Biomaterial and Stem Cell-Based Strategies for Skeletal

Muscle Regeneration
Andrew Dunn, Muhamed Talovic, Krishna Patel, Anjali Patel, Madison Marcinczyk, Koyal Garg
Department of Biomedical Engineering, Parks College of Engineering, Aviation, and Technology, Saint Louis University, Saint Louis, Missouri
Received 12 June 2018; accepted 13 December 2018
Published
Published online
online 14 February
in Wiley 2019
Online in Wiley
Library Online Library (wileyonlinelibrary.com).
(wileyonlinelibrary.com). DOI 10.1002/jor.24212
DOI 10.1002/jor.24212

ABSTRACT: Adult skeletal muscle can regenerate effectively after mild physical or chemical insult. Muscle trauma or disease can
overwhelm this innate capacity for regeneration and result in heightened inflammation and fibrotic tissue deposition resulting in loss
of structure and function. Recent studies have focused on biomaterial and stem cell-based therapies to promote skeletal muscle
regeneration following injury and disease. Many stem cell populations besides satellite cells are implicated in muscle regeneration.
These stem cells include but are not limited to mesenchymal stem cells, adipose-derived stem cells, hematopoietic stem cells, pericytes,
fibroadipogenic progenitors, side population cells, and CD133þ stem cells. However, several challenges associated with their isolation,
availability, delivery, survival, engraftment, and differentiation have been reported in recent studies. While acellular scaffolds offer a
relatively safe and potentially off-the-shelf solution to cell-based therapies, they are often unable to stimulate host cell migration and
activity to a level that would result in clinically meaningful regeneration of traumatized muscle. Combining stem cells and biomaterials
may offer a viable therapeutic strategy that may overcome the limitations associated with these therapies when they are used in
isolation. In this article, we review the stem cell populations that can stimulate muscle regeneration in vitro and in vivo. We also
discuss the regenerative potential of combination therapies that utilize both stem cell and biomaterials for the treatment of skeletal
muscle injury and disease. ß 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1246–1262, 2019.

Keywords: Skeletal muscle; stem cells; biomaterials; regeneration

Skeletal muscle is the most abundant tissue in the
body and constitutes approximately 40% of the total
body mass in humans.1 Each muscle cell (or myofiber)
is a multi-nucleated contractile unit surrounded by the
basal lamina.2 The basal lamina is a specialized layer
of the extracellular matrix (ECM) between the sarco-
lemma of each myofiber and the endomysium which
surrounds each muscle fiber. The basement membrane
consists of the outer reticular lamina and the inner
basal lamina. The outer reticular lamina is composed
mainly of fibrillar collagen (type I, III, VI) and
fibronectin whereas the inner basal lamina is com-
posed of an interconnected network of nonfibrillar
collagen (type IV) and laminin. Laminin is a cross-
shaped, heterotrimeric glycoprotein with an a-, b-, and
g-subunit.3 It is the primary ligand for the dystrophin-
associated glycoprotein complex (DGC) and a7b1
integrin receptor (Fig. 1).4,5 Both these receptors
associate with laminin in the extracellular matrix and
actin in the cytoskeleton and are essential for the
transfer of mechanical force from the inside of the
myofiber to the outer ECM during contraction.6,7 Any
disruption to the expression of these components can
impair the sarcolemma and myofiber cytoskeletal
integrity resulting in various forms of muscular dys-
trophy.4,5
Satellite cells are muscle resident stem cells that
are located in between the plasma membrane of the
muscle fiber and the surrounding basal lamina.3,4,8
Skeletal muscle’s outstanding regenerative capacity is
derived from the satellite cells. These cells persist
throughout life, although age and disease can lead to a
Grant sponsor: Saint Louis University Start up Funds.
Correspondence to: Koyal Garg, (T: 314.977.8200; F: 314.977.8288;
E-mail: koyal.garg@slu.edu)
# 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
decline in regenerative capacity.2 Satellite cells reside
in a specialized microenvironment known as the niche,
which contains stem cells, stromal cells, soluble
factors, ECM components, neural inputs, vascular
networks, and cell adhesion molecules.2,9–12 Satellite
cells are located near blood vessels, and their density
is reported to be higher around neuromuscular and
myotendinous junctions.9,13 The satellite cell niche is
responsible for protecting the inactive, physiologically
quiescent stem cell population from diminishing and
also for regulating its activation in response to various
stressors such as exercise, injury, or disease.9 Once
activated, satellite cells migrate out of stem cell niche
undergo asymmetric division to form myoblast
(Pax7þMyoDþ) and satellite cells (Pax7þMyoD) which
can either continue to proliferate or return to a
quiescent state.14 The myoblasts can either fuse with
one another or with existing myofibers to promote
muscle regeneration or repair, respectively.15 The
dynamic interaction between satellite cells and their
niche components is crucial for the maintenance of the
satellite cell pool and muscle mass. Any perturbations
in the cross-talk between the satellite cells and their
niche can result in impaired regeneration and fibrotic
tissue deposition.4,14,16,17
A variety of stem cell populations have also been
identified in skeletal muscle and the satellite cell niche
including, mesenchymal stem cells,18 pericytes,19 side
population cells,20,21 fibro/adipogenic progenitors
(FAPs),22,23 and interstitial stem cells.24 These cell
types share many features with one another such as
multipotency and cell-surface marker expression and
are known to proliferate in response to muscle
injury.18 Transplantation studies and in vitro co-
culture experiments13,25–29 have shown that these
stem cell populations can influence the self-renewal,
proliferation, and differentiation of satellite cells.

