Functional muscle regeneration with combined
delivery of angiogenesis and myogenesis factors
Cristina Borsellia,b, Hannah Storriea, Frank Benesch-Leec,d, Dmitry Shvartsmana, Christine Cezara, Jeff W. Lichtmane,
Herman H. Vandenburghc,d, and David J. Mooneya,1
aSchool of Engineering and Applied Sciences and Wyss Institute, Harvard University, Cambridge, MA 02138;bDepartment of Material Engineering and
Production, University of Naples Federico II, Naples 80138, Italy;cMyomics Inc., Providence, RI 02906;dDepartment of Pathology, Brown Medical School/
Miriam Hospital, Providence, RI 02912; andeDepartment of Molecular and Cellular Biology, Center for Brain Science, Harvard University, Cambridge,
Edited by Stephen F Badylak, University of Pittsburgh Medical Center, Pittsburgh, PA, and accepted by the Editorial Board October 22, 2009 (received for
review April 15, 2009)
Regenerative efforts typically focus on the delivery of single
factors, but it is likely that multiple factors regulating distinct
aspects of the regenerative process (e.g., vascularization and stem
cell activation) can be used in parallel to affect regeneration
of functional tissues. This possibility was addressed in the context
of ischemic muscle injury, which typically leads to necrosis and loss
of tissue and function. The role of sustained delivery, via injectable
gel, of a combination of VEGF to promote angiogenesis and
insulin-like growth factor-1 (IGF1) to directly promote muscle
regeneration and the return of muscle function in ischemic rodent
hindlimbs was investigated. Sustained VEGF delivery alone led to
neoangiogenesis in ischemic limbs, with complete return of tissue
perfusion to normal levels by 3 weeks, as well as protection from
hypoxia and tissue necrosis, leading to an improvement in muscle
contractility. Sustained IGF1 delivery alone was found to enhance
muscle fiber regeneration and protected cells from apoptosis.
However, the combined delivery of VEGF and IGF1 led to parallel
angiogenesis, reinnervation, and myogenesis; as satellite cell
activation and proliferation was stimulated, cells were protected
from apoptosis, the inflammatory response was muted, and highly
functional muscle tissue was formed. In contrast, bolus delivery of
factors did not have any benefit in terms of neoangiogenesis and
perfusion and had minimal effect on muscle regeneration. These
results support the utility of simultaneously targeting distinct
aspects of the regenerative process.
alginate|insulin-like growth factor-1|tissue engineering|VEGF|satellite
Under normal conditions, skeletal muscle can repair itself by
removing damaged myofibers and synthesizing new muscle fibers
to restore functional contractile properties (1). After necrosis of
damaged muscle fibers, an inflammatory response is initiated (2)
leading to the phagocytosis of the injured myofibers and the
activation of the normally quiescent population of satellite cells
(3, 4). The activated satellite cells proliferate, migrate to the site
of injury, fuse and, differentiate to form new myofibers (5, 6).
However, muscle degeneration in the context of tissue ischemia,
advanced age (7), severe injuries, or in the context of genetic
defects [e.g., muscular dystrophy (8)], may lead to impaired
healing, permanent loss of muscle mass, disease progression, and
functional deficiency. Given that 6 million Americans are diag-
nosed with musculoskeletal diseases each year, the potential for
improved skeletal muscle repair strategies is significant (9, 10).
The main strategies currently pursued for skeletal muscle
regeneration consist of cell therapies, drug delivery strategies, or
a combination of both approaches. Cell therapies, either by
direct injection of cells into the damaged tissues (11) or the
transplantation of progenitor cells on polymeric scaffolds (12,
13), are typically limited by the death of the majority of the
transplanted cells and/or poor integration of the templated tis-
he long-term goal for muscle regeneration strategies is to
recover the contractile properties of the injured muscles.
sues with the host tissue. Most of the drug delivery strategies thus
far have yielded limited success, most likely related to rapidly
depleted local concentrations, inappropriate gradients, and/or
loss of bioactivity of growth factors (GFs) resulting from bolus
drug delivery and rapid degradation in the inflammatory in vivo
environment of the damaged tissue. Biodegradable polymeric
systems have been developed to provide localized and sustained
GF release (14). However, it may be necessary to deliver mul-
tiple morphogens acting in distinct aspects of the tissue re-
generation process to drive muscle regeneration to completion,
as previously reported for other tissues (15).
The hypothesis underlying this study is that localized and
sustained presentation of factors that modulate both angio-
genesis and myogenesis can stop or reverse muscle injury
resulting from tissue ischemia. Many previous studies have
examined vascular regeneration after ischemic injury, but the
purpose of this study was to investigate instead the effect of GF
delivery on the regeneration of the skeletal muscle. The sus-
tained release of VEGF from polymeric delivery systems has
been widely implicated in neovascularization and was previously
demonstrated to enhance blood vessel formation and perfusion
within ischemic muscle tissue (15, 16). Several recent studies
have demonstrated that VEGF leads to better maintenance of
skeletal muscle tissue in mouse models of muscular dystrophy,
suggesting that VEGF may also play a role in muscle re-
generation after injury (17–19). Other findings highlight the
possibility that VEGF’s ability to promote vascularization could
increase the availability of blood vessel–associated stem cells
capable of participating in muscle regeneration (20–22). How-
ever, a direct improvement in muscle regeneration has not been
documented. In addition, although VEGF is a well-established
proangiogenic regulator, its presence is likely not sufficient to
recapitulate the complex muscle regeneration process, because
this requires the activation and differentiation of the muscle’s
stem cell population to generate new muscle.
