Model for Muscle Regeneration around Fibrotic Lesions in Recurrent Strain Injuries

Abstract

The purpose of this study was to establish an in vivo model for muscle regeneration after strain injury in the presence of a fibrotic discontinuity.
The musculus soleus of 5-wk-old male rats was exposed, completely lacerated, and sutured together with or without a collagen scaffold in between the muscle ends. The scaffold represents a fibrotic discontinuity in the muscle. Muscle healing was evaluated after 14 d by general histology and staining for myofibroblasts, satellite cells (activated), and inflammatory cells.
Around all wounds, satellite cells were activated. Inside the collagen scaffolds, satellite cells were absent, indicating that muscle regeneration was impaired. In the wounds without a collagen scaffold, the lacerated and the sutured myofibers contacted and had already started to regenerate, whereas this did not occur with an implanted scaffold.
A fibrotic discontinuity, such as an implanted collagen scaffold, delays muscle regeneration in skeletal muscle. This model is suitable to study skeletal muscle regeneration in the presence of a fibrotic lesion and to evaluate new treatment modalities for muscle strain injuries.

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Methodological Advances
Model for Muscle Regeneration around
Fibrotic Lesions in Recurrent Strain Injuries
SANDER GREFTE
1
, ANNE MARIE KUIJPERS-JAGTMAN
1
, RUURD TORENSMA
2
,
and JOHANNES W. VON DEN HOFF
1
1
Department of Orthodontics and Oral Biology, and
2
Department of Tumor Immunology, Radboud University
Nijmegen Medical Centre, Nijmegen, THE NETHERLANDS
ABSTRACT
GREFTE, S., A. M. KUIJPERS-JAGTMAN, R. TORENSMA, and J. W. VON DEN HOFF. Model for Muscle Regeneration around
Fibrotic Lesions in Recurrent Strain Injuries. Med. Sci. Sports Exerc., Vol. 42, No. 4, pp. 813–819, 2010. Purpose: The purpose of this
study was to establish an in vivo model for muscle regeneration after strain injury in the presence of a fibrotic discontinuity. Methods:The
musculus soleus of 5-wk-old male rats was exposed, completely lacerated, and sutured together with or without a collagen scaffold in
between the muscle ends. The scaffold represents a fibrotic discontinuity in the muscle. Muscle healing was evaluated after 14 d by general
histology and staining for myofibroblasts, satellite cells (activated), and inflammatory cells. Results: Around all wounds, satellite cells were
activated. Inside the collagen scaffolds, satellite cells were absent, indicating that muscle regeneration was impaired. In the wounds without
a collagen scaffold, the lacerated and the sutured myofibers contacted and had already started to regenerate, whereas this did not occur
with an implanted scaffold. Conclusions: A fibrotic discontinuity, such as an implanted collagen scaffold, delays muscle regeneration in
skeletal muscle. This model is suitable to study skeletal muscle regeneration in the presence of a fibrotic lesion and to evaluate new
treatment modalities for muscle strain injuries. Key Words: MUSCLE STRAIN INJURIES, FIBROSIS, MUSCLE HEALING,
SATELLITE CELLS
Muscle strain injuries occur regularly in profes-
sional athletes as well as in the general popula-
tion (14). The hamstring is the most common
muscle group affected and is characterized by a recurrence
rate of 30% within the first year after injury. This indicates
that full recovery of a hamstring strain injury is often not
obtained (7,34,35). Magnetic resonance imaging analysis
shows that during the healing of a hamstring injury, fibrotic
tissue is formed, preventing full recovery (10,38).
