite way.
3. Heterodimerization, where photoreceptors are fused to nat-
urally dimeric or artificially split effector molecules and in
response to light, reattach the split inactive halves into a
single active heterodimer. It can also be used to transport an
effector molecule intracellularly or into exosomes; one piece
of a photoassociating pair is attached to the desired cellular
membrane using a guide, and the other is fused to the effector,
leading to the light-activated membrane accumulation of the
effector.
4. Intramolecular dimerization, where two photoreceptors are
fused to the two ends of a single malleable effector, hiding
the active site in a closed loop, and when stimulated the
photoreceptor pair disassociates to reveal the active site of the
effector.
5. Conformational changes, where a single photoreceptor is
fused with the effector in a way that blocks the active site
of the effector, and in response to light, the photoreceptor’s
molecular conformation changes to reveal the effector’s
active site. Such photoreceptors are also compatible with
blocking guides such as nuclear-localization tags, as this
tag can be hidden inside the photoreceptor until it unravels
in response to light, at which point the effector will be
transported into the nucleus.
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FIGURE 2 Five ways photoreceptors can work in an optogenetic system. (A) Ion Flux. A channel opsin is activated by light stimulation and allows
the passage of ions. (B) Homooligomerization. Photoreceptors (PR) fused to effectors (E) are activated by light and attach to each other, bringing the
effectors together in a homooligomer which has a higher affinity for the effector targets. (C) Heterodimerization. (1) Photoreceptors are activatedbylight
and bind to their pair-proteins (PP), bringing together two inert halves (EAand EB) into a single active effector. (2) Photoreceptors are activated by light
and bind to their pair-proteins, which are attached to a membrane by a guide molecule (G), moving an effector to a target cellular area. (D) Intramolecular
dimerization. Two photoreceptors fused to each end of an effector are bound in a dimer and disassociate in response to light stimulation, revealing the
active site of the effector. (E) Conformational changes. A photoreceptor is fused to an effector in a way that prevents its activity, but in response to light,
it goes through a conformational change that releases the effector’s active site and allows it to activate once more. Created using BioRender.com.
Guides may or may not be included in an optogenetic system
depending its type and purpose. Guide molecules are usually
fused with a photoreceptor monomer, and they’re most often
tasked with leading the optogenetic system to the intracellular
position where it will function properly. There are two types of
guides: gene guides, used to position the system beside the target
genes, and localization guides, used to position the system at the
target cellular location.
Gene guides can target endogenous genes or exogenous genes.
Calcium-activated Nuclear Factor of Activated T-cells (NFAT)
proteins [23, 24], transcription activator-like effectors (TALEs)
[25] and nuclease-deactivated cas9 (dCas9)/small guide RNA
(sgRNA) [26] all attach to endogenous genes without altering
them, allowing for the effectors to find the selected gene.
Endogenous gene-targeting guides are more physiological and
better for genome editing and epigenetic modification but they
are less orthogonal and can suffer from endogenous interference
or nonspecific gene expression [27, 28]. On the other hand,
exogenous gene-targeting guides are based on an exogenous
operator gene inserted near the target gene, which the guide binds
with great fidelity [5]. There are many guide molecule/exogenous
gene pairs used for optogenetic systems, such as Gal4/UAS [29],
CarH/CarO [30], EL222/C120 [31].
Localization guides can be plasma membrane localizing, such
as transmembrane domains [32] or molecules containing a C-
terminal CaaX prenylation motif [33], nuclear localizing signal
molecules of appropriate strength such as a mutant c-Myc [34],
or chromatin localizing such as histones [35]. Some photore-
ceptors naturally contain guide domains, such as bcLOV4, a
LOV protein extracted from a fungus, which demonstrates single-
component localization to the plasma membrane under blue light
stimulation [36].
3.1.1 Photoreceptor Selection
Much of optogenetics’ potential comes from the untapped variety
of tools it offers. For a photoreceptor to be considered for use in
TERM, it must first be proven to embody all six of these traits:
(1) functional in mammal cells, (2) not toxic, (3) not leaky when
switched between on- and off-states, (4) reasonably operable with
currently available optical equipment, (5) linearly expressive for
linearly increasing input signal, and (6) not cross-interfering with
other optogenetic systems [5]. These criteria being considered,
there are still an ever-increasing number of different systems,
many of which have yet to be put to use in TERM.
It is possible to categorize the predominantly used photoreceptors
of TERM into two: First are channel opsins, and second are the
non-opsins.
Channel opsins are channel proteins that can be opened or closed
using light stimulation at a specific frequency range (color).
They are photoactivable transmembrane ion channels and do
not lead to specific protein interactions. Opsins were the first
optogenetic molecules to be discovered and are utilized predom-
inantly in neuroscience. They have two main types; microbial
opsins (type I) such as channel rhodopsins, halorhodopsins and
bacteriorhodopsins, and animal opsins (type II) [22]. Opsins that
are frequently used outside of neuroscience are the blue light
activated excitatory nonspecific cation channel rhodopsin ChR2,
the inhibitory chloride channel halorhodopsin NpHR [37, 38].
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These tools that allow for reversible and dynamically controllable
ion transfers have a place in tissues beyond the CNS, such as
peripheral nerves [17, 39], muscles [40, 41], blood vessels [42], and
immune cells [43].
Opsins with various alterations have been engineered to reach
different tissue depths, activate at different frequencies of light,
or activate and deactivate within different durations and patterns.
Especially blue light activated opsins such as ChR2 have been
transformed into an extensive number of red and far-red light
activated versions [44–46]. Furthermore, although opsins have a
typical activation pattern of an initial peak photocurrent followed
by a plateaued ion passage in the presence of continued light stim-
ulation, and then a characteristic deactivation time, engineered
versions have been created with different peaks, plateaus and
activation/deactivation times [22].
Non-opsins are light-responsive molecules found in a variety of
species such as algae, plants, and bacteria, which control more
complex cellular functions compared to opsins. The following are
commonly used non-opsin optogenetic photoreceptors:
LOV flavoprotein domains (LOV) which have the property of
either dimerizing or allosterically changing in blue light [47, 48].
LOV proteins require the cofactor, FMN. There are many types of
LOV proteins in nature that have been extracted for use, but they
can typically be categorized as “fast-cycling” with high intensity
light requirement but rapid on-off cycling or “slow-cycling” with
little light requirement but deactivation kinetics that measure in
minutes instead of seconds [49].
LOV proteins are the most common optogenetic tool applied to
the control of intracellular systems [50] and they are typically
utilized in one of two methods: fused to an effector that is
unblocked by light-controlled conformational changes or homod-
imerization. However, heterodimerizing photoswitches have also
been designed using LOV-domains [51, 52].
Cryptochromes, of which the most frequently used is CRY2
which dimerizes in blue or UV-A light. CRY proteins require
the cofactor FAD which is commonly found in mammals. can
homodimerize or cluster, or they can heterodimerize with the
pair-protein CIB1 (Cryptochrome-Interacting Basic-helix-loop-
helix) or CIBN in whose presence homodimerization can be
suppressed [5, 50, 53]. CRY2-CIB1 dimerization happens within
milliseconds and the dimer has a half-life of 5 minutes in
darkness [50].
They’re most commonly used as homooligomers with effectors
that activate when in dimers or clusters such as RTKs but have
also been used in a system with clustering inactivation [54].
CRY-CIB1 heterodimerization is also frequent in light-activated
relocalization of effectors [55]. Due to its light-induced clustering,
when used in the nucleus CRY proteins—and the effectors
attached—are cleared out from the nucleus in what could be an
adverse result [53].
UV Resistance Locus 8 (UVR8), which is a UV receptor that
rests in a homodimer state in the dark but monomerizes in
UV-B light at which point it either remains monomeric until
light stimulation ceases or heterodimerizes with the pair-protein
COP1 if it’s expressed in the cell [56, 57]. UV-B is a cytotoxic
light which is only used in pulses to ensure minimal damage
to the DNA [5], though UVR8 is capable of responding to a
low light intensity that is below the levels required for damage
serious enough to alter cell proliferation [58]. However, as it
only responds to UV-B light and not visible or laser light, UVR8
remains the optogenetic tool with the smallest noise and most
suitability to multichromatic systems [59]. UVR8 is one of the
two non-opsins that do not require a cofactor and can operate
in mammalian systems without the addition of any exogenous
chromophores [35].