Various stem cell populations are capable of building new
muscle fibers (8, 23–25), but satellite cells, an adult stem cell
population associated with myofibers and localized within the
basal lamina of the muscle fibers, are believed to be primarily
responsible for muscle regeneration (6). The activation of sat-
ellite cells is finely regulated via a number of biochemical and
biomechanical cues, including both inflammatory cytokines (e.g.,
Author contributions: C.B., J.W.L., H.H.V., and D.J.M. designed research; C.B., H.S., D.S.,
and C.C. performed research; C.B. and F.B.-L. contributed new reagents/analytic tools; C.B.
and H.S. analyzed data; and C.B. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission. S.F.B. is a guest editor invited by the
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
| February 23, 2010
| vol. 107
| no. 8
IL-4, leukemia inhibitory factor, TGF-β, IL-6, and TNF-α) (26),
and GFs, including hepatocyte growth factor, fibroblast growth
factor-2 (25), insulin-like growth factors (1), and GDF8/myo-
statin (27). Insulin-like growth factor-1 (IGF1) plays a key mod-
ulatory role in muscle growth and regeneration, acting during all
of the stages of regeneration (27). The binding of IGF1 to type I
receptors has been shown to activate three different intracellular
signaling pathways—the MAP kinase pathway, the PI3-K pathway,
and the calcium–calmodulin-dependent protein kinase pathway—
leading to the activation and proliferation of the satellite cells,
their terminal myogenic differentiation (indicated by MyoD and
myogenin levels), increased protein synthesis, myofiber survival,
and myofiber hypertrophy (27).
This study investigated a potential interplay between VEGF and
IGF1 in ischemic muscle regeneration, and the possibility that dual
sustained delivery of these two critical morphogens could induce
the regeneration of functional muscle in ischemic hindlimbs. The
impact of the distance of the muscle from the factor delivery site
on the regeneration process was also examined by analyzing dis-
tinct muscles in the hindlimbs. As targets for these experiments, we
chose the gracilis and tibialis muscles, respectively corresponding
to the muscle site of injection and a muscle distant to the site of
polymer placement. The ultimate goal of this approach is to pre-
serve the local progenitor cells from apoptosis and necrosis during
the degeneration process, and instead to activate the progenitor
cells to enter the proliferative phase and differentiate into con-
tractile muscle fibers to regenerate functional tissue.
Sustained VEGF and IGF1 Presentation Enhance Muscle Size and Limb
Vascularization. An ischemia injury was selected for these studies
after analysis of the spontaneous recovery of muscle mechanical
function subsequent to various types of injuries, including partial
laceration, cryoinjury, and notexin injection (Fig. S1). Ischemia
led to the greatest loss of muscle function, as compared with the
other injury models, and the least spontaneous return of function.
Mice were treated at the time of ischemia induction with an
injectable, degradable (28) alginate gel (Fig. 1A). In vitro, after an
initial burst, VEGF was released in a sustained manner over time,
as previously described, whereas IGF, owing to its smaller size (7.5
kDa) and its non–heparin-binding nature, showed a faster release;
≈80% of the total IGF loaded was released in the first 24 h (Fig.
1B). The following five interventions were analyzed: (i) blank al-
ginate gel, (ii) alginate gel delivering VEGF (3 μg), (iii) alginate
gel delivering VEGF and IGF1 (3 μg each), (iv) alginate gel de-
livering IGF1 (3 μg), and (v) bolus delivery of VEGF and IGF1 (3
μg each) in PBS.
Significant muscle loss was noted at 7 weeks after surgery with
blank gel treatment (Fig. S2 E and F), whereas injured muscles
treated with gel containing both GFs were grossly larger. Quan-
tification of the weight of these muscles revealed insignificant
changes with gel releasing either VEGF or IGF alone, or with the
saline bolus treatment, whereas statistically significant increases of
26% ± 11% and 30% ± 22% occurred for the tibialis (distant to
gel injection) and gracilis muscles (site of gel injection) (Fig. 1 C
and D), respectively, receiving gel releasing both GFs as compared
with the blank treatment. The large standard deviations in the
gracilis muscle analysis were due to the difficulty in isolating the
gracilis muscle from the other tightly associated muscles.
Because the effects of VEGF delivery on muscle regeneration
were likely mediated by its effects on angiogenesis, the level of
muscle hypoxia, perfusion of ischemic tissues, and tissue necrosis
were next analyzed. Immunohistochemical analysis of tibialis and
gracilis muscle tissues revealed that alginate/VEGF and alginate
VEGF/IGF1 increased muscle blood vessel densities, as com-
pared with injection of a blank vehicle or bolus delivery of VEGF/
IGF (Fig. S2 A and C). In particular, at 7 weeks, VEGF delivery
from the gels resulted in an approximately 2-fold increase in
vessel density in tibialis and 3-fold increase in the gracilis muscle,
as compared with the ischemic hindlimb treated with the blank
alginate (Fig. S2 B and D). IGF delivery alone had no significant
effect on vascularization in the gracilis muscle (Fig. S2D) and a
modest effect in the tibialis (Fig. S2B). The bolus delivery had no
effect on blood vessel densities, as compared with controls.