In muscle strain injuries, the muscle is sheared, which
results in a total rupture of the myofibers and their plasma
membrane (21,24). At this site, necrosis of the myofibers
begins but is restricted to the injury site by contraction
bands inside the myofibers (19). After injury, satellite cells,
which are located between the sarcolemma and the basal
lamina of the muscle fibers (29,32), are released, activated,
and migrate to the site of injury. There they proliferate,
differentiate, and fuse to each other or to damaged myo-
fibers to regenerate the skeletal muscle (8,37). However,
blood vessels are also torn, and a hematoma is formed,
filling the gap between the damaged muscle ends. This
forms a primary matrix not only for inflammatory cells but
also for fibroblasts, which synthesize extracellular matrix
components (21,24). These fibroblasts firstly produce
fibronectin, followed by collagen type III, and finally
collagen type I (22). This might lead to a fibrotic tissue
that inhibits growth of muscle fibers and thus impairs
regeneration and muscle function (15,18,24,25). It has been
shown that recurrent muscle strains occur in proximity of
this fibrotic discontinuity, probably because of its different
stiffness and contractility properties (34,38). Furthermore,
recurrent injuries are also more severe and take a longer
time to heal than primary strain injuries (7,27). It is there-
fore important to prevent or to minimize the formation of
such a fibrotic discontinuity to reduce the risk of recurrence.
To reduce fibrosis and to optimize muscle regeneration,
several strategies have been evaluated. The injection of
growth factors such as insulin-like growth factor, fibroblast
growth factor 2, nerve growth factor, and granulocyte
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Address for correspondence: Johannes W. Von den Hoff, Ph.D.,
Department of Orthodontics and Oral Biology, Radboud University
Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The
Netherlands; E-mail: H.vondenHoff@dent.umcn.nl.
Submitted for publication June 2009.
Accepted for publication August 2009.
0195-9131/10/4204-0813/0
MEDICINE & SCIENCE IN SPORTS & EXERCISE
Ò
Copyright Ó2010 by the American College of Sports Medicine
DOI: 10.1249/MSS.0b013e3181beeb52
813

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colony-stimulating factor improves muscle regeneration
(25,36,39). More importantly, the administration of decorin,
an inhibitor of transforming growth factor A, reduces fi-
brosis (12,36). The direct delivery of isolated muscle cells
is another approach (9,16,31). Although the latter yields
promising results, a major problem is the poor cell survival
and limited migration of the injected cells (3,11). Alterna-
tively, several different scaffold materials have been used
for improving muscle regeneration but with varying results
(5,17,26,41). However, a model to study impaired healing
in the presence of a fibrotic lesion is not yet available.
Therefore, the aim of this study is to establish an in vivo
model for a fibrotic discontinuity in healing skeletal muscle
by implanting a collagen scaffold.
MATERIALS AND METHODS
Animals. All animal experiments were approved by the
Animal Experiments Committee of the Radboud University
Nijmegen Medical Centre in accordance to the Dutch laws
and regulations on animal experiments, which conform to
the American College of Sports Medicine animal care
standards. Twenty-four 5-wk-old male Sprague–Dawley
rats (Janvier, Le Genest, France) were used for the
experiments. The rats were housed under normal laboratory
conditions, but in the first week after the experimental
procedure, they were housed individually. All the rats were
fed normal rat chow and water ad libitum. Before the start
of the experiments, the rats had been acclimatized to the
animal facility for 1 wk.
Preparation of the collagen scaffolds. The collagen
scaffolds were prepared and chemically cross-linked as
previously described (6). Briefly, a 1% (w/v) homogenized
collagen suspension was prepared using insoluble type I
collagen from bovine Achilles tendon (Sigma Chemical Co.,
St. Louis, MO). The collagen suspension was degassed to
remove air bubbles, frozen overnight at j25-Cinaluminum
trays, and lyophilized. The dried collagen scaffolds were
cross-linked using 1-ethyl-3-(3 dimethylaminopropyl) car-
bodiimide hydrochloride and n-hydroxy-succinimide (43).