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Figure 1. The structure of (A) sarcolemmal proteins and basal lamina, and the (B) sarcomere. Reproduced with permission from
Rockefeller University Press via copyright clearance center from Fedik Rahimov, and Louis M. Kunkel J Cell Biol 2013;201:499–510.
During muscle repair, satellite cells also interact with
immune cells and endothelial cells. Following injury,
the capillary density in damaged muscle tissue
increases initially and then returns to baseline 4
weeks after injury.30,31 Co-culture experiments have
shown that endothelial cells promote the proliferation
of the satellite cells by secreting mitogenic and anti-
apoptotic factors, such as vascular endothelial growth
factor (VEGF). A reciprocal relationship has been
observed where differentiating myogenic cells also
secrete VEGF which regulates the proangiogenic func-
tion of endothelial cells. These findings indicate that
there might be a feed-forward mechanism through
which VEGF modulates myogenesis and angiogenesis
in the stem cell niche.10
Injury to muscle signals an inflammatory process
that removes damaged cells, coordinates the regenera-
tive response, and restores tissue homeostasis.2,9,11,12,32
The immune response involves interactions between
leukocytes and satellite cells. In resting conditions,
multiple leukocyte populations reside in the adult
skeletal muscle, the most abundant being mast cells
and macrophages. Mast cells and macrophages are
resident cell types, and in conjunction with circulatory
monocytes, sense distress and secrete chemoattractive
molecules following muscle injury. Damage activated
mast cells instantly begin to secrete tumor necrosis
BIOMATERIALS AND STEM CELLS 1247
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factor-a (TNF-a), histamine, and tryptase which ini-
tiates de novo synthesis of other cytokines like interleu-
kin-6 (IL-6). Low levels of TNF-a, tryptase, and IL-6
promote the activation and proliferation of satellite
cells.33 The initial release of cytokines and chemokines
attracts circulating granulocytes which consist of neu-
trophils and eosinophils.10 Neutrophils recruit mono-
cytes which differentiate into macrophages.
Macrophages can be divided into two main phenotypes
(M1/M2). There is an initial wave of pro-inflammatory
M1 macrophages which is followed by a second wave of
anti-inflammatory M2 macrophages.34 M1 and M2
macrophages, respectively, represent the early and late
stages of myogenesis. Studies show that M1 macro-
phages are found close to proliferating myogenic cells
while M2 macrophages interact with differentiating
myocytes.10 Prolonged and heightened inflammation
characterized by the persistent presence of M1 macro-
phages, often seen in orthopedic trauma, dysregulates
the regenerative process and impairs satellite cell
activity.35,36
Therefore, a greater understanding of satellite cells
and their niche components- resident stem cells and
ECM proteins is of considerable therapeutic value.
This review highlights the studies that have explored
the functional capacity of stem cells and bioengineered
scaffolds for skeletal muscle regeneration.
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STEM CELLS FOR SKELETAL MUSCLE
REGENERATION
Skeletal muscle repair and regeneration is a compli-
cated process which is reliant not only on the muscle
resident stem cell population but also on a vast
number of other stem and stromal cell populations
(Fig. 2). The microenvironment of skeletal muscle is
heterogeneous and contains a diverse population of
cells which are influenced by both structural and
biochemical cues. The following section will review
these cell types and their contributions to muscle
repair in several animal models of disease and
injury.37
Satellite Cells
The most important and well-characterized stem cell
in the context of muscle repair is the satellite cell.38
The best way to identify satellite cells in skeletal
muscle is through their expression of the canonical
marker, Pax7.39 However, satellite cells are known to
exhibit heterogeneity and researchers have isolated
and transplanted different satellite cell subpopulations
based on their diverse molecular signatures. For
instance, Sacco et al. showed that transplantation of a
single luciferase-expressing muscle stem cell (a7 integ-
rinþ CD34þ) into the muscle of mice underwent
extensive proliferation, self-renewal, and differentia-
tion in vivo.40 In another study, a unique combination
of cell surface markers (CD45-Sca-1-Mac-1-CXCR4þ
b1-integrinþ) was used to purify a distinct population
of stem cells from the satellite cell pool.41 When
transplanted into dystrophic mice, these cells contrib-
uted to muscle regeneration both by fusion with
existing myofibers and by de novo myogenesis. Resto-
ration of dystrophin expression and improvement in
muscle function was also observed. Human satellite
cells also display heterogeneity and have been identi-
fied as NCAMþCD45CD31 cells,42 and also by the
variable expression of Pax7, c-Met, and Dlk1.43
Although the transplantation of autologous muscle
satellite cells may seem like a viable therapeutic
strategy, this approach has met limited clinical suc-
cess. Challenges of satellite cell transplantation in-
clude the reduction of the regenerative capacity of
cultured cells compared to freshly isolated cells, poor

Figure 2. (A) The relative presence and contribution of non-myogenic cell types in muscle regeneration. (B-D) Immunofluorescence
micrographs of tissue sections from regenerating mouse muscles. Pax7-positive satellite cells (green) are in close proximity to various
non-myogenic cell types (red): (B) CD11b-positive leukocytes; (C) Sca1-positive interstitial cells; and (D) VE-Cad-positive endothelial
cells. ECM is shown in orange and nuclei are labelled with DAPI (blue). Scale bar, 10 mm. Reproduced with permission from John
Wiley & Sons, Inc, from C Florian Bentzinger et al. EMBO Rep. 2013;14:1062–1072.