A laser Doppler perfusion imaging (LDPI) system was used to
quantify perfusion (Fig. 2A, Fig. S3A). The regional blood flow
was reduced immediately after surgery to ≈20% of normal in all
conditions, as expected (Fig. 2A). Alginate gel–only treatment led
to a slow increase in reperfusion over time, and the ischemic limbs
for the most part remained necrotic (Fig. 2B). Bolus delivery re-
sulted in little difference from the no-treatment control or blank
alginate injection. In contrast, VEGF and dual GF delivery from
the vehicle led to a final recovery of, respectively, 80% and 95% of
normal limbs. In particular, animals treated with alginate gels
delivering VEGF/IGF1 showed a marked increase in blood flow
starting around the 4th week after the injury, and an additional
20% increase at 7 weeks compared with the control (Fig. 2A).
The level of tissue necrosis (Fig. 2B) was also quantified by
visual observation. Hindlimb ischemia led to severe toe or foot
gangrene in control animals, but treatment with alginate gel with
VEGF and VEGF/IGF largely spared the limbs from necrosis.
Protection of myofibers from hypoxia was also observed with
alginate gel VEGF and VEGF/IGF delivery (Fig. S3B).
VEGF and IGF1 Induce Myoblast Proliferation and Protect Against
Apoptosis. Immunostaining of tissue sections against the pro-
liferation-associated protein Ki67 was performed to determine
cell proliferation activity at early (2 weeks) and late (7 weeks)
vitro. (C and D) Weight of the nonoperated (control) tibialis (C) and gracilis (D) muscles at 7 weeks, compared with the muscles after ischemic injury and
treatment with blank alginate gel (alginate gel), alginate gel delivering VEGF (gel/VEGF), alginate gel delivering VEGF and IGF1 (gel/VEGF+IGF), alginate gel
delivering IGF1 (gel/IGF), and bolus delivery of VEGF and IGF1 in PBS (bolus).Values represent mean ± SD (n = 6) in all graphs. At *P < 0.05 level the means are
significantly different compared with the control and the blank alginate.
(A) Photograph of the injectable alginate gel (color due to medium used to reconstitute alginate). (B) VEGF and IGF1 release kinetics from gels in
| www.pnas.org/cgi/doi/10.1073/pnas.0903875106Borselli et al.
times. Abundant expression of Ki67 was detected in muscle tis-
sues receiving alginate gels releasing VEGF alone and VEGF/
IGF1 in both tibialis (Fig. S4 A and B) and gracilis muscles (Fig.
S4 C and D) at 2 weeks (Fig. S4 A and C) and 7 weeks (Fig. S4 B
and D). A less-pronounced increase was observed with alginate
gel delivering IGF, whereas no proliferation was observed in
muscles treated with the blank vehicle. Furthermore, triple im-
munofluorescence for CD31 (red), Ki67 (green), and DAPI
(blue) for nuclear staining suggests that endothelial cells and
other cell types, presumably myoblasts, proliferated at early
stages of the regenerative process (Fig. S5). TUNEL analysis was
performed to measure apoptosis in the regenerating muscles at 2
weeks after ischemia. Whereas significant apoptosis was ob-
served in the blank vehicle group (Fig. S6A), apoptosis was re-
duced in the muscles treated with alginate delivering VEGF (Fig.
S6B) and was significantly lower with vehicles delivering IGF
alone (Fig. S6C). The combination of the two GFs was partic-
ularly effective in combating ischemia-induced apoptosis (Fig.
S6D). Apoptosis was virtually absent in contralateral normo-
perfused muscles (Fig. S6E), as expected.
Muscle Regeneration Enhanced by VEGF and IGF1, Along with
Reduced Fibrosis. To directly analyze muscle regeneration, the
mean diameter of regenerated myofibers and number of centrally
located nuclei in the resolving muscle tissue were quantified (Fig.
3). The mean diameters of muscle fibers were quantitatively
greater in muscles treated with alginate delivering both GFs
compared with alginate delivering only VEGF or IGF1 or the two
GFs in bolus saline, in both tibialis (Fig. 3 A and B) and gracilis
muscles (Fig. 3 D and E). The tibialis muscles treated with alginate
delivering VEGF or IGF1 alone showed an ≈10% increase in
average diameter, whereas codelivery of both GFs led to a 25%
increase in the diameter of regenerating fibers compared with the
blank alginate gel, and a 19% increase compared with gel/VEGF
(P < 0.05) (Fig. 3A). An increase was also observed in gracilis
muscle with VEGF/IGF delivery from the alginate gels (Fig. 3D).
At 2 weeks after injury the tibialis muscle fibers in the injury group
treated with VEGF or IGF1 alone also showed an ≈40% increase
in centrally located nuclei, vs. a lesser increase of 30% with bolus
factors delivery as compared with the blank. The two factors in
combination with alginate delivery led to a 53% and a 39% in-
crease in centrally located nuclei, as compared with the blank
alginate or alginate delivering VEGF alone (Fig. 3B). The number
of centrally located nuclei in the gracilis fibers (Fig. 3E) treated
with alginate delivering both GFs increased ≈70% and 20%, re-
spectively, when compared with either the blank alginate or with
alginate delivering VEGF only. Representative cross and longi-
tudinal microsections of tibialis tissue highlight the increase in
centrally located myonuclei in the ischemic muscles treated with
alginate delivering both GFs (Fig. 3C). Analysis of the muscle
fiber types confirmed an active regenerative process induced by
GF delivery. Type IIC fibers were noted at early times (3 days)
after injury with delivery of GF from the gel but were not present
in uninjured control muscles or uninjured muscles treated with
gel/GF (Fig. S7). Furthermore, analysis of injured muscle treated
with gel delivering VEGF revealed a significant increase in my-
ogenin-positive cells, which contrasts with few myogenin-positive
cells in control, uninjured muscle (Fig. S8), also supporting an
active muscle regeneration process. Furthermore, analysis for the
expression of the activated satellite cell marker Pax7 demon-
strated an increase in Pax7-positive cells in the injured muscle
perfusion analysis of C57 mice hindlimbs treated with blank
alginate gel, alginate gel delivering VEGF, alginate gel de-
livering VEGF and IGF1, alginate gel delivering IGF1, and
bolus delivery of VEGF and IGF1 in PBS. *P < 0.05 vs. blank
alginate gel and bolus; mean values are presented with SD.