UVR8 is most often utilized in mainly two ways; as a light-
monomerizing deactivator or as a light-heterodimerizing activa-
tor alongside COP1. UVR8 has been applied to light-controlled
genetic engineering, control of gene transcription, protein relo-
calization or detachment (see [35]) as well as to the extracellular
control of biomaterial properties (see Section 3.1.3).
DRONPA145N (DRONPA) which is a mutated coral photoreceptor
that homotetramerizes in violet light and disassociates in cyan
light [60]. As it disassociates very slowly in darkness (hours)
compared to cyan light (seconds), it can provide a distinct
additional dimension of control to the system [61]. Some of
its advantages are that it provides its own fluorescence, self-
reporting its state of activation, and that it is the second non-opsin
that doesn’t need a cofactor [5].
Furthermore, a widely-used altered version of DRONPA is pho-
todisassociating dimeric DRONPA (pdDRONPA) which dimer-
izes in violet light and darkness while monomerizing in cyan
light. It is also dimeric instead of tetrameric and is easily fused to
the two ends of an active ligand to turn it into a light-controlled
system [62].
Cobalamin-binding domains (CBDs) like CarH which rests as
homotetramers in the darkness and disassociates in green light.
This photoreceptor is cobalamin (vitamin B12) dependent and
although mammals naturally have this vitamin it may not be
sufficient to supply both the optogenetic system and the physi-
ological processes and thus must be given as supplements [63].
CarH activates and deactivates in minutes to tens of minutes,
which is significantly slower than most other photoreceptors and
may limit its applicability [64].
Phytochromes, like the frequently used PhyB with its pair-protein
PIF6 and BphP1 with its pair-protein PpsR2, are red light het-
erodimerizing and far-red light disassociating photoreceptors.
Although they can also disassociate in darkness, this can take
minutes instead of the seconds it takes in far-red light [59, 65].
Phytochromes all require exogenous biliverdin IXa (BV) to be
supplied so they can function [65].
Phytochromes are used often in optogenetic approaches that
involve deep tissues due to the tissue-penetrating property of red
and far-red light [66]. With their heterodimerization property,
phytochromes have been applied to many systems from control
of gene expression to the creation of light-controlled biomaterials
[65, 66].
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3.1.2 Intracellular Optogenetic Systems
Intracellular systems encompass a range of components, includ-
ing membranes, cytoskeletal structures, organelles, enzymes, and
other elements. For the sake of simplicity, intracellular sys-
tems contributing to intercellular interactions, such as exosome
formation, will also be regarded within the broad category of
intracellular systems. Opsins such as ChR2 can also be considered
in this category as the opening or closing of a channel is often an
initial step in the activation of intracellular pathways and indeed,
beyond their electrochemical effects, the more long-term cellular
effects of optogenetically induced channel protein activation have
been demonstrated to be mediated through various intracellular
pathways [17, 67].
Intra/Intercellular systems can be manipulated by using light-
controlled signal pathway alteration [68, 69], light-controlled
modulation of gene expression [25, 31, 70], and light-controlled
genetic engineering [71, 72].
Light-controlled signal pathway alteration is the use of optically
controllable intracellular proteins that act on a relevant enzyme,
membrane component, or protein to modulate the intra- or
intercellular pathways of the target cell. In their review, Zhang
and Cui (2015) categorized the multiple ways optogenetic systems
could control signal pathways as, activating specific parts in the
metabolism of proteins, trafficking proteins to different places,
and controlling genetic transcription. Here, we will consider
the first two to be part of light-controlled signal pathway
modulation and the latter to be light-controlled modulation
of gene expression, similar to Hu et al. [5] who differentiated
light-controlled signal pathway modulation from light-controlled
genetic engineering by the lack of light control of genetic
transcription, even though in both categories there is the addition
of exogenous genes that code the optogenetic system itself.
Light-controlled signal pathway modulation systems typically
only use a photoreceptor and an effector, but a localization guide
may also be included.
The effectors utilized in signal pathway modulation have a diverse
range of abilities. A typical example of these effectors are recep-
tor tyrosine kinases (RTKs), which can alter different cellular
pathways at various points of their cascade. Fibroblast growth
factor receptor 1 (FGFR1), epidermal growth factor receptor
(EGFR) rearranged during transfection (RET) [73], Eph Receptor
B2 (EphB2) [74], and tropomyosin receptor kinase A (TrkA)
[75] are all examples of RTKs that have been incorporated into
optogenetic systems. Furthermore, small GTPases that might be
associated with these RTKs or not can also be used as effectors
when fused with a photoreceptor; for example, the Raf/ERK
pathway associated with TrkA can also be manipulated through
Rac1 or Ras, small GTPases that have also been applied to
individual optogenetic systems [75, 76].
Effectors can be used with photoreceptors in various ways for sig-
nal pathway modulation. Homodimerization or homooligomer-
ization can easily promote the action of effectors such as RTKs
which activate strongly in dimers [77]. Heterodimerization can
be used to reactivate artificially split effectors or to activate
physiologically heterodimer effectors such as the receptor of the
Nodal signal pathway, which is naturally split into two proteins,
Acvr1b and Acvr2b [78]. Heterodimerization is also useful in
the selective localization of effectors in target areas, especially
for cell activities like migration where intracellular direction
matter [79]. Conformational changes of photoreceptors (most
commonly LOV) can be used to sterically inhibit an effector
or a localization guide. Intramolecular Dimerization with the
DRONPA photoreceptor system can theoretically be used to
control any eucaryotic kinase [5].
Another type of effectors newly introduced to light controlled sig-
nal pathway modulation are optogenetic intracellular antibodies
(optobodies). These are photoreceptor-fused intracellular anti-
bodies which are used for the modification of cellular function
by decreasing the functional amount of their target endogenous
protein. Intracellular antibodies are advantageous over gene-
knockout and inhibitory RNA methods because of their high
target specificity, ability to target functionally similar but sequen-
tially or genetically differing isoforms of proteins, proficiency
in recognizing diverse splice variants and post-translational
modifications, and sole localization at a single subcellular space
such as the nucleus or endoplasmic reticulum [80]. Optogenetic
modification of these intracellular antibodies has been made by
Yu et al. [81] when they split a Green Fluorescent Protein (GFP)
Antibody into two monomers and attached them to photoassoci-
ating receptors, exemplifying a heterodimerization module. The
result was the light-activated appearance of complete (dimeric)
GFP antibodies identified by the decrease of green fluorescence
in the cell as the GFP antibodies successfully sequestered away
the available GFPs.
Antibodies used for probing proteins have led to the creation of
some of the most widely used medical systems, such as ELISA and
Wes ter n Blot [ 82]. Up to now, there have been few ways to control
the actions of these antibodies, and most of those involved chem-
ical expression or degradation rather than reversible inactivation
[81]. Although the currently available research on optobodies is
limited, with the advantages of optogenetics meeting those of
antibody probes, they show great potential.
Light-controlled modulation of gene expression is the reversible
and photoactivable control of genetic expression. Light-
controlled genetic engineering systems typically require all
three elements; a gene guide, effector, and photoreceptor, and
may sometimes also use a localization guide such to bring the
system into the nucleus or onto the chromatin.
Effectors used for modulation of gene expression can directly
affect gene transcription or alter its expression epigenetically.
Activation domains like VP16 [31], p65 [83], VP64 [25]can
promote nearby gene expression, and epigenetic modifications
can be made by a variety of enzymes such as histone deacetylases,
methyltransferases, acetyltransferase inhibitors, or HDAC and
HMT recruiting proteins [25, 84].
A typical system used for this purpose may be an effector fused
with a photoreceptor monomer with the other photoreceptor
monomer fused to a gene guide, which come together under light
stimulation.
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Light-controlled genetic engineering is photoactivable genetic engi-
neering. Light-controlled genetic engineering systems require all
three elements of guide, effector, and photoreceptor, and can also
have a localization guide.