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survivability and migration of the transplanted cells,
and adverse immune reactions.39,44,45 For instance,
Montarras et al.46 demonstrated that ex vivo expan-
sion of satellite cells for as little as three days severely
reduces their proliferative capacity and increases the
fraction of differentiated cells, resulting in limited
regeneration upon transplantation in vivo. Improved
results have been reported by the transplantation of
larger number of cells (105 myoblasts/cm of injection)47
and the use of immunosuppressive drugs.48,49
As a way to circumvent these challenges, many
researchers are attempting the delivery of satellite
cells through methods other than direct transplanta-
tion. For example, a study that transplanted single,
intact myofibers into a radiation-ablated muscle dem-
onstrated that even as few as seven satellite cells from
the myofibers could regenerate more than 100 new
myofibers with thousands of nuclei.50 This method has
also been used in animal models of aging,51 and heart
failure52 and beneficial effects have been reported.
However, even transplanting satellite cells through a
single myofiber has its own challenges. The expertise,
technical skill, and regulatory approval required to
implement this process successfully could prevent the
clinical translation of this strategy.
Mesenchymal Stem Cells
Mesenchymal stem cells (MSCs) are the focus of
several cell-based therapies for a wide range of
diseases.53,54 They exhibit features that are attractive
for use in tissue engineering, including self-renewal,
multipotency, immunomodulatory properties, and se-
cretion of growth factors and cytokines.18 Further-
more, these cells can be easily isolated from a variety
of adult tissues including the bone marrow or adipose
tissue. MSCs have been shown to differentiate into
mesodermal lineages in vitro including bone, cartilage,
and fat.55
MSCs represent less than 0.01% of the bone marrow
mononuclear cell fraction but can be easily cultured
and genetically modified in vitro.56,57 Delivering these
bone marrow-derived MSCs (BMMC) intramuscularly
has shown to improve muscle function in a dose-
dependent manner in rodent models of crush
trauma.58,59 When BMMCs were delivered to a rodent
crush model intra-arterially, the muscle function was
restored. Interestingly, this systemically delivered cell
population was not observed localized to the injured
muscle. Thus the authors conjecture that the improve-
ments in function were likely due to the paracrine
action of the injected BMMCs.60 Corona et al. worked
with a lineage depleted (Lin-) population of bone
marrow cells to increase the stem and progenitor cell
populations in a mouse ischemia-reperfusion (I/R)
model. They found that when injected intramuscularly
cells survived up to 1-month post-transplantation, but
did not improve function.61 However, when injected
intravenously to avoid damage that can occur during
intramuscular injection, the cells homed to the site of
BIOMATERIALS AND STEM CELLS 1249
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injury and resulted in improvements in regeneration
and muscle function.62 The exact mechanisms of MSC
mediated tissue repair are unclear, and some contro-
versy exists regarding their capacity for myogenic
differentiation.57 For instance, intramuscular trans-
plantation of human BMMCs in dystrophic mice
restored dystrophin expression but did not improve
contractile function post engraftment.56 The authors
suggested that the presence of dystrophinþ myofibers
in transplanted mice may result exclusively from the
fusion of donor cells with resident myofibers rather
than from the reprogramming of BMMCs into myo-
genic progenitors.56
Besides bone marrow, muscle-derived MSCs have
also been investigated in recent studies. Muscle-resi-
dent MSCs (Sca1þ/CD45) delivered intramuscularly
were shown to increase Pax7þ satellite cell quantity,
myofiber hypertrophy, and arteriogenesis in mouse
hindlimb muscles damaged by eccentric contraction
injury.18,25,63–66 These studies have suggested that the
primary contribution of MSCs to the repair and
regeneration process is the trophic support they pro-
vide through the release of soluble mediators such as
growth factors and cytokines.18
Adipose-derived stem cells (ASCs) have increasingly
gained popularity in regenerative medicine due to
their morphological and phenotypic similarities to
MSCs with the added bonus that they can be readily
isolated with minimally invasive procedures.28 These
cells have been reported to have myogenic potential in
vitro, and myogenic progenitors that were ASC derived
have shown to successfully engraft into skeletal mus-
cle and promote myofibers synthesis in dystrophic
mice (mdx).67 Studies have reported that compared to
bone marrow, MSCs derived from adipose tissue have
a greater tendency for myogenic differentiation.68,69
Similar to MSCs, ASCs can aid in muscle regeneration
indirectly as well, largely through their modulation of
inflammation and fibrosis, which has been demon-
strated in mdx mouse models.70 In one study, the
delivery of ASCs with an anti-fibrotic medication
called losartan to dystrophic muscles downregulated
TGF-b1, inhibited fibrosis, and also improved muscle
regeneration and hypertrophy.71 Thus from these
studies, it can be seen that ASCs can support muscle
regeneration either directly or indirectly through
differentiation into myogenic progenitors or via the
modulation of inflammation and fibrosis, respectively.
Hematopoietic Stem Cells
Hematopoietic stem cells (HSCs), originating in adult
bone marrow have been shown to home to injured
muscle sites with guidance from the stromal cell-
derived factor-1 (SDF-1)/chemokine receptor 4
(CXCR4) and chemokine receptor 2 (CCR2) coupling.72
Other studies have also implicated HGF/c-Met signal-
ing in the homing of HSCs to injured muscle.73
Multipotent bone marrow-derived HSCs were first
reported to mediate muscle regeneration in vivo in 1998,
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and since then studies have repeatedly reported sup-
porting evidence of their regenerative capacity.26 For
example, one study showed that intravenously injected
HSCs could contribute to the myogenesis and restore
dystrophin expression in lethally irradiated mdx mice.74
Another study showed that following irradiation, the
satellite cell niche could be reconstituted by bone
marrow-derived HSCs that expressed satellite cell
markers such as a7 integrin, c-Met, and Myf5.75
Furthermore, these bone marrow-derived satellite cells
were demonstrated to undergo myogenic differentiation
in vitro and myofiber regeneration in vivo.75
While HSCs are quiescent in myofiber repair under
normal physiological conditions, it has been shown
that in response to damage or injury they do contrib-
ute to myogenic events. One such study showed that
the capacity of hematopoietic progenitors to fuse with
myofibers is increased significantly following muscle
damage.76 Sacco et al. identified insulin-like growth
factor 1 (IGF-1) as a damage related molecule that
substantially augmented the fusion of bone marrow-
derived cells with adult skeletal muscle.77 In another
study, Camargo et al. suggested that the fusion of
HSC-derived progenitors with damaged myofibers sup-
ported regeneration following injury.78 Xynos et al.
used a cardiotoxin injury model to demonstrate that
upon interaction with the muscle microenvironment,
HSCs undergo profound yet incomplete myogenic
reprogramming, differentiation, and incorporation into
muscle myofibers to participate in muscle regenera-
tion.79 However, the ability of these cells to undergo
reprogramming post-fusion has been challenged by
studies that have reported that myofibers with donor-
derived nuclei failed to express sarcoglycan80 or dys-
trophin81 and only temporarily turn on myogenic
genes. Negative results with transplantation of bone
marrow-derived cells have also been reported in a rat
model of volumetric muscle loss (VML), where these
cells only contributed to limited de novo myofiber
regeneration and did not show significant changes in
muscle function or myogenic gene transcription82
Overall, the efficacy of HSCs in supporting muscle
regeneration is controversial. In addition, it is unclear
if muscle repair is facilitated by HSC derived myogenic
cells, or if the HSCs themselves undergo reprogram-
ming after fusion with damaged myofibers.
Pericytes
Pericytes are perivascular stem cells that form
intimate connections with adjacent capillary endothe-
lial cells. These contractile connective tissue cells
reside beneath the microvascular basal lamina and
regulate capillary blood flow.83,84 These cells are
known to affect migration, proliferation, permeabil-
ity, and contractility of endothelial cells. This multi-
potent cell population shares many similarities with
MSCs. For instance, pericytes have been isolated
from various tissues and are known to possess
differentiation potential for adipogenic, chondrogenic,
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osteogenic, and myogenic lineages.19,85–87 These cells
also express canonical markers of MSCs such as
CD29, CD44, CD73, CD90, and CD105.88
In contrast to satellite cells, pericytes generally do
not express Pax7 or MyoD. These cells only express
myogenic markers post-differentiation, and this capac-
ity has been demonstrated both in vitro and in
vivo.19,83,87,89 Two major subpopulations of pericytes
have been identified: Type 1 (Nestin-NG2þ) and type 2
(NestinþNG2þ). While type 1 pericytes are reported to
contribute to fat accumulation, type 2 pericytes are
known to support new muscle formation.90 Type 2
pericytes, which are Nestinþ/NG2þ, have been identi-
fied in the satellite cell niche and are known to
support the new formation of muscle tissue.90
Depletion of pericytes has resulted in insignificant
hypotrophy and a slight increase in total Pax7þ cells.91
Co-culture of pericytes and satellite cells resulted in
either myogenic differentiation through the secretion
of insulin-like growth factor (IGF-1) or quiescence
through the secretion of angiopoietin 1 (ANGPT1).91
Another study demonstrated that when transplanted
into immunodeficient dystrophic mice, pericytes can
generate dystrophinþ myofibers.19 Further, this study
also showed that when delivered systemically the
pericytes can cross the vessel wall to colonize the
injured muscle. To support these findings, Lorant
et al. found that when injecting perivascular stem cells
into cryoinjured muscles of scid mice the cells inte-
grated into host tissue and contributed to the produc-
tion of structural proteins associated with
differentiated myofibers.92 In contrast, when Meng
et al. delivered muscle-derived pericytes intra-arteri-
ally to mice, they found that they did not contribute to
the regeneration of muscle.93 These authors believed
the discrepancy in results were due to the differences
in experimental techniques, animal models, and out-
come measurements. Despite these studies, the mech-
anisms that regulate the myogenic differentiation of
pericytes is largely unknown, and to fully understand
the role of pericytes in muscle regeneration it is
necessary to perform more comprehensive studies.
Fibro/Adipogenic Progenitors
Skeletal muscle resident PDGFR-aþ Sca-1þ CD34þ
fibro/adipogenic mesenchymal progenitor (FAPs) are
bipotent cells that primarily contribute to fibrotic and
fatty tissue deposition in skeletal muscle.22,23,94,95
According to some reports, the differentiation of these
interstitial cells depends on the severity and mecha-
nism of muscle injury. For instance, Uezumi et al.,
showed that PDGFR-aþ cells isolated from glycerol
damaged skeletal muscle did not differentiate into
adipocytes when transplanted into cardiotoxin (CTX)
injured muscle.96 However, PDGFR-aþ cells isolated
from CTX injured muscle readily accumulated into
glycerol damaged areas and differentiated into lipid
laden adipocytes (C/EBPaþ PPARgþ).22 Therefore,
these results suggest that the differentiation of FAPs