Experimental procedures. At the day of surgery, the
rats received 0.02 mgIkg
j1
body weight buprenorphine
(Temgesic; Schering Plough, Brussels, Belgium) subcuta-
neously as an analgesic and also at the next 2 d with a
12-h interval. Under 5% (induction) followed by 2%–3%
(continuation) isoflurane anesthesia (Pharmachemie BV,
Haarlem, The Netherlands), the left lower limb of the rats
was shaved. After a longitudinal incision in the skin and
underlying fascia, the musculus soleus was gently exposed
and transversally lacerated. The two ends were sutured
together using a 7-0 Polysorb suture (Tyco Healthcare UK,
Gosport, UK) with or without the collagen scaffold in
between. Before implantation, the collagen scaffolds were
sterilized by immersion in 70% ethanol for 1 h and then
washed three times with sterile phosphate-buffered saline
(PBS). The animals were divided into four groups of six
rats according to the suturing method and the presence of a
collagen scaffold: A) knot suturing without collagen
scaffold, B) knot suturing with collagen scaffold, C) con-
tinuous suturing without collagen scaffold, and D) contin-
uous suturing with collagen scaffold. The easiest method
for suturing is with one continuous suture around the
muscle. However, if this one suture breaks, the wound
opens and the scaffold might be lost. To be sure, we also
used a method with multiple sutures. However, none of the
sutures had broken, and there was no different response
between the two suturing methods. We therefore decided to
group the animals together (A + C and B + D). The fascia
and the skin were closed with 5-0 Polysorb and 5-0 Vicryl
sutures (Johnson-Johnson, Langhorne, PA), respectively.
To minimize muscle tension, the paw was splinted with an
aluminum strip at an angle approximately 45-with respect
to the tibia for 1 wk. In group B, the paws were swollen and
reddish when the aluminum strips were removed. These rats
therefore received 1 mgIkg
j1
enrofloxacin two times a day
(Bayer Healthcare, Brussels, Belgium) for 7 d. After 14 d,
the rats were sacrificed according to the standard CO
2
/O
2
protocol.
Histology and immunohistochemistry. After sacri-
fice, the left (wound) and the right (internal control) musculus
soleus of three rats of each group were fixed in freshly pre-
pared 4% paraformaldehyde in PBS for 4–6 h and processed
for paraffin embedding. The left and the right musculus
soleus of the other three rats of each group were immediately
frozen in optimal cutting temperature compound (OCT)
embedding compound (CellPath, Newtown, UK) using
isopentane precooled in liquid nitrogen. The muscles were
cut longitudinally, and 5-Hm sections were collected on
superfrost plus slides (Menzel-Gla¨ser, Braunschweig,
Germany). For general morphology, paraffin sections were
stained with hematoxylin and eosin (H&E).
Paraffin sections were also stained with the following
antibodies: mouse anti-alpha-smooth muscle actin (>-SMA;
Sigma), rabbit anti-Ki67 (Research Diagnostics Inc., Flan-
ders, NJ), mouse anti-ED1 (CD68, Serotec; DPC, Breda, The
Netherlands), and mouse anti-MyoD (DAKO; Dakopatts,
Glostrup, Denmark). Briefly, the sections were deparaffinated,
rehydrated, treated with 3% H
2
O
2
for 20 min to inactivate
endogenous peroxidase, and postfixed with 4% formalde-
hyde in PBS. For >-SMA and ED1 staining, the sections
were heated in citrate buffer (pH 6.0) for 10 min at 70-C. For
Ki67 and MyoD staining, the sections were heated to 100-C
for 10 and 40 min, respectively. After rinsing with 0.075%
glycine in PBS, the sections were preincubated with 10%
normal donkey serum (NDS; Chemicon, Temecula, CA)
followed by the antibodies against >-SMA (1:1600), ED1
(1:100), Ki67 (1:50), or MyoD (1:25) for 60 min. Subse-
quently, the biotinylated secondary antibodies goat-anti-
mouse immunoglobulin G (IgG; H + L) (1:500; Jackson
Labs, West Grove, PA) for >-SMA, ED1, and MyoD and
goat-anti-rabbit IgG (H + L) (1:500; Jackson Labs) for Ki67
were added. The bound antibodies were visualized using a
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preformed biotinylated horse radish peroxidase and avidin
complex (Vector Laboratories, Burlingame, CA).