(B) Ischemic hindlimbs treated with blank alginate gel, algi-
nate gel delivering VEGF, and alginate gel delivering VEGF
and IGF1 were visually examined to determine the severity of
hindlimb ischemia at 1, 3, and 7 weeks after ligation.
Blood perfusion and tissue necrosis. (A) LDPI blood
centrally located nuclei (B and E) of regenerating fibers at 2 and 7 weeks after
the induction of ischemia were quantified. ANOVA statistical tests were per-
formed on all data sets. *P < 0.05 vs. blank alginate gel. (C) Representative
photomicrographs of tibialis tissue sections from ischemic hindlimbs at post-
operative week 2, stained with H&E [cross and longitudinal (long) section,
respectively, of contralateral hindlimb and ischemic muscles treated with al-
ginate vehicle delivering VEGF/IGF1]. Scale bars, 50 μm.
Analysis of muscle regeneration. Diameter (A and D) and number of
Borselli et al.PNAS
| February 23, 2010
| vol. 107
| no. 8
tissue treated with gel delivering VEGF (Fig. S8), supporting
satellite cell activation with GF delivery.
Injured muscle tissue treated with blank alginate demon-
strated significant interstitial fibrotic tissue (Fig. S9). Control
(nonoperated) limbs demonstrated little fibrosis, as expected.
However, limbs treated with alginate gel delivery of both GFs
exhibited a significant decrease in fibrosis. A less-pronounced
reduction of fibrosis was observed with the two GFs delivered
alone. Conversely, in the bolus injection condition a large con-
tent of fibrotic tissue was formed.
Growth Factor Delivery Promotes Earlier Regeneration of Damaged
Neuromuscular Junctions. Induction of ischemia in the hindlimb
and treatment with a blank hydrogel led to a significant loss of
innervation at the neuromuscular junction (NMJ) in the tibialis
muscle 7 days after injury in control mice (Fig. 4A); by day 14
complete reinnervation had occurred, and NMJs appeared nor-
mal (Fig. 4E). In contrast, muscles treated with either VEGF
alone (Fig. 4B), IGF1 alone (Fig. 4C), or VEGF/IGF1 (Fig. 4D)
had completely reformed NMJs, and no damage to receptors or
muscle fibers was observed at 7 days.
Dual Gel Delivery of VEGF and IGF1 Enhances the Contraction Force of
Damaged Muscles. Finally, to test whether muscle changes induced
by GF delivery would correspond to increased function, the con-
tractile force of the muscles was analyzed. The weight normalized
tetanic force of the tibialis (Fig. 5A) and gracilis muscles (Fig. 5B)
were measured after maximal tetanic stimulation. Muscles treated
with gel delivering both GFs showed a significant increase above
normal values in the tetanic force at 2 weeks after surgery (2.3-
and 7.9-fold increase, respectively, for tibialis and gracilis muscles,
when compared with the blank) followed by a decrease toward the
normal value at 7 weeks. Animals receiving alginate delivering
VEGF alone showed a similar trend, but the increase in the force
of contraction was less pronounced. In particular, at 2 weeks a 1.6-
and 5.7-fold increase was measured in tibialis and gracilis muscles,
respectively, compared with alginate gel only. In contrast, the
animal receiving alginate gel without GFs had a markedly lower
contractile function at all time points.
The results from these studies suggest a beneficial interplay
between VEGF and IGF1, when delivered appropriately, in
enhancing skeletal muscle regeneration, revascularization, rein-
nervation, and gain of function after ischemic injuries. Past
therapies to regenerate ischemic tissues typically relied on bolus
delivery or systemic administration of single GFs. VEGF specifi-
cally has been widely used as a potent proangiogenic initiator in
many strategies to treat ischemic diseases (29). However, the
impact on salvaging and driving regeneration of ischemic muscle
has not been addressed (30). Moreover, an extensive body of lit-
erature supports a role for IGF1 in regulating the establishment
and maintenance of the mature muscle phenotype in normal and
regenerating muscle tissue both in vitro (28, 31) and in vivo (32,
33). In particular, IGF1 has been implicated in early and late
stages of muscle developmental processes, playing first a role in
inducing myoblast proliferation and subsequently promoting
myogenic differentiation (5, 34). Past approaches to exploit GF
signaling in muscle regeneration typically used bolus GF delivery,
which leads to rapid depletion of the factors in the target tissue.
Supraphysiologic concentrations of GFs are used in an effort to
offset this issue, potentially leading to unwanted side effects (35).
