Skeletal Muscle Regeneration: A Case Study
ABSTRACT: Skeletal muscle is able to repair itself through regeneration.
However, an injured muscle often does not fully recover its strength because complete muscle regeneration is hindered by the development of fibrosis.
Biological approaches to improve muscle healing by enhancing muscle regeneration and reducing the formation of fibrosis are being investigated.
Previously, we have determined that insulin-like growth factor–1 (IGF-1) can improve muscle regeneration in injured muscle. We also have investigated the use of an antifibrotic agent, decorin, to reduce muscle fibrosis following injury. The aim of this study was to combine these two therapeutic methods in an attempt to develop a new biological approach to promote efficient
healing …show more content…
and recovery of strength after muscle injuries. Our findings indicate that further improvement in the healing of muscle lacerations is attained histologically by the combined administration of IGF-1 to enhance muscle regeneration and decorin to reduce the formation of fibrosis. This improvement was not associated with improved responses to physiological testing, at least at the time-points tested in this study.
Muscle Nerve 28: 365–372, 2003
IMPROVEMENT OF MUSCLE HEALING THROUGH
ENHANCEMENT OF MUSCLE REGENERATION
AND PREVENTION OF FIBROSIS
KENJI SATO, MD,1,2 YONG LI, MD, PhD,1 WILLIAM FOSTER, BS,1
KAZUMASA FUKUSHIMA, MD,1,2 NEIL BADLANI, MS,1 NOBUO ADACHI, MD,1,2
ARVYDAS USAS, MD,1 FREDDIE H. FU, MD,2 and JOHNNY HUARD, PhD1,2
1
Growth and Development Laboratory, Department of Orthopaedic Surgery,
Children’s Hospital and University of Pittsburgh, Pittsburgh, Pennsylvania, USA
2
Department of Orthopaedic Surgery, Division of Sports Medicine, University of Pittsburgh,
Pittsburgh, Pennsylvania, USA
Accepted 24 April 2003
There are various types of muscle injury, including those that occur through direct trauma (e.g., laceration and contusion) and those attributable to indirect damage (e.g., ischemia, denervation, and strain), but the general process of muscle damage and repair is similar in most cases.2,10,12,19 –21,24 –26
Muscle fibers have the ability to regenerate following injury through the activation of satellite cells,13 but the healing process is very slow and often results in incomplete recovery of strength due to the development of scar tissue.12,24,25 The muscle healing …show more content…
process comprises several phases, including degeneration and
Abbreviations: ECM, extracellular matrix; bFGF, basic-fibroblast growth factor; GM, gastrocnemius muscle; H&E, hematoxylin and eosin; IGF-1, insulin-like growth factor–1; NGF, nerve growth factor; PBS, phosphate-buffered saline; TGF-1, transforming growth factor-1
Key words: decorin; fibrosis; IGF-1; muscle healing; regeneration.
Correspondence to: J. Huard, Growth and Development Laboratory, 4151
Rangos Research Center, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213-2583; e-mail: jhuardϩ@pitt.edu
© 2003 Wiley Periodicals, Inc.
Improvement of Muscle Healing
inflammation, regeneration, and scar formation (after severe muscle injury).12,24 Muscle regeneration occurs early in this process; it begins about 1 week after injury, peaks during the 2nd week, and then rapidly declines.3,12,16,20,24,25 We have shown previously that growth factors, including insulin-like growth factor–1
(IGF-1), basic-fibroblast growth factor (bFGF), and nerve growth factor (NGF), can improve muscle regeneration during this preliminary phase of healing.19 –21,26
Of these growth factors, IGF-1 was found to have the greatest effect on the healing of the injured muscle by increasing both the efficiency of muscle regeneration and muscle strength. However, none of the growth factors that have been investigated appears able to completely heal injured muscle, possibly due to the development of muscle fibrosis.
The development of fibrosis begins 2 weeks after muscle injury and continues over time. This process hinders muscle regeneration and prevents full strength recovery in the injured skeletal muscle.2,3,12,24,25 We have reported that myogenic cells can differentiate into myofibroblasts upon muscle
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laceration injury.23 We also have observed high levels of transforming growth factor-1 (TGF-1) expression in injured skeletal muscle.23 TGF-1 stimulates the deposition of collagens and overgrowth of the extracellular matrix, thereby leading to the accumulation of fibrotic tissue.4,31 Many studies have shown that upregulation and increased expression of the cytokine TGF- at the injured site constitute central events during fibrosis development through the activation of myofibroblasts.1,4,,6,7,22,23,27,28,31 Accordingly, we believe that TGF-1 plays a pivotal role in skeletal muscle fibrosis and have targeted TGF- inhibition via an anti–TGF- human proteoglycan, decorin,11,14 as an approach to preventing muscle fibrosis.8 We determined that direct injection of human recombinant decorin at 2 weeks after laceration effectively prevents muscle fibrosis and, in turn, enhances muscle regeneration.8
In this study, we investigated the combined use of
IGF-1 and decorin to enhance muscle regeneration, reduce fibrosis, and thereby facilitate improved healing of muscle injuries in vivo. To investigate this hypothesis, we developed a reproducible muscle laceration injury model in mice.8,12,23,25 After treatment of the injured muscles with IGF-1 and decorin, muscle healing and fibrosis were analyzed both histologically and physiologically to assess muscle regeneration and strength. Our primary goal was to improve muscle regeneration through the use of IGF-1 and reduce muscle fibrosis through the use of decorin, in the hope that these two substances would have an additive or synergistic effect on muscle healing and thus facilitate more complete recovery of muscle strength. MATERIALS AND METHODS
Twenty-four mice (C57BL10Jϩ/ϩ, 8 weeks old, 20 –30 g) were used in this experiment. The policies and procedures of the animal laboratory are in accordance with those detailed by the U.S. Department of
Health and Human Services. The Animal Research and Care Committee of the authors’ institutions approved the research protocol used for these experiments.