into adipocytes is highly dependent on the muscle
microenvironment. It was revealed that CTX injured
muscles show higher numbers of new myofibers com-
pared to glycerol injured muscles and the interaction
between regenerating fibers and FAPs may be neces-
sary for the inhibition of fat deposition. Consistent
with CTX injury results, FAPs did not undergo signifi-
cant adipocyte differentiation in response to notexin
injured muscles. However, they showed extensive
proliferation in the defect region and were found in
close proximity to the regenerating fibers, indicating
that FAPs may have a role to play in modulating
myogenesis.23 While FAPs do not directly differentiate
through myogenesis, they do produce several factors
such as IL-6, IL-10, IL-33, IGF-1, Wnt 1, Wnt3A, and
Wnt5A that are known to support myogenic activity.
23,97,98
The ability of these cells to accumulate and in
fibrotic areas and express fibrosis related molecules
has also been well-documented. In the diaphragm of
dystrophic mice and CTX injured skeletal muscle,
FAPs were found co-localized in collagen type 1 rich
regions.96 Studies have identified TGF-b1 and PDGF-
AA to be potent regulators of FAP mediated fibrosis in
skeletal muscle. Exposure to these growth factors in
vitro was shown to promote the proliferation and
expression of fibrosis related markers (e.g., Col1a1,
col3a1, CTGF, TIMP1, and a-SMA) in PDGFR-aþ
FAPs.96
Targeting these cells could provide new therapeutic
strategies for preventing pathological changes in mus-
cle following injury and disease. Genetic and pharma-
cological ablation of FAPs has been associated with
impaired muscle regeneration.99,100 Therefore, modu-
lation of FAP activity may hold the key for enhancing
muscle regeneration. For instance, by inhibiting TGF-
b1, FAPs were shown to become apoptotic in the
presence of macrophages, which correlated with de-
creased fibrosis in vivo.101 Cytokines such as IL-4 and
IL-15 were shown to prevent FAPs from undergoing
adipogenic differentiation.102,103 Future studies should
focus on elucidating the diverse differentiation mecha-
nisms of FAPs. Identification of precise factors and
molecules that can modulate FAP activity to promote
myogenic regeneration without fibrotic and fatty tissue
deposition are likely to offer great therapeutic benefits
to aging, diseased, or severely injured skeletal muscle.
Induced Pluripotent Stem Cells
Induced pluripotent stem cells (iPSCs) provide several
attractive features for myogenic research such as
capacity for self-renewal, suitability for genetic edit-
ing, ability to differentiate into multiple cells types
including myogenic cells while maintaining pathologi-
cal phenotypes.104,105 Myogenic cells have been derived
from iPSCs via genetic reprogramming or addition of
small molecules. Studies have reported that iPSC
derived myogenic cells can fuse with existing myofib-
ers following in vivo transplantation105–107 and can
BIOMATERIALS AND STEM CELLS 1251
3
also generate three-dimensional (3D) functional skele-
tal muscle in vitro.108–111 The breadth of lineages in
which iPSCs differentiate through allows for a variety
of cell types (neural, vascular, and muscular) to
populate these 3D tissue constructs.108 These func-
tional tissue constructs were able to survive, vascular-
ize, and function when transplanted in vivo,
suggesting that the can replace damaged or dysfunc-
tional muscle.111 Additionally, these constructs can be
used for disease modeling and drug screening applica-
tions.108
However, this revolutionary therapy is not without
its flaws. Despite the appeal of autografting, there
have been cases of immune rejection and the necessity
for immunosuppressive drugs.112 Tumor formation is
also a rare but observable problem with iPSCs trans-
plantation. 113,114
Other Stem Cell Populations
Several other stem cell populations have been identi-
fied in skeletal muscle, and there has been increasing
interest in understanding their role in the process of
muscle regeneration. The muscle side-population (SP)
is a heterogeneous cell population that is reportedly
positive for Sca1, ABGC2, Syndecan-4 but negative for
CD45.115 An interesting feature of these cells is their
ability to migrate and engraft into host myofibers
following systemic delivery.116 The myogenic potential
of these cells is under investigation. In one study,
direct injection of the SP cells in injured muscles
resulted in efficient engraftment into the satellite cell
niche. These cells also gave rise to satellite cells and
donated myonuclei to the regenerating fibers.115 How-
ever, in another study, the ABGC2þ SP cells were
reported to increase in muscle after injury, giving rise
to endothelial cells and pericytes but not contributing
significantly to myonuclei.117 Similar results were
obtained in a recent study where SP cells derived from
dystrophic or injured muscles gave rise to FAPs but
failed to undergo myogenesis.118 Therefore, more rig-
orous studies are needed to establish a clear role of
these cells in skeletal muscle regeneration.
CD133þ stem cells have gained increased popularity
in recent years. These cells have been tested in both
animal models and clinical trials to repair injured
tissues.119 A major advantage of this cell population
from a clinical standpoint is the ease with which it can
be purified from the peripheral blood. A study com-
pared the regenerative capacity of the human muscle-
derived CD133þ stem cells with human satellite cell-
derived myoblasts in a mouse model of cryoinjury.120
The results showed that the CD133þ stem cells out-
performed myoblasts in terms of migration, repopula-
tion of the satellite cell niche, and overall regenerative
capacity. A recent study demonstrated that human
CD133þ cells transplanted into mouse muscles are
functional and can give rise to satellite cells in vitro
and in vivo.121 However, similar to myoblasts, the
prolonged culture of donor CD133þ cells was shown to
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reduce their effectiveness in vivo. In another study,
human adult CD133þ stem cells were mobilized from
the peripheral blood by granulocyte colony-stimulating
factor (G-CSF) and intramuscularly injected into lacer-
ated muscles in athymic rats. The injected cells
differentiated into endothelial and myogenic cells and
improved the microenvironment of the injured tissue
by upregulating angiogenesis and downregulating fi-
brosis.122 Autologous muscle-derived CD133þ cells