Sustained VEGF delivery alone from alginate gels had a sig-
nificant impact on angiogenesis and tissue perfusion but a less
pronounced effect on muscle regeneration (17–19). These results
are in accord with previous reports that the sustained and con-
trolled release of VEGF from both a poly(lactide-co-glicolide)
(PLG) (15) and the same injectable alginate-based vehicle (16)
stimulated angiogenesis, returned perfusion to normal levels,
and prevented necrosis in ischemic hindlimbs. VEGF has also
recently been implicated in muscle regeneration (36) and muscle
reinnervation via a direct neuroprotective and neurodirecting
effect (37, 38). The contractile activity of skeletal muscle, and
hence its functionality, are regulated by the nervous system, and
loss of innervation leads to a decrease in satellite cell number
and muscle atrophy (38). The results of this study suggest that
delivery of VEGF alone has profound effects on muscle re-
generation, because increases in the diameter of regenerating
fibers and the number of centrally located nuclei in muscle fibers,
both hallmarks of regenerating myofibers (39, 40) (Fig. 3), were
found with gel/VEGF delivery. These results are consistent with
past reports that VEGF may play an important role in muscle
maintenance and regeneration (17–19). The contractile proper-
ties of the injured muscle (Fig. 5) were also improved with ap-
propriate VEGF delivery.
IGF1 delivery alone from alginate gels was found to have a
modest effect on muscle fiber regeneration and cell protection
from apoptosis. These data are consistent with previous studies in
which increased levels of IGF1 augmented tissue DNA content
(resulting from activation of satellite cells) (40–42) and muscle
protein synthesis within existing myofibers (43–46). Gel/IGF1
and treatment with blank alginate gel (A), alginate gel delivering VEGF (B), alginate gel delivering IGF1 (C), or alginate gel delivering VEGF and IGF1 (V+I, D).
(E) Quantification of the sites of overlap of motor neuron axon (yellow) and endplate (red) as a site of reinnervation. Values are mean ± SD. *P < 0.05.
Analysis of reinnervation. (A–D) Photomicrographs of the NMJ in the tibialis muscle stained with bungarotoxin at 7 days after induction of ischemia
terior tibialis (A) and gracilis (B) muscles of mice was measured at 2 and 7
weeks after induction of ischemia and treatment. Tetanic force was nor-
malized to each muscle’s weight to obtain weight-corrected specific force.
Mean values are presented with SD. *P < 0.05 vs. control limb and blank
alginate gel; **P < 0.05 level vs. blank alginate gel.
Functional properties of skeletal muscles. Tetanic force of the an-
| www.pnas.org/cgi/doi/10.1073/pnas.0903875106 Borselli et al.
delivery alone also induced neoangiogenesis in the tibialis muscle
and to a lesser effect in the gracilis muscle. This effect was likely
secondary to the effects of IGF1 on the muscle cells. The delivery
approach used in this study resulted in an initial burst delivery of
this factor, likely leading to a rapid diffusion of the factor from the
site of the injection. A more sustained delivery of IGF1 may more
significantly increase muscle regeneration.
Strikingly, dual VEGF/IGF1 delivery from gels had a syn-
ergetic effect on the regenerative parameters in both of the
analyzed muscles. In particular, both the mean fiber diameter
and the number of centrally located nuclei in the fibers (Fig. 3)
were significantly enhanced with alginate delivery of both GFs,
showing a more pronounced response in the muscle where the
gel was injected (gracilis). These results were qualitatively vali-
dated by an increased number of myoblasts found in an active
proliferative state, the presence of myogenin and Pax7-positive
cells (Fig. S8), type IIC muscle fibers (Fig. S7), and decreased
cell apoptosis (Fig. S6). These results suggest an enhancement in
myoblast recruitment for neomuscle formation, which is con-
sistent with the larger size and mass of these muscles (Fig. 1, Fig.
S2 E and F). However, the enhanced myogenic regeneration in
response to VEGF and VEGF/IGF sustained delivery could also
be explained by recent findings (20–22) suggesting the existence
of myogenic precursors distinct from satellite cells (e.g., pericyte-
derived cells and myoendothelial cells) endowed with multi-
lineage potential, including high muscle regenerative potential.
Stimulation of angiogenesis may increase the pool of myogenic
stem cells that are available to drive muscle regeneration. Fur-
thermore, the combination of VEGF/IGF1 was shown to alle-
viate ischemia, with a return to normal hemodynamic levels and
a better prevention of the necrosis associated with ischemia.
Previous in vivo studies, using this same animal model, con-
firmed that the sustained delivery of bioactive GFs (VEGF) from
this gel system led to long-term (>15 days) elevated muscle levels
(16). This contrasted with bolus delivery: the factor concen-
tration fell to undetectable levels within hours after that delivery
approach. The sustained presence of factors enabled by alginate
gel delivery correlated with the long-term alterations in the
vascular and muscle tissue noted in the present study with gel
delivery, as contrasted to bolus delivery. However, the precise
relationship between GF presence and regeneration remains
unclear. This relationship should be probed in the future by re-
moving the gels at various times after implantation and/or pro-
viding multiple gel injections at various time points.
Because the peripheral nervous system is also affected by ische-
NMJ were also examined. Ischemia is known to result in loss of
was observed in the injury model used in this study. In the absence
of GFs, axons required 2 weeks to fully regenerate. In contrast,
treatment with gels releasing either IGF1 alone, VEGF alone, or
amyotrophic lateral sclerosis, which is believed to be mediated by
(49), suggesting that the mechanism of IGF neuroprotection may
observed upon treatment with VEGF and IGF1 suggests that gel
delivery of factors may be useful in treating the neurologic com-
plications of chronic ischemia. Together these effects likely played
Most strikingly, tetanic force measurements of the tibialis (Fig.