The frozen sections were double stained with the antibodies
rabbit anti-collagen IV (Euro-Diagnostica BV, Arnhem, The
Netherlands) and mouse anti-Pax7 (Developmental Studies
Hybridoma Bank, Iowa City, CA). Briefly, the sections were
dried in air overnight and postfixed with 1% paraformalde-
hyde in PBS for 10 min. After rinsing with 0.05% Triton X-
100 in PBS, the sections were preincubated with 10% NDS
followed by rabbit anti-collagen IV (1:100) for 60 min.
Collagen IV was then detected using the biotinylated donkey
anti-rabbit IgG (H + L) (1:500; Jackson Labs) for 60 min and
an AlexaFluor-488-labeled avidin (1:500; Molecular Probes,
Eugene, OR) for 60 min. Thereafter, the sections were again
preincubated with 10% NDS and then incubated with mouse
anti-Pax7 (1:100) overnight at 4-C. Pax7 was detected using
an AlexaFluor-594-labeled goat-anti-mouse IgG (H + L)
(1:200; Molecular Probes). All sections were photographed
with the Zeiss Imager.Z1 together with the AxioCam MRc5
camera using the AxioVision 4.6.3 software (Carl Zeiss
Microimaging GmbH, Jena, Germany).
Statistical analysis. The numbers of Pax7- and
MyoD-positive cells were counted in 1) the control muscle
(C), 2) the cutting zone without the collagen scaffold (W),
3) the cutting zone with the collagen scaffold (W + S), 4)
inside the collagen scaffold (S), and 5) in the noninjured
muscle tissue of the wounded musculus soleus (NI). To
count the Pax7-positive cells, the images were divided into
50 squares. In five random squares, the total number of
Pax7-positive cells and DAPI-stained nuclei was counted.
The total number of MyoD-positive cells and nuclei of
every group was determined in three different fields of an
overview image. The numbers of Pax7- and MyoD-positive
cells were expressed as a percentage TSD of the total
number of cells. The differences in the percentages of Pax7-
and MyoD-positive cells were tested for significance using
a Kruskal–Wallis one-way ANOVA on Ranks followed by
Dunn’s method. A value of PG0.05 was considered to be
significant.
RESULTS
Of the 24 rats, 1 rat in group A without a collagen
scaffold did not survive the surgery. After an initial growth
arrest, all rats in every group had gained about 25% body
weight at the 10th day. The groups were not significantly
different. The immobilization of the left hind leg did not
affect the growth of the rats. Macroscopically, the wounded
muscle adhered partly to the surrounding tissues. Further-
more, the collagen scaffolds were not visible anymore and
appeared to be integrated into the muscle tissue. The su-
tures did not break, and the different suturing methods
had no effect on muscle morphology and gave the same
results regarding muscle regeneration. Therefore, the ani-
mals of groups A and C and groups B and D were grouped
together.
General histology. H&E staining (Fig. 1A) revealed
properly arranged longitudinal myofibers in the controls (C)
but not in the wounded muscles. Within the wounds,
regenerating myofibers were present, indicated by centrally
located nuclei (Fig. 1A, magnification). Some myofibers in
the cutting zone had fused properly in the group without the
collagen scaffold. On the contrary, the implantation of a
collagen scaffold prevented fusion of the myofibers (W + S).
The collagen scaffolds were surrounded by giant cells and a
cell layer (an interphase).
Immunostainings. Paraffin sections were stained with
antibodies against >-SMA, ED1 (CD68), Ki67, and MyoD
to identify blood vessels and myofibroblasts, inflammatory
cells, proliferating cells, and activated satellite cells,
respectively (Fig. 1B). In the controls (C), ED1-positive
inflammatory cells and Ki67-positive proliferating cells
were present. However, the controls hardly contained any
MyoD-positive nuclei (indicated by arrows). As expected,
>-SMA–positive cells were not present in the muscle tissue
of the controls but only in blood vessels. Without a collagen
scaffold (W), there was an increase in the number of ED1-
and Ki67-positive cells of which the majority surrounded
the sutures. More importantly, many MyoD-positive nuclei
were present (a few are indicated by arrows). The number
of >-SMA–positive blood vessels was also increased, and
there were also >-SMA–positive cells in the muscle tissue.