have also been transplanted in humans with Duch-
enne muscular dystrophy.123 While the procedure was
well-tolerated and safe, functional benefits were not
observed.
Therefore, it is clear that muscle function and
regeneration are impacted not only by myogenic cells
but also by a lot of other stem cell populations. The
heterogeneity of stem cells and their function further
demonstrates the complexity of skeletal muscle main-
tenance and regeneration. Studies that illustrate and
define the mechanism through which these stem cell
populations orchestrate muscle repair would be valu-
able for the development of cellular therapies.
BIOMATERIALS FOR SKELETAL MUSCLE
REGENERATION
While the quest for an ideal stem cell population for
stimulating muscle regeneration continues, some researchers
have diverted their attention toward finding an ideal bioma-
terial substrate to modulate tissue-resident stem cells,
deliver therapeutic molecules, and encapsulate cells to
protect them from the injured microenvironment and the
immune system.124
As mentioned previously, stem cells often constitute an
extremely rare population in tissues, and their ex vivo
expansion is reported to reduce their regenerative capacity
greatly [96–100]. Several studies have shown that freshly
isolated satellite cells or satellite cells derived from trans-
plantation of one intact myofiber contribute to muscle repair
more significantly than cultured myoblasts.2,40,125 Gilbert
et al. demonstrated that the stem cell niche supports freshly
isolated satellite cells in their ability to regenerate when
transplanted whereas satellite cells grown on standard tissue
culture plastic lose their stem cell capability by producing
progenitors with greatly diminished regenerative poten-
tial.125 Furthermore, matrix rigidity was shown to affect the
quiescence, proliferation, and differentiation of satellite cells.
The study showed that cultured on poly(ethylene glycol)
hydrogels with rigidity similar to native muscle tissue
(12 kPa), the satellite cells are capable of self-renewal and
maintenance of regenerative properties. Similar results were
obtained in another study where muscle progenitor cells
showed increased proliferation on polyacrylamide gels of
lower stiffness and adopted a differentiating phenotype on
substrates with higher stiffness.126 These results were
corroborated by Urciuolo et al. who also showed that satellite
cells grown on gelatin hydrogels of physiological (12 kPa)
stiffness maintained a large proportion of Pax7þMyoD cells,
which was dramatically decreased when satellite cells were
grown on structures of 7 kPa stiffness.127 The authors further
demonstrated the effect of ECM components on the myogenic
potential of satellite cells. Satellite cells derived from colla-
gen VI null mice (Col6a1/) showed a higher ability to
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maintain the Pax7þMyoD population when grown on puri-
fied collagen VI, with decreased apoptosis and increased
proliferation compared to cultures grown on Matrigel alone.
Taken together, these studies show that biomaterials
designed for supporting specific cellular functions have the
potential to overcome the limitations associated with cell-
based therapies. However, recent work has suggested that
acellular biomaterial-based therapies have several limita-
tions of their own as they have failed to recover
strength,128,129 recruit endogenous satellite cells to the defect
site,130,131 and regenerate nerves.132,133 Instead of using the
two strategies in isolation, researchers have investigated cell
and biomaterial combination therapies. Combining cells with
candidate biomaterials may overcome issues associated with
acellular scaffolds and cell-only therapies. Such combination
therapies have shown promising results in animal models
and may provide a potential therapy in the near future.134
The following section describe some of the promising scaffold
technology that has been developed in combination with
cellular therapies over the recent years.
Satellite Cells and Myoblasts Transplantation with Biomaterials
A scaffold-based strategy for satellite cell or myoblast
encapsulation and delivery has been attempted in various
studies. Rossi et al. reported improvement in muscle struc-
ture, the total number of new myofibers as well as muscle
function by delivering satellite cells encapsulated in a
hyaluronan-based photocrosslinkable hydrogel to partially
ablated tibialis anterior muscles.135 At 6 weeks post-injury,
the cell-loaded hydrogels had extensive and consistent mus-
cle regeneration with minimal scar tissue formation, while
the untreated and hydrogel only groups had a large deposi-
tion of fibrotic connective tissue and altered morphology.
In a study by Pollot et al., five commonly used natural
polymeric materials were used in skeletal muscle tissue
engineering grafts and were characterized for mechanical
and myogenic properties by seeding primary rat satellite
cells.136 The five materials were collagen I, agarose, fibrin,
collagen-chitosan, and alginate. Overall findings of this study
suggested collagen I was supportive of myogenesis, while
fibrin demonstrated the highest myogenic potential mea-
sured by the quantification of gene expression of MyoD,
myogenin, and myosin heavy chain.
Fibrin provides an excellent matrix for myoblast activity
and vascularization.137 Marcinczyk et al. studied the efficacy
of laminin-111 enriched fibrin hydrogels for skeletal muscle
regeneration.3 With the addition of increasing concentrations
of laminin-111, fibrin hydrogels displayed progressively
thinner, interlaced fibers. While lower concentrations of
laminin-111 supported myoblast differentiation, a higher
concentration of laminin-111 promoted myoblast differentia-
tion. Additionally, myoblasts seeded hydrogels displayed
responsiveness to electromechanical stimulation through
elongation, alignment, myokine secretion, and sirtuin
(SIRT1) expression.
Fibrin-based scaffolds have also been used in animal
models of muscle trauma with encouraging results. Page
et al. fabricated fibrin microthreads to serve as efficient
delivery vehicles for primary human muscle-derived cells
that expressed both pluripotent and myogenic markers in
culture.138 Cell-seeded fibrin microthreads were implanted in
a VML defect in immunodeficient mice. The microthreads
were reabsorbed at 1-week post injury, and new muscle
fibers were reconstituted at 2-weeks for microthread treated
 BIOMATERIALS AND STEM CELLS 1253
3