The commonly used effectors are the classic toolbox of genetic
engineering. CRISPR-Cas9 systems which assist in gene editing,
and Cre recombinases which are used for genetic recombination
[72] are all applicable in light-controlled genetic engineering as
well. Heterodimerization has been found applicable to Cas9 and
Cre recombinase systems both systems [26, 85] and so has confor-
mational changes, especially using LOV photoreceptors [72, 86].
Intramolecular dimerization has been utilized for CRISPR-Cas9
systems by fusing them directly to a dark-dimerizing photorecep-
tor (such as pdDRONPA) so that it blocks effector activity until
the light-induced disassociation of the dimer [87]. CRISPR-Cas9
systems have also been controlled indirectly by the addition of
optogenetic anti-CRISPR proteins which are inactivated by the
light-induced conformational change of the LOV-domain [71].
3.1.3 Extracellular Optogenetic Systems
Extracellular systems comprise elements situated beyond the cell
boundaries, encompassing structures like the extracellular matrix
(ECM) with all its fibrous components and externally introduced
tissue replacements like scaffolds. This category encompasses
optogenetic systems that regulate matrix proteins and membrane
receptors rather than directly influencing intracellular molecules.
Additionally, it includes intelligent biomaterials featuring inte-
grated optogenetic systems, exemplified by OptoGels.
Extracellular optogenetic systems do not require guides, and only
some require effectors; especially OptoGels utilize optogenetic
systems comprised entirely of photoreceptors bound to the bio-
material fibers themselves. These OptoGels are hydrogels that are
photoreceptive due to purified optogenetic proteins incorporated
into them. The first OptoGel was made by Zhang et al. [57]by
using UV-B Resistance 8 (UVR8), a UV-B light receptor, to link
nanofibers and peptides together. Since then, several more types
of OptoGels have been designed with a wide variety of light-
controlled properties such as stiffness, size, or phase changes
(see [88]). The optic controllability of such properties offers many
benefits in TERM, not only from a research perspective but also
from a clinical one. For example, the stiffness of a biomaterial
can define its use in wound filling while its porousness is
highly indicative of its ability to support cellular migration—
optogenetics can offer us an OptoGel whose stiffness can be con-
trolled by light without affecting its porousness [51]. Such Opto-
Gels can come with many variations, such as red light activation
instead of blue light using phytochromes instead of UVR8 [66].
Optogenetics can confer a biomaterial with the ability to change
states in a light-controlled manner, converting the gel into a
solution in a non-cytotoxic photoactivated manner, which is
promising for the future of 3-dimensional cultures, such as
organoids, since one of the major limitations of 3D culture is
harvesting the cultured tissue or cells without harming it [88].
The injection of wound-covering gels on difficult-to-heal injuries
is another example where light-controlled state changes can be
highly effective, as hydrogels which are liquid during injection
and solidify once on the wound site are especially valuable in
tissues such as the oropharyngeal mucosa [89].
Beyond biomaterials, extracellular optogenetic tools have also
been utilized in research on dynamic control of cell membrane
interactions. Jaeger et al. [90] have designed a phytochrome-
based T-cell receptor (TCR) stimulator that engages the TCRs
of non-engineered murine T-cells in vitro when triggered by
red light and successfully investigated the differences in T-
cell activation for spatiotemporal variations of TCR stimulation.
Following this, Armbruster et al. [91] have designed a simi-
larly phytochrome-based TCR stimulator for the activation of
non-engineered human T-cells. Unlike intracellular optogenetic
activation, extracellular receptor activation such as these does
not require the target cells to be engineered—rendering clinical
applications and research on physiological tissues possible.
Furthermore, extracellular optogenetic systems have also been
applied to the light-controlled genetic engineering of non-
optogenetic physiological cells through the use of optogenetic
viral capsids. Hörner et al. [92] have designed an adeno-associated
virus (AAV) capsid exposing PIF6, which when light-stimulus
is provided binds to a PhyB photoreceptor fused to an adapter
molecule that attaches to the target cell surface proteins, ensuring
that gene transduction will only occur in the presence of red
light. Their method could alter biology at the single-cell level
while also providing a non-cross-interfering way to dynamically
control the timing of different transgene transductions in a single-
culture of cells, which may be interesting for the transduction of
differentiation factor genes in TERM.
3.2 Integration of Optogenetic Systems into the
Body
Optogenetic systems have classically been used in conjunction
with transgenic animals or viral vectors. Transgenic animals are
used in research and can be useful for genetic designs that are too
large to fit into viral vectors with a packaging limit [93].
Viral vectors are the second most used method of optogenetic
system delivery. They are recombinant viruses whose replicative
ability has been suppressed to protect the researcher handling
them and to prevent cytotoxicity or systemic viral spread in the
subject being injected with them [94]. For the optogenetic system
to be expressed only in the correct cell types, the optogenetic gene
is fused with a cell-type specific promoter gene that only promotes
transcription in that type of cell. A gene encoding a fluorescent
protein is also frequently added to the package to confirm the
expression of genes in the target cells. Finally, the recombinant
virus containing these genes is given to the subject using either
local injection or intravenous systemic injection [95].
There are several viral vectors commonly utilized in optoge-
netic research, each with their advantages and disadvantages.
Lentiviruses are frequently used viral vectors; their advantages
lay in their capacity for carrying medium-sized genes, their low
cytotoxicity, and their ability to insert the genes they carry directly
into the host DNA, thus allowing for the transgenes to be passed
forward by mitosis [96, 97]. However, they only integrate one
transgene per cell, lowering opsin expression [94], and they’re
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usually obtained in low-titer compared to other viruses [98].
Adeno-associated viruses (AAVs) and their recombinant versions
(rAAVs) are the other types of frequently used viral vectors.
They’re advantageous due to the fact that they are not human
pathogens and have low cytotoxicity [99], come in a variety
of serotypes with different tissue tropisms [100], and provide
high-titer preparations [101]; their disadvantages are that the
transgenes they carry will remain extrachromosomal in the host
cell [93] and that they have a very small packaging capacity, which
poses a challenge when using complex optogenetic systems [102].
An interesting ability of some AAV serotypes is their ability to do
retrograde transmission [103] which allows for viral transduction
of deep nerves by injection into more superficial nerves or their
associated muscles, at which point they will transduce only
the nerves controlling that muscle group [104], or even the
intraperitoneal space [105]. Other viral vectors are Rabies Virus-
based vectors and Herpes Simplex Virus (HSV)-based vectors
both of which are capable of retrograde transmission but are not
commonly used due to their pathogenicity [102]. HSV vectors can
carry a very large gene packaging [106].
Beyond transgenic animals and viral vectors, other novel methods
of optogenetic system delivery are being researched. For example,
Hsieh et al. [107] developed a self-healing injectable hydrogel
system designed for the delivery of an optogenetic tool without
the use of viral vectors. They embedded both stem cells and opsin-
carrying plasmids into the hydrogel and then used it for injection,
during which the mechanical stress caused the plasmids to enter
the stem cells, granting optogenetic properties to the cells.
Unlike chemogenetics, which has less spatiotemporal precision
but can work without local light delivery [108], the activation
of optogenetic systems requires light by definition. While this is
what confers optogenetics its typical spatiotemporal precision, it
is also a disadvantage as light is difficult to get into the body.
Different wavelengths of light are able to reach different depths
in the body: low wavelength light penetrates less while higher
wavelengths can penetrate more. UV light has the most shallow
penetration; when separated to UVC (100–280 nm), UVB (280–
320 nm) and UVA(320–400 nm), only UVA can reach to the very
depths of the epidermis, which is the most superficial layer of
skin with a less than 1 mm of thickness [109]. Blue (400–470 nm)
and green (470–550 nm) light can only penetrate skin thickness
between 0.5 and 2 mm [110]; and these wavelengths activate
several important photoswitches like CRY2, LOV2, and ChR2
[111] which may lead to the necessity of an external light source,
causing difficulties in awake animals [112]. Yellow/orange (550–
630 nm) and red (630–700) light can penetrate deeper, between 1
to 6 mm skin thickness, reaching below the dermis even in thick
skin, and IR light (700–1000 nm) has the maximum penetration of
all light wavelengths, transmitting past even bone to reach deeper
tissues [110, 113]. Specific wavelength ranges to optimally reach
different areas of skin and connective tissue can especially be
found in literature of dermal phototherapy.