5A) and gracilis (Fig. 5B) muscles demonstrated a significant in-
crease to above-normal levels with dual delivery of GFs at 2
weeks, with a 2 fold- and an 8-fold increase in force for tibialis and
gracilis respectively, vs. the untreated (blank alginate) hindlimb,
indicating functional muscle regeneration. Conversely, a sig-
nificant decrease toward the normal value was observed after 7
weeks, likely indicating an adaptation to normal physiologic re-
quirements for these muscles. Increased muscle strength was also
associated with a decrease in fibrotic tissues (Fig. S9). Previous
studies have shown a role of IGF1 in finely modulating the balance
between inflammation and regeneration, which is crucial for ac-
celerating the functional recovery of injured muscle (52). After
muscle injury, an inflammatory response is activated, but pro-
longed accumulation of fibrotic tissue limits muscle cell replace-
ment, leading to less strength and functional depletion compared
with normal muscles. The increased force observed in muscles
with GFs delivery may also be related to enhanced reinnervation,
although the specific mechanisms by which these GFs influence
reinnervation remain to be defined (53, 54).
In summary, the dual delivery of VEGF/IGF1 from an
injectable biodegradable hydrogel leads to a complete functional
recovery of ischemic injured skeletal muscle. This strategy to
enhance skeletal muscle regeneration may provide a therapeutic
option for treatment of muscle damaged from a variety of causes.
In the future, additional factors that play roles in regulating the
proliferation and differentiation of satellite cells and cells could
also be incorporated and delivered with this system. More
broadly, the concept of simultaneously stimulating the regener-
ation of the vascular and the parenchyma of damage tissues will
likely be useful in many situations.
Materials and Methods
Growth Factor Incorporation and Release Kinetics. Ultrapure MVG alginate was
purchased from ProNova Biomedical. Gels were formed from a combination of
polymer molecular weights as previously described (16). Alginates were re-
constituted in EBM-2 (Cambrex) to obtain a 2% wt/vol solution before gelation
and cross-linked with aqueous slurries of a calcium sulfate (0.21g CaSO4/mL
dH2O) at a ratio of 25:1 (40 μL of CaSO4per 1 mL of 2% wt/vol alginate sol-
ution). Alginates were premixed with recombinant human VEGF165protein
(generously provided by the Biological Resources Branch of the National
Cancer Institute) and/or with recombinant human IGF1 (R&D Systems), at a
final concentration of 60 μg/mL for each protein; in vitro release kinetics were
measured using ELISA.
Animals and Surgical Procedures. Animal work was performed in compliance
with National Institutes of Health and institutional guidelines. Female C57BL/6J
mice (aged 6 to 7 weeks; Jackson Laboratories) were anesthetized with an i.p.
injection of ketamine 80 mg/kg and xylazine 5 mg/kg before all surgical pro-
cedures. Hindlimb ischemia was induced by unilateral external iliac and femoral
artery and vein ligation, as previously described (55). After the vessel ligation,
mice were injected with a total volume of 50 μL of alginate gel containing 3 μg
of VEGF165and/or 3 μg of IGF1, gel containing 3 μg of IGF1, gel with no GFs, or a
PBS solution containing 3 μg of VEGF165and 3 μg of IGF1 directly into the gracilis
muscle (1–3 mm inside the muscle).
For analysis of reinnervation, hindlimb ischemia and gel delivery were
carried out as described in transgenic C57BL/6 mice selectively expressing YFP
under control of a thy-1 promoter in motoneurons (56).
Ischemia and Perfusion. Measurements of the ischemic/normal limb blood flow
ratio were performed on anesthetized animals (n = 10) using an LDPI analyzer
Histologic Assessment of Skeletal Muscle. Micewerekilledand hindlimb muscle
tissues (n = 10 per time point per experimental condition) processed for his-
tologic analyses. The samples were stained with H&E, and fiber diameter and
the number of centrally located nuclei were analyzed as described (25). Vas-
cular endothelial cells were identified by immunostaining for mouse CD31 (BD
Biosciences Pharmingen). For measurement of capillary densities, histologic
analysis was performed in a blinded fashion as previously described (16). Im-
munostaining for Ki-67 (Ki-67 mouse IgG1; Dako) was performed to identify
cell proliferation. Qualitative analysis of apoptosis was assessed by TUNEL as-
say (Roche). Interstitial fibrosis was morphometrically assessed in Masson Tri-
chrome (Sigma Aldrich) stained sections.
Analysis of Reinnervation. Mice were anesthetized by an i.p. injection of ket-
amine/xylazine and fixed by transcardial injection of 4% paraformaldehyde.
The tibialis muscle was explanted and stained with Alexa594-bungarotoxin
Borselli et al.PNAS
| February 23, 2010
| vol. 107
| no. 8
(Invitrogen) to visualize acetylcholine receptors. Innervation at the NMJ was Download full-text
imaged using a Zeiss Pascal 5 LSM upright laser scanning confocal microscope
bungarotoxin at 543 nm. At least 50 NMJs were counted for each condition.
Statistical significance was determined using unpaired ANOVA analysis.
Mechanical Measurements. Intact gracilis and tibialis muscles were dissected
(n = 5/condition), mounted vertically midway between two cylindrical par-
allel steel wire electrodes (1.6 mm diameter, 21 mm long) attached by their
tendons to microclips connected to a force transducer (FORT 25, WPII) and
bathed in a physiologic saline solution (58) in a chamber oxygenated with 95%
O2/5% CO2at 25°C. Muscle length was adjusted until maximum twitch force
was achieved (100–300 Hz). A wave pulse was initiated using a custom-written
LabVIEW program and delivered to the stimulation electrodes via a purpose-
built power amplifier (QSC USA 1310). Contractions were evoked every 5 min.
Tetani were usually evoked at 300 Hz, 15–20 V, with constant pulse width and
train duration of 2 ms and 1 s, respectively. Peak tetanic force was determined
as the difference between the maximum force during a contraction and the
baseline level, and specific force calculated by normalization by muscle weight.