wound sites. In contrast, the untreated control wounds had
collagen as the predominant constituent of the healed
wound. After 4 months, the cell-loaded microthreads signifi-
cantly improved the recovery of muscle strength compared to
untreated controls.
Gilbert-Honick et al. engineered electrospun fibrin scaf-
folds to promote muscle regenerations and functional recov-
ery.139 Hydrogel microfiber bundles were created by
extruding both fibrinogen and alginate in parallel. Acellular
or C2C12 myoblast seeded scaffolds were implanted in a
hindlimb VML injury model in immunodeficient mice. While
the acellular electrospun scaffolds resulted in fibrosis and
lacked the presence of MHC myofibers, defects treated with
C2C12-seeded electrospun scaffolds showed an increase in
muscle mass, lack of fibrosis as well as high myofiber and
vascular density. Interestingly, both acellular and C2C12-
seeded scaffolds demonstrated maximum torque levels equal
to uninjured controls, while untreated defects showed a 30%
torque deficit at 2 and 4 weeks post-injury. In the absence of
muscle regeneration, this result could be explained by
“functional fibrosis”—a fibrosis mediated mechanical bridg-
ing effect that helps in force transmission and protection of
remaining muscle mass from reinjury.140
Another study evaluated the efficacy of muscle-derived
stem cells (MDSCs) combined with fibrin gel for VML
repair in immunodeficient mice.134 At 4 weeks post-injury,
this combined approach showed that MDSCs differentiated
into new myofibers and significantly increased muscle
mass along with a significant decrease in fibrotic tissue
deposition. The number of small fibers with centrally located
nuclei were reported to increase with treatment indicating
significant increase of regenerative fibers in the engrafted
area of treated muscles (Fig. 3). The MDSC seeded fibrin gels
also supported increased vessel formation and restored
satellite cell population in the injured muscles. However,
functional recovery of the VML injured muscle was not
assessed. Taken together, these studies suggest that cell-
loaded fibrin scaffolds can prevent the rapid progression of
fibrosis that impedes muscle regeneration and restore muscle
functionality.
It has also been shown that growth factors coupled with
cell or scaffold-based therapies can improve muscle regenera-
tion. Keratin hydrogels have been used for the controlled
release of growth factors in some recent studies, since their
degradation rate can be tailored easily. Tomblyn et al.
investigated a keratin hydrogel carrier system for the deliv-
ery of exogenous growth factors and muscle progenitor cells
(MPCs) to the site of VML injury.141 Two different isomers
were studied—oxidatively extracted keratin (or keratose)
and reductively extracted keratin, (or kerateine). Due to the
presence of covalent disulfide bonds within kerateine, its
degradation is slower compared to keratose. This difference
in degradation rates resulted in different release profiles of
exogenous growth factors such as vascular endothelial
growth factor (VEGF), insulin-like growth factor 1 (IGF-1)
and basic fibroblast growth factor (bFGF). Following in vivo
testing in a subcutaneous mouse model, the study suggested
that keratose hydrogels maintained the viability of MPCs
and the bioactivity of growth factors with minimal inflamma-
tory responses. The delivered muscle progenitor cells were
reported to form a very primitive muscle-like construct.
In another study, Baker et al. loaded kerateine hydrogels
with skeletal MPCs with or without IGF-1 or bFGF to treat
volumetric muscle loss (VML) injury in the latissimus dorsi
muscle in a mouse model.142 After 2 months, keratin hydro-
gels loaded with both growth factors but without MPCs
yielded a greater recovery of contractile force than the other
treatment groups and promoted extensive new muscle tissue