The intensity, beam width, and duration of the light beam utilized
can also affect the penetration depth of light. Increased beam
width can lead to greater penetration of the central photons up
to a limit (beam width of 10 mm) where the maximal penetration
is reached [111], while short pulses of light can in some circum-
stances reach deeper in the tissue than continuous light [110].
There are some methods to provide light delivery in deep tissues:
(1) the usage of multi-photon excitation which can provide
tissue-penetration to blue light but requires equipment that is
costly and bulky [50, 114], (2) the usage of red or far-red-shifted
photoswitches which are activated by the more penetrative red
spectrum light [115], (3) optic cuffs made of optic fibers and
wireless/wired LEDs which are used for optogenetic control of
peripheral nerves but suffer from a requirement for invasive
surgery [116] and, (4) bioluminescent-optogenetic systems where
genes encoding a light-producing enzyme such as luciferase is
added to the optogenetic system, forming a luminopsin that
can be activated by systemically given luciferase substrates but
undermines the characteristic spatiotemporal specificity of opto-
genetics [117]. Compared to CNS-based light delivery approaches,
though, methods for light delivery to other tissues in the body are
lacking and require more research.
3.3 Limitations of Optogenetic Systems
Optogenetics is primarily hindered by the disadvantages of its
methods of delivery. Transgenic animals bring difficulty because
they take a long time to breed and there may be unwanted gene
interruptions if the researcher doesn’t keep careful track of the
genome of the animals [93]. It is also impossible to depend on
transgenicity in clinical practice. However, viral vector delivery
is also not without issues. Firstly, no. matter how extensively
altered they are, viruses can still cause cytotoxicity and immune
response, so they should be closely monitored for the protection of
the host’s health [118, 119]. Moreover, some of the gene products
necessary for the generation of vectors are themselves cytotoxic
[120]. Finally, viral delivery is a highly variable method where
the number of infected cells and the expression level of the
optogenetic system cannot be stabilized over multiple hosts,
causing disparities between the results of different experiments
or patients [121, 122].
Light delivery is another limitation that affects the use of
optogenetic systems. Light penetration into deeper tissues can
be difficult to achieve for photoswitches with lower wavelength
activations, and as stated in the chapter above, light delivery
methods all have their individual advantages and disadvantages.
Beyond these, light can also have its own detriments. UV light
required for some photoreceptors, such as UVR8, is cytotoxic
and can only be used in short pulses [35]. Sustained light
exposure at non-UV frequencies can also cause an accumulation
of toxic waste due to the activation of endogenous photoreceptive
enzymes [123]. Furthermore, light of any color can damage cells
directly as well as through breaking cell media down in ways
that produce cytotoxic byproducts, and this toxicity has been
considered to be underestimated in both light-based cell imaging
and in vitro optogenetics. In vivo, there may or may not be
considerations for safety, because blood has been theorized to
carry away toxic byproducts and prevent their buildup [123].
Another safety concern arises from the fact that optogenetic sys-
tems may come with innate immunogenic properties. The viral
vectors used in the delivery of the system can cause long-lasting
humoral immune responses in ways that differ for vector type,
serotype, delivery method, delivery dosage, and the host animal
[124]. The innate and adaptive immune responses characteristic
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to each individual or system from muscle to nerve to liver can
create both a variable toxicity and a changing viral transduction
efficiency for each experiment, creating an unavoidable obstacle
on the path to clinical success [125]. Moreover, there is increasing
evidence that not only viral vectors and fluorescent proteins, but
also the photoreceptors, even clinically used opsins like ChR2,
can cause an immune response [126]. In fact, the phenomenon
of loss-of-expression of optogenetic systems in peripheral nerve
activation seems to be caused by T-cell mediated immunogenicity
against photoreceptor-expressing spinal cord neurons, leading
to neuron destruction and muscle wasting due to denervation,
which is supported by the fact that drug-induced and genetic
immune suppression can both prevent this characteristic loss-
of-expression of the optogenetic system [126, 127]. Leading from
this, a question arises on whether immunosuppressive drugs
should be provided alongside optogenetic treatments. Addition-
ally, most research on the immunogenicity of optogenetic systems
is on opsins and on nerve cells, and there is a need for the
immunogenicity of non-opsin photoreceptors on other relevant
tissues to be investigated before they can be applied in therapeutic
settings.
There is also an unknown risk of side effects that may arise from
the various intracellular pathways that are of interest to opto-
genetic regenerative medicine. Abnormal or over-expression of
BMP [128], AKT [129], TGF-β[130], or integrins such as αVβ3[131]
can lead to tumorigenesis. All these endogenous pathways have
been proposed as an area of interest for optogenetic approaches
to regenerative medicine and if they are to be used in a clinical
setting, their many patterns of activation and subsequent cellular
effects need to be extensively researched.
The final limitation of optogenetic research in TERM is that it
yet remains at an early stage. Most research is still done at less
developed levels such as in vitro or on fly and mouse systems,
and this is especially the case for non-opsin photoswitches. The
change from mouse- and fly-targeting optogenetic systems to
those applicable to primates is ongoing, and while primate-based
optogenetic research has begun taking off it is primarily only seen
in the field of neuroscience [132–134]. A vector’s functions can
be lessened or changed in unexpected ways when it is moved
from a mouse to a primate, and non-human primates and humans
themselves have differing reactions and innate immunities to
vectors [124, 135].
Before optogenetics in TERM can transcend research and enter
the clinics, there needs to be a standardized, streamlined
approach to the production and delivery of the system to
ensure patient comfort, decreased variances in effect, and fewer
immunogenic and cytotoxic side effects.
4 TERM with Optogenetics
A big part of TERM is tissue engineering, which focuses on
the actualization of artificially produced bio-adaptable matter.
Optogenetics is an invaluable tool in this production as it can
reveal, manipulate, or induce various cellular interactions on
the road to artificial tissue engineering. For example, optoge-
netics can be used to assess embryogenic cell migration. A
photoactivatable form of Rac, a type of small GTPase down-
stream of multiple pathways, was utilized to demonstrate cell
cluster movement in the direction of the cell with the highest
Rac concentration or, in this case, towards the source of light
[83, 136, 137].
It has also been used to probe the role of several pathways in fetal
development and cell differentiation; such as the work of Johnson
and Toettcher [18] on dynamic Erk signaling. Furthermore, tissue
morphogenesis, the change of shape of tissue by the coordinated
movement of cells that especially occurs in embryos, can also be
researched through the use of optogenetic tools; Guglielmi et al.
[55] worked on a CRY2/CIBN optogenetic system to control phos-
phatidylinositol phosphate (PI(4,5)P2) which is decisive on the
actin-related collective apical constriction of cells during tissue
invagination and successfully probed the relationship between
tissue folding, cellular communication and local geometrical
obstacles.
Optogenetics can also have a role beyond tissue engineering.
Regenerative medical treatment can greatly benefit from
optogenetics, and although clinically optogenetics is still newly
utilized in regenerative medicine, success stories do exist.
Especially in areas such as ophthalmology, where treatment to
restore vision by the insertion of channelrhodopsins after retinal
degeneration have gotten to the point that several clinical trials
are currently under way and, although still investigationally,
patients are being treated using optogenetic approaches to
regenerative medicine [16].
In areas of regenerative medicine that do not concern organs,
research has yet to reach such advanced stages. However, while
still at early stages, spatiotemporally precise control of cell activity
in vivo holds great potential for regenerative treatments and it can
assist in various ways from cell-lineage tracking in physiological
conditions to the promotion of wound regeneration instead of
scar-based repair which—while invaluable in the continuation
of life when faced with major tissue trauma—can be greatly
debilitating to the tissue’s function [1, 10]. For example, in the
cardiac cell model Toh et al. [138] have succeeded in promoting
wound closure through cardiomyoblast migration instead of
fibroblastic scar formation by using optogenetic systems. Table 1
provides a summary of significant advancements of optogenetics
in TERM for different systems, including brief descriptions of
studies mentioned in this section.
4.1 Skin and Connective Tissue
For the body to naturally heal a wound, four phases must
be achieved in accurate succession and duration: hemostasis,
inflammation, proliferation, and finally remodeling. However,
many factors may interfere with these phases, causing subpar
or improper wound healing or chronic wounds that don’t heal.