Statistical Analyses. All results are expressed as mean ± SD. Multivariate re-
peated-measures ANOVA was performed to test for interactions between con-
ditions. Differences between conditions were considered significant at P < 0.05.
ACKNOWLEDGMENTS. We thank the Biological Resources Branch of the
National Cancer Institute for providing VEGF for the studies. Supported by
National Institutes of Health Grants R01 DE013349 and R43AG029705), the
Italian Institute of Technology, the “Fondo degli Investimenti della Ricerca di
Base” (Italy), and European Molecular Biology Organization Long-Term Fel-
lowship ALTF 42-2008.
1. Chargé SB, Rudnicki MA (2004) Cellular and molecular regulation of muscle regeneration.
Physiol Rev 84:209–238.
2. Tidball JG (2005) Inflammatory processes in muscle injury and repair. Am J Physiol
Regul Integr Comp Physiol 288:R345–R353.
3. Mauro A (1961) Satellite cell of skeletal muscle fibers. J Biophys Biochem Cytol 9:
4. Schultz E, Gibson MC, Champion T (1978) Satellite cells are mitotically quiescent in
mature mouse muscle: An EM and radioautographic study. J Exp Zool 206:451–456.
5. Engert JC, Berglund EB, Rosenthal N (1996) Proliferation precedes differentiation in
IGF-I-stimulated myogenesis. J Cell Biol 135:431–440.
6. Wagers AJ, Conboy IM (2005) Cellular and molecular signatures of muscle regeneration:
Current concepts and controversies in adult myogenesis. Cell 122:659–667.
7. Grounds MD (1998) Age-associated changes in the response of skeletal muscle cells to
exercise and regeneration. Ann N Y Acad Sci 854:78–91.
8. Gussoni E, et al. (2002) Long-term persistence of donor nuclei in a Duchenne muscular
dystrophy patient receiving bone marrow transplantation. J Clin Invest 110:807–814.
9. Lubeck DP (2003) The costs of musculoskeletal disease: Health needs assessment and
health economics. Best Pract Res Clin Rheumatol 17:529–539.
10. McClatchey KD (2004) Musculoskeletal conditions affect millions. Arch Pathol Lab
11. Partridge TA, Morgan JE, Coulton GR, Hoffman EP, Kunkel LM (1989) Conversion of mdx
myofibres from dystrophin-negative to -positive by injection of normal myoblasts. Nature
12. Saxena AK, Marler J, Benvenuto M, Willital GH, Vacanti JP (1999) Skeletal muscle
tissue engineering using isolated myoblasts on synthetic biodegradable polymers:
Preliminary studies. Tissue Eng 5:525–532.
13. Levenberg S, et al. (2005) Engineering vascularized skeletal muscle tissue. Nat
14. Lee KY, Peters MC, Anderson KW, Mooney DJ (2000) Controlled growth factor release
from synthetic extracellular matrices. Nature 408:998–1000.
15. Richardson TP, Peters MC, Ennett AB, Mooney DJ (2001) Polymeric system for dual
growth factor delivery. Nat Biotechnol 19:1029–1034.
16. Silva EA, Mooney DJ (2007) Spatiotemporal control of vascular endothelial growth
factor delivery from injectable hydrogels enhances angiogenesis. J Thromb Haemost
17. Messina S, et al. (2007) VEGF overexpression via adeno-associated virus gene transfer
promotes skeletal muscle regeneration and enhances muscle function in mdx mice.
FASEB J 21:3737–3746.
18. Kärkkäinen AM, et al. (2009) Vascular endothelial growth factor-D transgenic mice
show enhanced blood capillary density, improved postischemic muscle regeneration,
and increased susceptibility to tumor formation. Blood 113:4468–4475.
19. Deasy BM, et al. (2009) Effect of VEGF on the regenerative capacity of muscle stem
cells in dystrophic skeletal muscle. Mol Ther 17:1788–1798.
20. Dellavalle A, et al. (2007) Pericytes of human skeletal muscle are myogenic precursors
distinct from satellite cells. Nat Cell Biol 9:255–267.
21. Zheng B, et al. (2007) Prospective identification of myogenic endothelial cells in
human skeletal muscle. Nat Biotechnol 25:1025–1034.
22. Crisan M, et al. (2008) Purification and culture of human blood vessel-associated
progenitor cells. Curr Protoc Stem Cell Biol Unit 2B.2.1–2B.2.13.
23. Ferrari G, et al. (1998) Muscle regeneration by bone marrow-derived myogenic
progenitors. Science 279:1528–1530.
24. LaBarge MA, Blau HM (2002) Biological progression from adult bone marrow to
mononucleate muscle stem cell to multinucleate muscle fiber in response to injury.
25. Hill E, Boontheekul T, Mooney DJ (2006) Regulating activation of transplanted cells
controls tissue regeneration. Proc Natl Acad Sci USA 103:2494–2499.
26. Tidball JG (2005) Mechanical signal transduction in skeletal muscle growth and
adaptation. J Appl Physiol 98:1900–1908.
27. Heszele MF, Price SR (2004) Insulin-like growth factor I: The yin and yang of muscle
atrophy. Endocrinology 145:4803–4805.
28. Shansky J, Creswick B, Lee P, Wang X, Vandenburgh H (2006) Paracrine release of
insulin-like growth factor 1 from a bioengineered tissue stimulates skeletal muscle
growth in vitro. Tissue Eng 12:1833–1841.