The implantation of a collagen scaffold (W + S) caused an
infiltration of ED1-positive giant cells and other inflamma-
tory cells, which surrounded the scaffold. Even inside the
scaffolds, inflammatory cells were present. Proliferating
Ki67-positive cells were present in the muscle tissue, in the
interphase, and also inside the collagen. Again, the muscle
tissue around the scaffold (W + S) contained many MyoD-
positive nuclei (a few are indicated by arrows), but all the
cells inside the scaffolds were negative for MyoD. The
expression pattern of >-SMA was similar to the wounds
without the scaffold (W). Inside the scaffolds, >-SMA–
positive blood vessels were also found.
To identify the resident satellite cells, cryosections were
stained with the Pax7 antibody (Fig. 2). In the controls (C),
only a few satellite cells were present, but around the
cutting zone in the wounded muscle tissue with (W + S) or
without (W) a collagen scaffold, the number of satellite
cells was increased. However, no satellite cells were present
within the collagen scaffolds.
Quantifications. The percentage of MyoD- and Pax7-
positive cells were determined on the paraffin (Fig. 1B) and
cryosections (Fig. 2), respectively (Fig. 3). The controls
contained only a low number of Pax7-positive satellite cells
(2.7% T0.4%), which significantly (PG0.05) increased to
7.2% T0.6% and 6.2% T0.6% in the wounded tissue
without (W) or with (W + S) the collagen scaffold, re-
spectively. The number of MyoD-positive cells also signifi-
cantly (PG0.05) increased from 6.2% T1.1% in the
controls to 16% T4.3% and 15.9% T4.9% in the wounds
without (W) or with (W + S) the collagen scaffold,
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FIGURE 1—Histology of the musculus soleus at 14 d after surgery. A. H&E staining of the control (C), wound without the collagen scaffold (W;
group C), and wound with the collagen scaffold (W + S; group D) revealed the disruption of the aligned myofibers at the cutting zone after laceration.
The implanted collagen scaffold is surrounded by an interphase and prevented myofiber fusion. B. Immunohistochemistry of the control (C), wound
without the collagen scaffold (W; group C), and wound with the collagen scaffold (W + S; group D) with antibodies directed against ED1, Ki67,
MyoD, and >-SMA. Only a few ED1-, Ki67-, and MyoD-positive cells (indicated by arrows) and >-SMA–positive blood vessels are present in the
control (C). In the wound (W), the number of these cells is higher and >-SMA–positive cells are present. The collagen scaffold (W + S) is surrounded
by an ED1-positive interphase. In the wounded muscles and around the scaffold, many Ki67-positive, MyoD-positive (a few are indicated by arrows),
and >-SMA–positive cells are present. The scaffold also contains ED1-, Ki67, and >-SMA–positive cells and blood vessels but no MyoD-positive cells.
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respectively. Furthermore, there was a slight but nonsig-
nificant increase of Pax7-positive (3.9% T0.5%) and
MyoD-positive cells (9.1% T1.9%) in the noninjured area
(NI) of the wounded muscles compared with the controls.
However, no Pax7- and MyoD-positive cells were found
inside the collagen scaffolds.
DISCUSSION
The successful treatment of muscle strains in sports
medicine is still a problem. Fibrotic lesions are often formed
during muscle regeneration, causing incomplete functional
recovery. More importantly, recurrent muscle injuries may
occur near this fibrotic tissue (10,34,38). Because fibrotic
tissue consists mainly of collagen type I (20,23), we
developed an in vivo model for a fibrotic discontinuity by
implanting a type I collagen scaffold between the lacerated
muscle ends. Using this method, it is possible to standardize
the wounds with a collagen scaffold, but it is important to
be aware that this is an extreme version of a muscle strain.
In this model, we evaluated muscle regeneration after a
2-wk healing period. The numbers of Pax7- and MyoD-
positive (activated) satellite cells or myoblasts were in-
creased about twofold in the wounded muscle tissue and
around the collagen scaffolds compared with the control
muscle. This indicates that the muscle fibers were regener-
ating and that the scaffold did not inhibit the activation of
satellite cells in the adjacent muscle tissue. However, inside
the collagen scaffold, these cells were absent. Thus, in the
presence of a fibrotic discontinuity, the skeletal muscle
cannot regenerate properly because activated satellite cells
do not migrate into the fibrotic tissue. Similar to our results,
others have also shown that after a strain injury, inflamma-
tion occurs, followed by the production of fibrous tissue,
which could eventually develop into a fibrotic lesion (14,33).