The wound’s vascularization and oxygenation, infection, the
hormonal state of the patient, mechanical stresses, and some
conditions such as diabetes can interfere with these phases and
impair wound healing [159].
Diabetic chronic wounds are one example of wounds that require
medical interventions to heal, as the body cannot balance the four
healing phases correctly and effectively [159]. However, medical
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FIGURE 3 Current state of optogenetic approaches to TERM of skin and connective tissue. (A) Healing exosomes are formed when light stimulus
leads to the binding of the eNOS-mCherry-CRY2 fusion with the membrane-attached CIBN-EGFP-CD9 fusion. (B) Light stimulus causes the VVD
pairs to heterodimerize, leading to Tiam1 and thus Tiam1-Rac ligands building up in the direction of light input, creating directional filopodia. (C) This
OptoGel can change its porousness and stiffness in response to light stimulus, which unlatches SNAP-tags from their LOV-domain cages and allows
them to bind each other. (D) Through an intricate system that depends on doxycycline and light stimulus, theincreased expression of a Cre-recombinase
gene leads to subsequent genetic engineering. Created using BioRender.com.
science is still insufficient in their definitive treatment and more
diabetic patients are suffering from delayed wound healing every
year [160]. Hyperglycemic microenvironment caused by glucose
imbalance, prolonged inflammation, secretion of reactive oxygen
species (ROS), and impaired nitric oxide (NO) production are
also pathophysiological factors that cause chronic wounds in
diabetes [161]. The current innovative solution to these problems
is either direct mesenchymal stem cell (MSC) injection or the
application of exosomes full of healing factors derived from
MSCs [162].
To improve the healing of diabetic chronic wounds, Zhao et al.
[160] have taken up the optogenetic toolkit to create “healing
exosomes” with greater yield per MSC, more effectiveness per
exosome, and added healing factors in each produced exo-
some (Figure 3A). The selected healing factor was eNOS, a
NO-synthesizing enzyme for the increased oxygenation and
angiogenesis of the open wound. Their approach was to use
light-controlled modulation of intracellular systems to produce
exosomes loaded with eNOS by inducing the production of
two proteins: CIBN-EGFP-CD9 and eNOS-mCherry-CRY2. Here,
EGFP and mCherry are fluorescent proteins used for the eval-
uation of protein production. In the presence of light, CRY2
and CIBN heterodimerize on the plasma membrane and bring
the exosome-formation promoter CD9 in place, forming an
exosome around the loaded eNOS [163]. Zhao et al.’s exper-
iments showed that the application of “healing exosomes”
to cells in a hyperglycemic environment elicited a remark-
able improvement in their biological functions and a notable
decrease in inflammatory factors and ROS-induced apoptosis.
In vivo, they observed that the diabetic mice that received the
treatment had increased wound healing, vasculogenesis, and
matrix remodeling.
Another approach using light-controlled modulation of intra-
cellular systems involves a process known as “wound contrac-
tion”. In embryos, wounds are closed by the formation of an
actin string around the open area, which then contracts like
a “purse-string” and closes the wound. Meanwhile, in adults,
wounds are closed by actin-based cell migration where small G
proteins work in tandem with RhoA controlling stress fibers,
Rac1 controlling lamellipodia formation and Cdc42 controlling
filopodia formation [164, 165]. Using optogenetic tools, these
proteins and their respective pathways can be controlled. Ueda
and Sato [32] created an optogenetic system for Tiam1, which
controls lamellipodia formation through the Tiam1-Rac1 axis
(Figure 3B). In their study, light stimulus caused the photore-
ceptor VVD to be attracted to its oppositely charged magnetic
pair on the plasma membrane, the presence of Tiam1 on the
membrane leading to actin-based protrusions in the direction
of the light. On the other hand, Valon et al. [79]usedthe
RhoA protein in their optogenetic system, using light-induced
heterodimerization by CRY2/CIBN to transport RhoA to the
plasma membrane where it upregulated cellular tension and
tissue compaction, which reversed easily in darkness when
the system separated and the RhoA was sequestered onto the
mitochondrial membrane. Optogenetic regenerative medicine
incorporates light stimulus as a method to manually control actin
formation and direct cell migration, tissue contraction and wound
closure.
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An important TERM treatment is for wounds that are too
wide or deep to regenerate naturally. This can be achieved by
dressing the wound with biomaterials to keep the structural
stability and provide a scaffold for the migration of cells as the
wound heals [166], or by tissue grafts or flaps using surgical
techniques [167]. Optogenetics is a valuable tool for both of these
extracellular approaches.
To improve the outcomes of tissue grafting, a necessary surgical
intervention for wounds too large to naturally heal, TERM
strategies focus on the creation of an adhesive to attach the
tissue surface to gauze for easy transportation and placement
of the graft on the wound surface. This gauze will, however,
need to be removed after the graft starts healing—and herein
lies the problem. If the adhesive is not degradable, the removal
will be painful, and there may be damage to the graft as well
[168]. Moreover, the material must be flexible and durable, with
biocompatibility and non-antigenicity [169]. A hydrogel wound
dressing is also indicated in wounds that are difficult to close,
like those in the wet and mobile environment of the oropharynx
which creates a challenge for the treatment of oral mucosal
diseases. In this case, especially, there is a need for a hydrogel
that can be injected in a liquid state before being converted
into a solid to cover the wound. The most effective method
for this is light-controlled state changing of the hydrogel, as
heat and chemicals are difficult to use in state changes, a heat-
related chemical process itself. Photocrosslinkable hydrogels are
currently in use for this [89] but they are recommended to
be used with caution, as the irreversible crosslinking-induced
state change caused by light exposure produces ROS and free
radicals, which have a potential cytotoxicity [170]. Comparatively,
optogenetically controlled state-changing hydrogels such as the
one designed by Zhang et al. [57], have low phototoxicity and
high reversibility and can thus provide a much-needed solution to
this issue.
OptoGels also provide various changeable biomechanical proper-
ties to encourage different responses from cells, acting similar to
the ECM which provides cells with a variety of mechanic signals.
The hydrogel produced by Hörner et al. [66] using phytochromes
was capable of such biomechanical control and demonstrated the
ability of optogenetic mechanical changes to alter the migration
patterns of T cells and the gene expressions determining the
cell fate decisions of MSCs. Following them, Hopkins et al. [51]
have developed an OptoGel from nonsynthetic collagen polymers
and OptoSNAP, a purified LOV-domain containing protein fused
to a SNAP-tag (Figure 3C). This OptoGel provides an example
for enhancing the uses of biomimetic materials through the
integration of optogenetic systems.
Lastly, optogenetics has provided a new approach to TERM
beyond clinically used materials; optogenetics is instrumental
in developmental research for skin and flesh. The epidermis
is the first barrier between the body and the exterior environ-
ment, and thus it is harmed quite often and must be repaired
quickly. Researching skin is essential in researching repair and
regeneration, as it is one of the few tissues that demonstrates
both with such frequency. As such, spatiotemporally precise
reversible control of the skin cell genome in vivo is a worthwhile
technology. Li et al. [10] have utilized optogenetics to create a
light-controlled genetic engineering tool that induces transcrip-
tion in the presence of blue light and doxycyclin, named Light
Inducible rtTA (Li-rtTA) (Figure 3D). Li-rtTA is comprised of
CRY2 fused with the transcriptional promoter VP16 and CIB1
fused with the doxycycline-responsive rTetR and exogenous gene
guide TetO. rTetR and CIB1 attach to TetO in the presence of
doxycycline and to CRY2 and VP16 under the light stimulus.
By adding a gene for a Cre recombinase next to TetO, they’ve
created a system that will promote genetic recombination upon
light and doxycycline stimulation. Li et al. [10] used their
optogenetic tool on the normally hairless plantar hind paws of
mice to delete an endogenous gene locus that inactivates Wnt.