Figure 3. Therapeutic efficiency of MDSC/fibrin gel casting method for muscle defect repair. (A) Representative histological images of
muscle cross sections from different experimental groups show different levels of fibrosis versus engraftment. (B) Quantification of
muscle regeneration and fibrosis. Combination therapy of MDSC/fibrin gel significantly increased muscle regeneration and decreased
fibrosis. (C) Quantification of fibers with centrally located nuclei and their cross section area (CSA) indicates significant increase of
regenerative fibers in engrafted area of treated muscles compared to non-engrafted native regions. Reprinted from “Volumetric muscle
loss injury repair using in situ fibrin gel cast seeded with muscle-derived stem cells (MDSCs)” by Matthias et al., Stem Cell Research,
volume 27 pages 65–73, Copyright 2018, with permission from Elsevier.

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formation. These results were unexpected as the inclusion of
MPCs did not provide functional improvement. The authors
speculated that likely reasons for this result include a lower
cell density of MPCs and lack of preconditioning in a
bioreactor.143 Similar results were obtained in a subsequent
study conducted in a rat VML model.144 The exact mecha-
nism responsible for the enhanced performance of the kera-
tin hydrogel with the addition of growth factors needs to be
investigated in future studies.
Mesenchymal Stem Cells Delivery with Engineered Matrices
Several studies have delivered MSCs via hydrogels to the
site of muscle injury. In a model of repeated laceration
injury, BMMC were suspended in Matrigel and transplanted
in the soleus muscles.145 Compared to non-treated muscles,
the BMMC treated muscles showed fewer fibers with a
centrally located nucleus, and larger muscle fiber cross-
sectional area, but no differences in fibrotic tissue deposition
were reported. In another study, transplantation of BMMC
suspended in a fibrin matrix in a rodent model of muscle
laceration restored muscle function. The cells did not differ-
entiate into myotubes or fuse with existing myofibers,
suggesting an alternate mechanism for repair.146 In a recent
study, BMMCs were delivered with recombinant growth
factors in an alginate cryogel to injured soleus muscles, a
strategy that enhanced paracrine signaling in MSCs. This
approach resulted in improved muscle function, remodeling
of scar tissue, and increase in new myofiber regeneration.147
MSCs have also been used with decellularized ECM
scaffolds for the regeneration of skeletal muscle. Merritt
et al. injected BMMCs at the site of VML injury 1 week after
the implantation of muscle-derived ECM.148 After 42 days of
recovery, the BMMC injected ECM group showed more blood
vessels, regenerating skeletal myofibers, and functional
recovery than ECM treatment alone. Qui et al. repaired
VML injured rat hindlimb muscles with human umbilical
cord MSCs and decellularized porcine cardiac ECM scaf-
folds.149 The treatment groups included decellularized ECM
powder, MSC aggregates, or the combination of the two. At 8
weeks post-injury, the compound group showed improved
muscle regeneration and functional recovery along with
reduced collagen deposition compared to the other treatment
groups. The study further reported that treatment with
either MSCs or ECM scaffold alone was immunomodulatory
and that this effect was more pronounced when the two
therapies were combined. The combination of MSCs and
decellularized ECM scaffolds promoted the pro-regenerative
M2 macrophage phenotype, while simultaneously suppress-
ing the pro-inflammatory M1 phenotype. These promising
results need to be replicated and confirmed by other
researchers in the field in order to establish the use of MSCs
with biomaterials as a viable therapeutic strategy for trau-
matized muscle tissue.
Co-Administration of Adipose-Derived Stem Cells with Scaffolds
Human adipose tissue-derived adult stem cells (ASCs)
were co-administered with bFGF loaded gelatin-poly(ethyl-
ene glycol)-tyramine (GPT) hydrogels in a mouse model of
muscle laceration.150 At 4 weeks post-injury, the combined
treatment resulted in significant improvements in muscle
regeneration, functional recovery, revascularization, and
reinnervation with a concomitant decrease in fibrosis com-
pared to other treatment groups (Fig. 4). However, the exact
mechanism of repair was unclear, and the authors attributed
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the observed positive effects to the possibly enhanced para-
crine action of ASCs.
In another study, ASCs and their conditioned medium
were delivered to the VML defect site in a collagen hydrogel.
The results showed that this treatment approach accelerated
muscle repair, improved vascularization, and blood perfu-
sion, while simultaneously reducing inflammation and fibro-
sis.151 The authors attributed these effects to the enhanced
release of anti-inflammatory cytokines by the ASCs which
were able to polarize the macrophages in the defect site
toward an M2 phenotype.
Aurora et al. combined PEGylated-platelet free plasma
hydrogels with a decellularized muscle ECM scaffold to
deliver human ASCs in a rat VML injury model.152 At 2
weeks post-injury, the study showed that the composite
scaffolds are viable cell delivery vehicles as shown by the
increased presence of ASCs in the defect region. These
delivered cells were also reported to home to the perivascular
space. The composite treatment resulted in increased vascu-
larization but did not result in appreciable muscle regenera-
tion. The authors speculated that perhaps vascularization
alone is not enough and a myogenic cell source is necessary
to support muscle regeneration.
Co-Delivery of Minced Muscle Grafts with Biomaterials
An alternative approach to delivering a specific stem cell
population is to deliver bundles of minced muscle fibers to
transfer not just satellite cells but also other cell types
in their niche with an intact ECM. Corona et al. have
repeatedly demonstrated appreciable muscle regeneration
by transplantation of minced muscle autografts in both
rodent130,153,154 and porcine155 models of VML. Since this
approach has the obvious limitations of inadequate availabil-
ity and potential donor site morbidity, Corona and coworkers
have investigated various biomaterials to deliver the autolo-
gous minced muscle grafts to the VML defect site.156 Since
collagen type I is the primary skeletal muscle ECM constitu-
ent, it was evaluated as a potential expansion biomaterial.
The VML injured hindlimb muscles received a 25 or 50%
autologous minced graft suspended in collagen hydrogel,
with the 100% minced muscle graft treatment serving as the
positive control. After 8 weeks post-injury, only the 50 and
100% minced graft treatment restored promoted significant
de novo fiber regeneration and improved muscle function. It
was also noted that the regenerated tissue associated with
the 50% graft had greater fibrotic and fat tissue deposition.
In another study, Goldman and Corona found that the
regenerative capacity of minced muscle grafts used for the
treatment of VML injuries is impeded with the co-delivery of
urinary bladder matrix (MicroMatrixTM, ACell Inc., Colum-
bia, MD).157 MicroMatrixTM was either used alone or in
conjunction with the minced muscle graft (1:1). After 8 weeks
of treatment, the compound group showed little to no muscle
regeneration but improved functional capacity, possibly due
to functional fibrosis.140 An aggravated immune response to
the decellularized MicroMatrixTM was reported contrary
to several previous reports of a favorable immune
response.132,133,158–162
In a subsequent study, Corona and coworkers investi-
gated a laminin-111 supplemented PEG-hyaluronic acid-
based hydrogel as a delivery vehicle for minced muscle grafts
for VML injuries.163 At 8 weeks, the hydrogels were found to
be wholly intact and did not support cellular infiltration into
the defect site. The lack of cellular infiltration was associated
 BIOMATERIALS AND STEM CELLS 1255
3

Figure 4. (A) Representative histological images from Masson’s trichrome staining of lacerated gastrocnemius muscle 4 weeks after
surgery. Experimental treatment groups included saline (C), the h-ADSCs (AD), the bFGF hydrogel (bH), and the bFGF hydrogel
containing h-ADSCs (AD/bH group). (B) Quantified area of muscular fibrosis. AD/bH group revealed significantly lower muscular
fibrosis induced by muscle laceration (scale bars, 100 mm,  p < 0.05 compares with the laceration group; #p < 0.01 compares with the
laceration group). Reprinted from “Combination therapy of human adipose-derived stem cells and basic fibroblast growth factor
hydrogel in muscle regeneration” by Hwang et al., Biomaterials, volume 34 issue 25 pages 6037–6045, Copyright 2013, with permission
from Elsevier.

with impaired muscle regeneration and function. Therefore,
an optimal carrier material for the expansion and delivery of
minced muscle grafts needs to be investigated intensively.
CHALLENGES WITH EXISTING TECHNOLOGIES
AND FUTURE TRENDS
A successful strategy for muscle regeneration cur-
rently does not exist. The two main obstacles to muscle
regeneration following trauma are fibrotic tissue depo-
sition and persistent inflammation.36,130,153,157,164–166
A regenerative strategy for traumatic injuries such as
VML must support the migration, proliferation, and
differentiation of cell populations that can promote
myofiber synthesis, angiogenesis, immunomodulation,
and neurogenesis while simultaneously suppressing
fibrotic tissue deposition.
Decellularized scaffolds have been widely used for
skeletal muscle repair and regeneration.167 Recent
work has also described encouraging clinical outcomes
following the implantation of decellularized scaffolds
in patients with VML injuries (reviewed in37). How-
ever, acellular ECM based therapies have a set of
challenges that must be overcome in order to promote
clinically relevant levels of skeletal muscle regenera-
tion. A common problem with acellular treatments is
that they do not produce a meaningful regeneration of
muscle tissue and get remodeled into a fibrotic
scar.128,131,140,168–170 These scaffolds completely rely on