29. Conway EM, Collen D, Carmeliet P (2001) Molecular mechanisms of blood vessel
growth. Cardiovasc Res 49:507–521.
30. Khurana R, Simons M, Martin JF, Zachary IC (2005) Role of angiogenesis in
cardiovascular disease: A critical appraisal. Circulation 112:1813–1824.
31. Vandenburgh HH, Karlisch P, Shansky J, Feldstein R (1991) Insulin and IGF-I induce
pronounced hypertrophy of skeletal myofibers in tissue culture. Am J Physiol 260:
32. Musarò A, et al. (2004) Stem cell-mediated muscle regeneration is enhanced by local
isoform of insulin-like growth factor 1. Proc Natl Acad Sci USA 101:1206–1210.
33. Pelosi L, et al. (2007) Local expression of IGF-1 accelerates muscle regeneration by
rapidly modulating inflammatory cytokines and chemokines. FASEB J 21:1393–1402.
34. Rosenthal S-M, Cheng Z-Q (1995) Opposing early and late effects of insulin-like
growth factor I on differentiation and the cell cycle regulatory retinoblastoma
protein in skeletal myoblasts. Proc Natl Acad Sci USA 92:10307–10311.
35. Bass J, Oldham J, Sharma M, Kambadur R (1999) Growth factors controlling muscle
development. Domest Anim Endocrinol 17:191–197.
36. Arsic N, et al. (2004) Vascular endothelial growth factor stimulates skeletal muscle
regeneration in vivo. Mol Ther 10:844–854.
37. Jin KL, Mao XO, Greenberg DA (2000) Vascular endothelial growth factor: Direct
neuroprotective effect in in vitro ischemia. Proc Natl Acad Sci USA 97:10242–10247.
38. Schratzberger P, et al. (2000) Favorable effect of VEGF gene transfer on ischemic
peripheral neuropathy. Nat Med 6:405–413.
39. Hawke TJ, Garry DJ (2001) Myogenic satellite cells: Physiology to molecular biology.
J Appl Physiol 91:534–551.
40. Adams GR, McCue SA (1998) Localized infusion of IGF-I results in skeletal muscle
hypertrophy in rats. J Appl Physiol 84:1716–1722.
41. Chakravarthy MV, Davis BS, Booth FW (2000) IGF-I restores satellite cell proliferative
potential in immobilized old skeletal muscle. J Appl Physiol 89:1365–1379.
42. Coleman ME, et al. (1995) Myogenic vector expression of insulin-like growth factor I
stimulates muscle cell differentiation and myofiber hypertrophy in transgenic mice.
J Biol Chem 270:12109–12116.
43. Bark TH, McNurlan MA, Lang CH, Garlick PJ (1998) Increased protein synthesis after
acute IGF-I or insulin infusion is localized to muscle in mice. Am J Physiol 275:
44. Barton-Davis ER, Shoturma DI, Sweeney HL (1999) Contribution of satellite cells to
IGF-I induced hypertrophy of skeletal muscle. Acta Physiol Scand 167:301–305.
45. Musarò A, McCullagh KJ, Naya FJ, Olson EN, Rosenthal N (1999) IGF-1 induces skeletal
myocyte hypertrophy through calcineurin in association with GATA-2 and NF-ATc1.
46. Semsarian C, et al. (1999) Skeletal muscle hypertrophy is mediated by a Ca2+-dependent
calcineurin signalling pathway. Nature 400:576–581.
47. Musarò A, Dobrowolny G, Rosenthal N (2007) The neuroprotective effects of a locally
acting IGF-1 isoform. Exp Gerontol 42:76–80.
48. Dobrowolny G, et al. (2005) Muscle expression of a local Igf-1 isoform protects motor
neurons in an ALS mouse model. J Cell Biol 168:193–199.
49. Dobrowolny G, Aucello M, Molinaro M, Musarò A (2008) Local expression of mIgf-1
modulates ubiquitin, caspase and CDK5 expression in skeletal muscle of an ALS mouse
model. Neurol Res 30:131–136.
50. Mack T-G, et al. (2001) Wallerian degeneration of injured axons and synapses is
delayed by a Ube4b/Nmnat chimeric gene. Nat Neurosci 4:1199–1206.
51. Coleman MP, Ribchester RR (2004) Programmed axon death, synaptic dysfunction
and the ubiquitin proteasomesystem. Curr Drug TargetsCNS NeurolDisord 3:227–238.
52. Mourkioti F, Rosenthal N (2005) IGF-1, inflammation and stem cells: Interactions
during muscle regeneration. Trends Immunol 26:535–542.
53. Vergani L, et al. (1998) Systemic administration of insulin-like growth factor decreases
54. Caroni P, Grandes P (1990) Nerve sprouting in innervated adult skeletal muscle induced
by exposure to elevated levels of insulin-like growth factors. J Cell Biol 110:1307–1317.
55. Couffinhal T, et al. (1999) Impaired collateral vessel development associated with
reduced expression of vascular endothelial growth factor in ApoE-/- mice. Circulation
56. Lichtman JW, Sanes JR (2003) Watching the neuromuscular junction. J Neurocytol 32:
57. Chen RR, Silva EA, Yuen WW, Mooney DJ (2007) Spatio-temporal VEGF and PDGF
delivery patterns blood vessel formation and maturation. Pharm Res 24:258–264.
58. Duty S, Allen DG (1994) The distribution of intracellular calcium concentration in
isolated single fibres of mouse skeletal muscle during fatiguing stimulation. Pflugers
| www.pnas.org/cgi/doi/10.1073/pnas.0903875106Borselli et al.