Under light and doxycycline stimulation, their optogenetic tool
successfully removed the Wnt-inactivating gene, leading to the
abnormal growth of hair follicles on the applied area. This showed
the workability of Li-rtTA for in vivo spatiotemporally precise
genetic modifications. A significant area where such tools may be
applicable is “lineage tracing”, one of the most recent methods of
researching skin regeneration. By labeling cells to follow the fates
of their descendants the researcher may identify the functions of
various cells and the migrative patterns of the tissue in general,
and this method is particularly advantageous due to its ability to
demonstrate cell fates in their physiological environment rather
than in culture or transplantation [171] which provides valuable
insights for research on tissue engineering.
4.2 Vessels
Optogenetics has been applied to the control of vessels for years
now. Most of such research was interested in the manipulation of
vasoconstriction and vasodilation, giving scientists and medical
practitioners an effective tool in the spatiotemporally specific
control of local blood flow in various tissues [42, 172, 173].
Research aiming to increase angiogenesis and vessel regeneration
using the various benefits of optogenetic systems does, however,
also have a considerable impact.
One such research is by Müller et al. [65] who designed the
first red light/far red light controlled bi-stable mammalian gene
expression modulation system and applied it into studying the
possibility of temporally controlling neovascularization in chick
embryos. Their phytochrome PhyB-based system was made out
of three components the gene guide TetO inserted before a VEGF
gene, the TetO-binding TetR fused with PIF6, and PhyB fused
with the transcription factor VP16. In the presence of red light,
PhyB would heterodimerize with PIF6 and bring VP16 in range
to promote VEGF transcription, while far-red light would trigger
quick disassociation and the cease of gene promotion. They
successfully demonstrated VEGF-induced increased neovascu-
larization and angiogenesis in the chorio-allantoic-membrane of
chick embryos in vitro.
Liao et al. [52] have focused instead on understanding the
true angiogenesis-related interaction of kindlin-2, an integrin-
interacting intracellular effector [174], and αVβ3, an integrin-type
transmembrane protein that has a vital role in endothelial cell
adhesion, migration and survival and is high in concentration
in proliferating endothelial cells during both wound healing
and pathological angiogenesis [175] but which has also been
found to be unrelated to angiogenesis in some other models
[176]. Liao et al. [52] use an optogenetic system where the
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LOV-domain photoreceptor LOVpep and its heterodimerization
binding partner ePDZb1 were respectively fused to αVβ3and
kindlin-2. This system allows for the LOVpep to dimerize with
ePDZb1 when activated by blue light, thus making kindlin-2
interact with αVβ3. In their in vitro endothelial cell assay, Liao
et al. [52] successfully demonstrated that the optogenetically-
induced interaction of αVβ3 and kindlin-2 in the endothelium
can lead to endothelial cell migration, as well as the formation of
podosomes and angiogenic sprouts, all of which are important in
angiogenesis. They, however, also noted that the αVβ3/kindlin-
2 interaction is not an isolated angiogenic trigger and requires
at least 5 other binding partners to promote the meaningful
processes that lead to angiogenesis. An important cytoplasmic
protein, talin, was later shown to be one such binding partner
that enhances integrin (and thus αVβ3) function and increases
angiogenesis and sprouting [140]. Liao et al. [140] used talin fused
with CRY2 alongside plasma membrane-bound CIB1 to induce
blue light activated membrane localization of talin, leading to
increased αVβ3 activation and endothelial cell migration.
A study was performed by Yamanaka et al. [141] on a human
blue light-activated Ca2+channel switch 2 (hBACCS2) to increase
intracellular calcium levels of endothelial cells in vitro by induc-
ing an extracellular calcium influx, increasing concurrently the
activities of endothelial NO, NFAT and NF-κB. Yamanaka et al.
[141] mentioned a theoretical basis for such optogenetic calcium
influx to stimulate angiogenesis and identified it as an area that
needs further research. Another optogenetic study on endothelial
intracellular signaling was by Zhou et al. [142] where they used
optoAkt1, a CRY2/CIB1-based membrane-translocation system
that when activated leads to Akt1 accumulating on the plasma
membrane and thus getting phosphorylated by the membrane-
bound PDK1. They interrogated the different signaling meth-
ods of phosphorylated-Akt1—pulsed versus sustained, differ-
ent durations, different intensities—and discovered the diverse
downstream activities triggered by such signals. Their research
provides an important basis for future research on the effects of
Akt1 signaling, a pathway known to regulate angiogenesis [177]
and its downstream substrates on endothelial cells.
Mahlandt et al. [143] designed an optogenetic system to control
Rho GTPase, an intercellular pathway enzyme that regulates the
connection between cells and the endothelial barrier strength of
vessels, named optoRhoGEF. In their design, they fused GEF, a
Rho GTPase activator, with iLID, a light-responsive dimerizing
molecule designed by Guntas et al. [178]fromaLOV2domain
with an increased light-induced change in affinity of more
than 50-fold. Mahlandt et al. [143] confirmed that optoRhoGEF
allowed them to control cell size, morphology, contraction and
local extension as well as vascular endothelial barrier strength in
a reversible and temporally precise manner.
4.3 Bone
The base and support of the musculoskeletal system, bone, has
the unique ability to regenerate itself, allowing the skeletal system
to heal itself from fractures and even small amounts of lost
tissue. Despite this regenerative ability, though, traumatic loss,
tumor-related lysis or surgical removal causes defects that may
be debilitating without surgery and even with it may lead to
disability, financial deficits, and secondary morbidities such as
infection, pain, or fibrotic non-union [179]. Considering donor-
site morbidity associated with bone grafts and the immune
rejection and lack of regeneration associated with other options
such as allografts, xenogenic tissue grafts, and synthetic prosthet-
ics [180], it is clear why tissue engineering is instrumental for the
future of orthopedics.
The usual method of TERM applied to bone defects is the
insertion of a scaffold seeded with osteoblasts, chondrocytes
and MSCs that are obtained from the patient’s tissues and later
expanded or differentiated in culture, allowing the cellular prolif-
eration and function to continue while the scaffold architecture
eventually shapes the regenerating bone and cartilage [181]. In
this manner, the cells within the scaffold provide more cells
through proliferation and secrete necessary chemical signals
for the inflammatory modulation, migration and proliferation
of nearby cells [179, 182]. Such signals activate intracellular
pathways such as Lhx8, which promotes MSC proliferation but
inhibits osteogenic differentiation, and BMP, which promotes
osteoblast differentiation and so disallows proliferation [48].
There has been some important research concerning the opto-
genetic manipulation of these pathways. Humphreys et al. [47]
worked on the creation of a light-homodimerizing BMP like
receptor (optoBMP) which would homodimerize and activate
in the presence of light and go on to phosphorylate SMARD1
and SMARD5, the ligands of BMP. On the other hand, Huang
et al. [144] used light-controlled genetic engineering to promote
the transcription of Lhx8 with an optogenetic tool made of a
LOV-domain fused to the effector VP16, and the LOV-ligand
GIGANTEA (GI) fused to the guide molecule Gal4. The Lhx8
gene was added exogenously alongside the 5xUAS exogenous
gene that Gal4 attaches to. Under the light stimulus, LOV and
GI came together, making the transcription promoter VP16 reach
Lhx8 and increase its transcription. The precise control of Lhx8
promotion is instrumental as generalized Lhx8 underexpression
can lead to insufficient cellularity and impair regeneration, but
its overexpression can cause cell overpopulation, which may
lead to early bone aging and fragility [183]. Not only that, but
the timing of Lhx8 expression is also vital: Huang et al. [144]
demonstrated that late Lhx8 activation can have deleterious
consequences in bone regeneration because it downregulates
osteogenesis, and when applied to a calvarial defect modeled on
mice, the optogenetic Lhx8 promoter used to upregulate Lhx8
in the early stages of the injury resulted in the greatest bone
regeneration and healing for the mice.
One step beyond these, Wang et al. [48] engineeredan optogenetic
system which promotes Lhx8 until stimulated, at which point it
silences the Lhx8 gene and promotes BMP2 instead (Figure 4).