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the migration and proliferation of host satellite cells to
infiltrate the defect area and restore function to the
injured site. Degradation products of these ECM
scaffolds termed as “cryptic peptides” are known to
exert chemotactic and mitogenic effects on multipoten-
tial progenitor cells in vitro171 and in vivo.172 In
animal models, implantation of ECM scaffolds has
resulted in the recruitment of several stem cell
populations such as Sca1þ cells,130,158 perivascular
stem cells,173,174 pluripotent adult progenitors
(Sox2þ),175 CD133þ progenitor cells,132 and neural
stem cells.176 However, several studies have shown
that satellite cells or myogenic progenitors fail to
substantially repopulate the acellular construct,130,131
resulting in the formation of small muscle fiber islands
(<50 fibers) in the defect region of VML that are
unable to restore functional use of the injured mus-
cle.161 Furthermore, each laboratory decellularizes
tissues using different protocols and processing techni-
ques. The variable and heterogeneous composition of
the resulting matrix not only influences their perfor-
mance in vivo but also makes it difficult to compare
and conclude published work. Future studies should
strive to meticulously present decellularization meth-
ods as well as the protein and DNA composition of the
ECM scaffolds.
The capability of stem cells and ECM scaffolds to
modulate the immune response at the site of injury is
another aspect that is crucial to muscle regeneration.
Of particular interest, VML injury results in a height-
ened and persistent inflammatory response that over-
whelms its innate capacity for regeneration.36,164 In a
recent study, it was shown that muscle trauma
prompts robust and persistent overexpression of in-
flammatory transcripts, which contributes to fibrotic
tissue deposition and may impair satellite cell-medi-
ated repair.35 It has been suggested that both M1 (pro-
inflammatory) and M2 (anti-inflammatory) macro-
phage phenotypes are necessary for muscle regenera-
tion.33,130 M1 macrophages promote the activation,
migration, and proliferation of satellite cells.34,160,177
M2 macrophages are needed to mitigate the immune
response and support the differentiation and matura-
tion of satellite cells into myotubes and myofibers.33
However, early recruitment of M2 macrophages can
dysregulate satellite cell activity and delay regenera-
tion.160,177 On the other hand, the persistent M1
phenotype is likely to exacerbate muscle damage and
support fibrotic tissue deposition. Therefore, these
macrophage populations need to be activated for an
optimum duration and at an ideal intensity. Immuno-
genic concerns have been reported with decellularized
scaffolds,157 poly(ethylene glycol),178 and hyaluronic
acid179 based materials. Immunomodulation via immu-
nosuppressant drugs180 or paracrine action of stem
cells181 may be able to regulate the intensity and
duration of macrophage phenotypes.
There are several ongoing clinical trials with stem
cells for a variety of different conditions.182 While
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these trials are highlighting the roles these stem cell
populations play in replacing damaged tissue and
replenishing trophic and extracellular factors, con-
cerns about these treatments have also been
expressed. Several factors must be considered for
selecting a suitable stem cell population for skeletal
muscle regeneration which include availability and
abundance of the cell source, susceptibility to genetic
manipulation, ease of delivery, and finally the ability
to undergo functional myogenesis in vivo.183 Regard-
ing stem cell therapies, uncontrolled differentiation is
a potential concern. For instance, heterotopic ossifica-
tion—an aberrant growth of bone within a soft tissue
has been associated with orthopedic trauma.184,185
This debilitating condition has been linked to
MSCs,186,187 pericytes,188 FAPs,189 and decellularized
scaffolds.190 Pre-treating cells to promote their com-
mitment down a particular lineage prior to injection is
one avenue that that could be explored to potentially
evade these issues. Some research has been done to
“prime” cells prior to injection.191,192 In several stud-
ies, preconditioning of myoblasts seeded on 3D scaf-
folds has accelerated muscle tissue regeneration and
function when implanted in vivo.143,193–195 In other
studies, pre-conditioning MSCs with mechanical strain
has resulted in enhanced myogenesis and secretion of
beneficial growth and immunomodulatory factors in
vitro.192,196–198 However, a positive effect on in vivo
myogenesis was not observed following the injection of
“preconditioned” MSCs in aged muscles.191 The poten-
tial of preconditioning stem cells is immense, and
further research in this field is necessary. Culturing
cells on tissue engineered scaffolds with electrical and
mechanical stimulation is an avenue currently being
explored in various studies.3,199
Recently, the paracrine action of stem cells is being
investigated via exosomes. Extracellular vesicles, i.e.,
exosomes, allow for cell-cell communication and could
be a key to unlocking many of the challenges associated
with the delivery and engraftment of stem cells.
Exosomes can be released from paracrine cells and
myofibers, containing mRNA, miRNA, and proteins
that can influence gene expression and cellular func-
tions after being endocytosed by the host cell.200 These
nanospheres could be used to trick the host tissue into
a more pro-regenerative state. Using specific myogenic
media, cultured MSCs will release exosomes with a
specific myogenic load. Exosomes can be collected using
ultracentrifugation and could supplement the injection
of myogenic stem cells. C2C12 cells showed increased
proliferation and migration when exposed to MSC
exosomes.200 Similar results have been seen in myo-
blast derived exosomes.201 In a murine TA laceration
model, muscles treated with myoblast-derived exosomes
showed increased regeneration and decreased fibrosis
compared to untreated controls. Despite convincing
evidence for this cell-free therapeutic approach, the
identification of exact factors in exosomes that play key
roles in muscle regeneration remains unclear.

Overall, it is becoming increasingly clear that tissue
engineering solutions to muscle trauma can be im-
proved by combining bioengineered scaffolds with
stem cells, growth factors, and potentially electrical/
mechanical stimulation to guide muscle tissue growth.
Although a combination therapy of cells and biomate-
rials seems promising, the complexity of this approach
is likely to impact regulatory approval, clinical trans-
lation, and off-the-shelf availability to patients.
AUTHORS’ CONTRIBUTIONS
AP drafted the Introduction. AD and MM drafted
section 2, Stem cells for skeletal muscle regeneration.
MT and KP drafted section 3, Biomaterials for skeletal
muscle regeneration. AD drafted section 4, Challenges
with existing technologies and future trends. KG and
AD reviewed and revised each section. All authors have
read and approved the final version of the submitted
manuscript.
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