Another study on rectus femoris strain in humans showed a
chronic inflammation and a mixture of regenerating muscle
fibers and fibrotic tissue in the wound (40). Although
muscle regeneration was only evaluated after 2 wk in this
initial study, collagen scaffolds can persist in the muscle
tissue for up to 50 d (28). Therefore, our model can be used
to evaluate treatment strategies for recurrent muscle strains.
Optimal treatment should diminish or prevent the forma-
tion of fibrotic tissue and reduce the risk of recurrence. We
and others (1,30) observed that suturing the lacerated
muscle ends directly together allows full regeneration of
the muscle. Currently, the treatment principle of muscle
strains consists of rest, ice, compression, and elevation (22).
With specific compression, which could serve as a splint, it
may also be possible to bring the muscle ends to each other
and diminish the onset of fibrosis. Surgical treatment to
suture the muscle ends together is only indicated in cases
with extensive injury to the muscle (22). If a fibrotic tissue
FIGURE 2—Fluorescent immunohistochemistry of the musculus soleus at 14 d after surgery. The control (C), wound without the collagen scaffold
(W; group A), and wound with the collagen scaffold (W + S; group B) were stained with the antibody directed against Pax7. In the control, only a few
Pax7-positive cells are present, whereas in the wound (W), the number of these cells is increased. In the wounded muscle tissue around the collagen
scaffold (W + S), the number of Pax7-positive cells is also increased. On the contrary, these cells are absent in the interphase and the collagen
scaffold.
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from a previous injury is already present, additional
treatment with matrix metalloproteinase 1 (MMP-1) might
offer a solution. Previous research has shown that treatment
with injection of MMP-1 improves muscle regeneration and
that a fibrotic lesion can be partially resolved (4,23). Thus,
combining the injection of MMP-1 with specific compres-
sion therapy might diminish a preexisting fibrotic discon-
tinuity or minimize the risk of a secondary fibrosis.
In this study, the musculus soleus in rats is used as a
wound model because all the myofibers run parallel. How-
ever, the musculus soleus consists mainly of type I (slow)
fibers (42), whereas the hamstring, which is the most
common muscle group affected in muscle strains, consist of
type II (fast) fibers (13). It has been shown that type II
muscles regenerate better than type I muscles, which more
often develop fibrotic lesions (2). This indicates that the
results obtained in this study may differ from a hamstring
injury, in which the regeneration process could be more
efficient. However, it also demonstrates that the musculus
soleus is a good model to study the effects of the presence
of a fibrotic discontinuity on muscle regeneration.
In this study, we only analyzed 14 d after surgery be-
cause satellite cell activation is a relatively early event in
muscle healing (8,37). In future studies, analysis at later
time points is necessary to exclude the possibility that im-
plantation of a collagen scaffold only delays muscle regen-
eration. In addition, it is important that functional studies
are performed to further evaluate this model.
In conclusion, we generated a model for the regeneration
of skeletal muscle in the presence of a fibrotic discontinuity.
This model can be used to evaluate new treatment strategies
for recurrent muscle strains.
The Pax7 antibody developed by Atsushi Kawakami was ob-
tained from the Developmental Studies Hybridoma Bank developed
under the auspices of the National Institute of Child Health and
Human Development and maintained by The University of Iowa,
Department of Biological Sciences, Iowa City, IA 52242. This study
was supported by a grant from the Radboud University Nijmegen
Medical Centre, The Netherlands. For this research, we did not
receive any funding from the National Institutes of Health, the
Wellcome Trust, the Howard Hughes Medical Institute, or any other
organization. The results of the present study do not constitute
endorsement by the American College of Sports Medicine.
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satellite cells. The number of Pax7- and MyoD-positive cells is
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