To achieve this, they also used two proteins, LOV fused with
VP16, and GI fused with Gal4. Additionally, they introduced
two exogenous genes; the gene Lhx8, and the fusion of the
gene BMP2 with 5xUAS (binding sequence of Gal4) and shLhx8
(binding sequence that silences Lhx8). With this setup, Lhx8
was constitutively activated in darkness, while light stimulation
would recruit the photoactivatable transcription promoter (LOV-
VP16) to the guide (Gal4-GI bound to 5xUAS) in order to activate
BMP2 and shLhx8 instead. Their study on the optimal timing
to activate this system in a calvarial bone defect model of mice
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FIGURE 4 A calvarial injury model mouse is injected with viral vectors carrying an optogenetic system designed for bone regeneration. After the
exogenous genes are in place and the optogenetic proteins have been synthesized by the mouse cells, the exogenous gene Lhx8 is expressed, promoting
cell proliferation. At blue light stimulation, the exogenous gene BMP2- shLhx8 is expressed, silencing Lhx8 and promoting bone maturation. Created
using BioRender.com.
demonstrated up to 80% reduction in the bone wound size for the
optimally timed light stimulus (during the days 9 to 14 after the
injury) compared to less than 20% reduction in the least effective
timing (during days 2 to 7) and around 5% reduction in the control
group without any BMP2 or Lhx8 stimulation (Figure 3).
4.4 Cartilage
Bone is not the only tissue that requires new tissue engineering
approaches; cartilage is a vital component of the musculoskeletal
system that can be harmed by various illnesses or injuries
throughout life and as it is an avascular tissue with little self-
healing ability, it is a prime option for tissue engineering-based
treatments [184]. However, it is challenging to produce cartilage
in a laboratory. It’s a tissue with a gradient of histochemically
varying zones, which is difficult to achieve using chemical
promoters, especially in diffusion-limited tissue constructs and
pseudo-vascularized systems like organs-on-chip [146] because
TGF-b, the predominant inducer of cartilage differentiation in
vitro, has been shown to promote the close to physiological
organization of zones in the cartilage tissue only if produced in
a spatiotemporally controlled manner [185].
To this end, Wu et al. [146] have demonstrated that the optoge-
netic control of TGF-b promotion can form the zonal structure
of cartilage from human MSCs. They used the two parts of
the innate heterodimer TGF-b Receptor (TGFBR), TGFBR1 and
TGFBR2, and fused those with CRY2 and CIBN to achieve a
blue light activatable inducer of the TGF-b signaling pathway.
With this system, they successfully achieved three-dimensional
physiologically similar cartilage tissue as well as cartilaginous
differentiation of genetically modified human MSCs without
differentiation of the unmodified MSCs in a single shared envi-
ronment. Wu et al. also used the TFG-b system to promote the
formation of smooth muscle and tendon tissues from human
MSCs in vitro.
4.5 Muscle
Research concerning skeletal muscle growth is hampered by the
lack of an optimized method to stimulate muscle contraction
in vivo. The two most common methods available both have
significant drawbacks; muscle contraction by forced exercise has
been shown to increase anxiety and lead to several undesirable
effects such as systemic inflammation and neuronal or cardio-
vascular damage [186, 187], while muscle contraction by electrical
stimulation using subdermal needle electrodes leaves the muscle
open to infection and further poses a physical obstacle to the
limb’s movement. Optogenetics has been proposed as a new
method to stimulate muscle contraction noninvasively and with-
out any superfluous systemic effects. Bruegmann et al. [147]have
used genetically modified mice with a plasmid containing ChR2
promoted by a chicken-β-actin promotor which only activates in
skeleton muscle, and successfully demonstrated the possibility of
using optogenetics to contract muscle cells in vivo.
Since then, optogenetics has been applied to muscle systems
for research relevant to the field of regenerative medicine, such
as evaluating the effects of muscle loading on the Achilles
tendon’s insertion on the calcaneus bone. It has also been used
in the control of 3D muscular structures, as it is a simple
method for simulating movement in such 3D tissue cultures
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[149, 150]. Through the culture of ChR2-expressing muscle
cells and vasculature in a 3D model, Osaki et al. [41]have
demonstrated the connection of muscle contraction with nearby
angiogenesis; both an angiogenic effect—with increased vessel
sprouting—and an anti-angiogenic effect—causing some regres-
sion of vascular tip cells as if due to abnormal angiogenesis.
Furthermore, optogenetics is in consideration as a regenerative
treatment to reduce muscular atrophy after traumatic or chronic
nerve injuries or diseases. Some of the current treatments are
the stimulation of muscle through the peripheral nerves (indirect
stimulation) or the electrode-based stimulation of muscle fibers
(direct stimulation) [188–190]. Another less relevant treatment is
the transplantation of embryonic or iPSC-derived motor neurons
into the site, which carries tumorigenic risk [151]. Electrical stim-
ulation of atrophied muscle groups can facilitate their future use
as well as strengthen them [191], and it can even restore muscle
function and improve bone density loss after debilitating injuries
such as spinal cord injuries [192]. However, electrode-based direct
stimulation has some distinct disadvantages: (1) Due to the insu-
lation between muscle fibers, their direct stimulation consumes
great energy, leading to the production of large amounts of
toxic waste [193, 194], (2) direct stimulation is often painful or
uncomfortable due to the co-activation of nociceptive and other
neurons [195], and (3) the electrical charge is indiscriminative
and activates both fast and slow fibers, creating a muscle fatigue
that differs between muscle groups and people, which makes it
difficult to account for [196].
Comparatively, optogenetic direct stimulation of muscle cells
allows for more selective activation of specific muscle fibers
without concurrent nerve activation and is a relatively non-
invasive treatment. It is also unlike electrode-based treatments
in that it follows the physiological order of muscle recruitment,
leading to less fatigue; optogenetically created muscle tension
has been shown to last 20+minutes, compared to electrically
created tension which disappeared completely after 4 minutes
[40]. Optogenetic direct stimulation has already shown promise
in the treatment of denervation atrophy after peripheral nerve or
spinal cord injuries, demonstrated through light stimulation in
genetically altered mice with ChR2 in their muscle cells [151,152]
as well as in wild-type mice injected with AAV viral vectors that
carry ChR2 [116].
4.6 Peripheral Nerves
Optogenetic modification of peripheral nerves has been used in
a multitude of ways, from the motor control of breathing [197]
and research on digestion [198] to the control of somatosensory
and pain reception [199–201]. In the field of regenerative science,
optogenetic control of peripheral nerves has been used for both
injured nerve regeneration and the regeneration of the motor unit
of muscle cells connected to a specific nerve.
In the restoration of skeletal muscle control after traumatic
or disease-associated paralysis, optogenetics has not only been
proposed to take the place of direct stimulation of muscle
fibers but also indirect stimulation through the activation of the
attached peripheral nerves. Indirect stimulation using electrodes
has some of the same disadvantages as direct electrode-based
stimulation; concurrent afferent nerve activation eliciting pain
and discomfort [202] and the unphysiological recruitment of
larger motor units before smaller ones causing increased muscle
fatigue [189]. Optogenetic systems show the potential to overcome
the disadvantages of indirect muscle stimulation.
Most studies on the stimulation of peripheral nerves to activate
skeletal muscle have focused on the widely used optogenetic
photoreceptor ChR2; since 2010, in animals that express this
molecule transgenically in their motor nerves [40] and since
2013, in those that have been injected with viruses encoding
the molecule [116]. Although ChR2-encoding viruses can be
given systemically, local injection of a specific muscle with
peripheral-nerve targeting viral vectors carries significance due
to viruses such as AAV and HSV which are capable of retrograde
transduction. Gundelach et al. [189] have emphasized this point
and proposed that, as some vectors are capable of encoding single-
muscle-specific neurons in a large nerve and as there are various
photoreceptors with activation at different light frequencies, the
retrograde transduction of several muscles in a limb with different
photoreceptors can allow the optogenetic control of a single large
nerve to differentially stimulate various muscle groups.
Even without muscle wasting, peripheral nerve injuries and
illnesses can cause debilitating problems for both the livelihood
and the survival of a patient. Although peripheral nerves have
some regenerative potential unlike central nervous system neu-
rons, their regeneration is still often too slow, too insufficient,
or misguided, and incomplete recovery can lead to neuroma
formation or neuropathic pain even after surgical intervention
[203]. Electrical stimulation is known to improve nerve regener-
ation [204], and optogenetics has recently entered this field as a
non-invasive, less painful alternative to electrode-based methods.
Park et al. [17] have shown significant results in the optogenetic
stimulation of the regeneration of peripheral nerves in vitro,
using Dorsal Root Ganglions (DRGs) from transgenic mice
with ChR2-expressing DRG. The light stimulation of the ChR2-
DRGs resulted in larger total neurite outgrowth areas (around 3
times more) as well as longer neurites (around 1.5 times more)
compared to unstimulated ChR2-DRGs and wild-type DRGs.
Intriguingly, they also noted that when two DRGs were paired
up and then subjected to optical stimulation, wild-type DRGs in
the presence of a ChR2-DRG also had their growth enhanced and
directed towards the neighboring stimulated ChR2-DRG. They
suggested this to be caused by the fact that optogenetic depo-
larization results in an increase of soluble neurotrophic factors,
BDNF and NGF, as well as an increase in Schwann cell migration,
all of which contribute to peripheral nerve growth. Although the
optogenetic stimulation in short bursts (30 minutes to 3 hours)
led to enhanced growth, stimulation at the same frequency for
longer durations (1 day to 3 days) did not lead to any significant
growth compared to the control groups—this was proposed to be
due to TrkB-downregulation-related desensitization to prolonged
stimulation that was previously discovered similarly regarding
electrical stimulation by [148, 205]. In their research, Park et al.
[17] identified the method leading to maximal neurite outgrowth
to be 1-hour optogenetic stimulation of 5 to 20 Hz frequency
alternating between 1-second stimulation and 1-second rest. Ward
et al. [154] applied this method at 20 Hz frequency and tested
the advantages of optogenetic stimulation on peripheral nerve
injuries in vivo, using ChR2 transgenic mice with transected
16 of 24 Nano Select,2024

FIGURE 5 After cutting the axon of a transgenic peripheral nerve expressing optoRaf or optoAKT, three different illumination methods are tried.
When given no light stimulation, the peripheral nerve grows little and shows no directional growth. When given generalized light stimulation, the nerve
grows more, but preferentially away from the injury site which disallows it from reaching the correct location. When given directional light guided
towards the injury site, the nerve grows more and towards the injury site, allowing it to reach the correct location. Created using BioRender.com.
sciatic nerves. They saw that the mice that received a one-time
optogenetic stimulation in their peripheral nerves had more
muscle activity, larger axon regenerations, and a bigger number
of reinnervations at target muscles after 4 weeks of rest. It was
later shown that light-induced neuronal activity was effective in
the regeneration of both sensory and motor neurons [39].
Following these, a bioluminescent-optogenetic system compro-
mised of a luciferase enzyme fused with a ChR2 was applied to
peripheral nerve regeneration in vivo. English et al. [156]used
both wild-type mice infected with AAV-vectors that carried the
luminopsin and transgenic mice, then gave them a single dose of
coelenterazine (a luciferase substrate whose interaction luciferase
results in light formation), and in both types they discovered a
significant increase in nerve regeneration compared to the control
mice after 4 weeks of rest. Similar results were shown in both
sensory and motor nerves, both when optogenetic stimulation
began before or after the nerve injury [155].
There are some relevant notes on the use of ChR2 in nerve
regeneration. A recent study on the axon-growing effects of short-
term ChR2 light stimulation revealed that the neurostimulatory
regenerative effects of such optogenetic stimulation are only
specific to permissive (not containing any inhibitory substances)
environments in vitro and to peripheral nerves such as sciatic
nerves in vivo [206]. Another study has emphasized the impor-
tance of using a control without ChR2 molecules which is still
subjected to light as, they discovered, blue light like the type that
activates ChR2 is capable of increasing neuronal activity even in
the absence of any optogenetic molecules [207].
Some more specific optogenetic mechanisms have also been
applied to peripheral nerve regeneration. Calcium signaling from
voltage-gated calcium channels was shown to be a major effector
of axon growth and guidance in ChR2-lead depolarization events
[208] but conversely, calcium influx is known to be an inhibitor
of axon growth [209] and optogenetic stimulation at higher
frequencies leading to excessive calcium influx resulted in axon
retraction rather than growth [210]. The calcium-specific channel
rhodopsin, CatCh, has been tested on transfected Schwann cells
instead of neurons, and their optogenetic stimulation elicited an
increase in Schwann cell proliferation, differentiation and myeli-
nation which presents an alternative to the currently available
Schwann cell therapies for peripheral nerve injuries [153].
Xiao et al. [157] measured the changes in zebrafish peripheral
nerve regeneration when affected by increased cAMP, using the
optogenetic system bPAC, which is a bacterial adenylyl cyclase
enzyme activated when homodimerized by blue light. They
demonstrated that while the regeneration of the central axon of
the nerve benefitted from cAMP increases, the peripheral axons
regenerated at the same speed with or without bPAC stimulation.
A zebrafish’s central axon regeneration is dependent on cAMP-
dependent protein kinase-A while its peripheral axons are not,
and this is similarly witnessed in adult mammalian DRGs where 1
day after a peripheral nerve injury cAMP levels triple to inactivate
the endogenous inhibitors of regeneration [157, 211].
Deleted in Colorectal Cancer (DCC), an effector that oligomerizes
when triggered by Netrin-1—a major neuron growth guidance
molecule—and activates a downstream pathway that leads to
axon growth, has also been converted to a photoactivable form.
Using CRY2 fused with DCC, Endo et al. [158] demonstrated light-
guided growth in the nerves of chick dorsal root ganglion neurons
in vitro and of Caenorhabditis elegans in vivo.
Raf/MEK/ERK and AKT, two signaling pathways that are highly
active in regeneration [212, 213], can be manipulated using the
17 of 24

optogenetic systems optoRaf and optoAKT, which activate when
the Raf and AKT proteins fused to CRY2 cluster on the plasma
membrane thanks to the light-triggered heterodimerization of the
membrane-bound CIBN and the free-floating CRY2-Raf/CRY2-
AKT [145, 214], Wang et al. [145] demonstrated the efficacy of
these systems in transgenic fruit fly (Drosophila) larvae periph-
eral nerve regeneration. OptoRaf and OptoAKT both increased
peripheral neuron axon growth when activated, and OptoRaf
resulted in a response in a threshold-gated way while OptoAKT’s
results showed a graded response. Wang et al. [145] also showed
that both pathways could grant regenerative potential to nerve
cells without such ability.A noteworthy facet of their research was
on nerves that preferentially grew away from the injury site where
their original path crossed to reach the correct connections; Wang
et al. [145] showed that these nerves grew negligibly when wild-
type or when in the dark, while generalized light would cause
them to grow outward in all directions. Comparatively, light
guidance towards the injury site would cause the nerves to grow
following the directed light on the correct pathway, proving the
ability of OptoRaf and OptoAKT systems to provide guided axon
growth (Figure 5).
5 Conclusion
The intersection of tissue engineering, regenerative medicine,
and optogenetics presents a frontier of unparalleled potential in
reshaping the landscape of healthcare and therapeutic interven-
tions. As we delve deeper into the intricate dance of stem cell
development, scaffold engineering, and ligand modulation, the
advent of optogenetic tools has emerged as a revolutionary force,
enabling unprecedented control especially over time-dependent
cellular pathways. While there are inherent challenges and
nuances associated with optogenetics, it is impossible to overlook
the remarkable strides made in regenerative and tissue engi-
neering research. From unraveling the mysteries surrounding
elusive cell proteins to orchestrating precise, time- and frequency-
dependent cellular activities, optogenetics opens a realm of
possibilities that were once deemed unimaginable.
As we stand on the cusp of the future, the untapped opportunities
presented by optogenetics in the realm of TERM are bound to
unfold in ways that will undoubtedly captivate and inspire. The
forthcoming years promise not only to witness the refinement of
existing techniques but also the emergence of novel applications,
propelling the field into uncharted territories. The journey ahead,
propelled by the fusion of optogenetics with tissue engineer-
ing, holds the promise of transforming how we perceive and
address physiological development, offering hope and innovative
solutions that can positively impact the lives of millions. The
evolution of this interdisciplinary frontier is poised to be nothing
short of exciting, as researchers continue to push the boundaries
of what is achievable in the quest for regenerating soft and hard
tissues.
Acknowledgments
The completion of this review article was undertaken without the support
of external funding. The authors did not receive financial assistance for
the review process or related activities.
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