The muscle laceration model developed in mice entailed laceration of the gastrocnemius muscle
(GM) of both legs based on previously described studies.8,12,19,25,26 The mice were anesthetized by intramuscular injection of 0.03 ml ketamine (100 mg/ ml) and 0.02 ml xylazine (20 mg/ml). A surgical blade (#11 SteriSharps, Mansfield, Massachusetts) was used to lacerate each GM at 60% of its length
Animal Model of Muscle Laceration.
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from its distal insertion through the lateral 50% of muscle width and 100% of muscle thickness. PDSII
5-0 suture (Ethicon, Somerville, New Jersey) was placed at the medial edge of the lacerated site, with
4 mm of the suture remaining visible on the posterior of each leg as a marker for the lacerated site.
After laceration, the skin was closed with black silk
4-0 suture (Ethicon).
Injection of IGF-1 and Decorin following Laceration.
The mice were divided into three groups (eight mice per group). After laceration, IGF-1 (human recombinant IGF-1; Gibco BRL, Carlsbad, California) and decorin (from bovine articular cartilage; Sigma, St.
Louis, Missouri) were injected along the suture material using a micro-syringe (Hamilton, Reno, Nevada).
In group 1, repeated injection of IGF-1 (100 ng/injection) was performed in the right GM at different time points (1, 3, and 5 days postlaceration), while the left
GM was injected with phosphate-buffered saline (PBS) to serve as the control. In group 2, decorin (50 g) was injected into the right GM at 2 weeks postlaceration, while the left GM was injected with PBS as the control.
In group 3, the right GM was injected three times with
IGF-1 (100 ng/injection at days 1, 3, and 5 postlaceration) and once with decorin (50 g at 2 weeks postlaceration), and the left GM served as the lacerated control. Four weeks after laceration, all animals were sacrificed to evaluate muscle healing and regeneration.
The GMs were isolated and frozen in 2-methylbutane precooled in liquid nitrogen.
The harvested muscles were divided into five groups: group N contained the normal, nonlacerated muscle; group C contained the lacerated, shaminjected muscle; group IG contained the lacerated,
IGF-1–injected muscle; group D contained the lacerated, decorin-injected muscle; and group IGϩD contained the lacerated muscle injected with IGF-1 and decorin. Four muscles from each group were evaluated by quantitative histological staining and quantitative physiological analysis.
Characterization of Muscle Regeneration following Injection. Hematoxylin and eosin (H&E) staining was
used to evaluate histologically the degree of muscle regeneration following laceration. We monitored the number and diameter of the centronucleated, regenerating myofibers within the injured injection sites and control groups. As previously described,8,19,20,23,25 five random fields within the injured area were selected in each sample. In brief, low-power images (10ϫ) were obtained with a Nikon
Eclipse E800 microscope (Nikon, Melville, New
York) connected to the Spot Image capture system
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September 2003
(Spot Image, Chantilly, Virginia). Using the Spot software calibrated with a micrometer, the number and diameter (m) of the centronucleated myofibers (i.e., the regenerating myofibers) were measured directly from the images of the injured area.
The number and diameter of the centronucleated myofibers were expressed as number of centronucleated myofibers per field and diameter of myofibers, respectively. Characterization of Muscle Fibrosis following Injection.
As previously described,8,19,20,23,25 immunohistochemical techniques that measure the expression of vimentin under a fluorescence microscope were used to evaluate scar tissue formation within the injured muscle. The intermediate filament vimentin was used as a marker for fibrosis in this study because it is expressed mainly in monocytes and fibrocytes and is expressed at high levels within the scar tissue of injured muscle.5,8,19,20,23,25 Vimentin is expressed by myofibers early in development and shortly after injury. However, mature myofibers—as well as regenerating myofibers present in injured muscle 2– 4 weeks after injury— do not express vimentin.5,19,20,23,25 Cryosectioned muscles were used for the vimentin staining. In brief, slides were blocked in
2% horse serum for 1 h. After washing, anti-vimentin conjugated Cy3 (Sigma C-9080) antibody then was applied for 1 h at a dilution of 1:250. The total vimentin-positive area was measured in 10 random fields from each sample (n ϭ 4) of each group.
Images were collected using an Olympus Provis epifluorescence microscope (Olympus Optical, Tokyo,
Japan) and a Sony 970 3 chip CCD camera (Sony,
Tokyo, Japan). These images then were digitized using a Coreco (San Jose, California) frame grabber board and were rendered to monochrome. To evaluate the number of vimentin-positive pixels, the images were collected, converted to grayscale, and a threshold was set. The National Institutes of Health
(NIH) image software package expressed the vimentin-positive area as the number of square pixels. The area of fibrosis (i.e., the vimentin-positive area) was compared among the groups.
We also performed Masson modified trichrome staining (IMEB Inc., San Marcos, California) according to the manufacturer’s protocol to further confirm that the vimentin-positive area was indeed collagenous scar tissue. This staining procedure, which stains collagen blue, muscle red, and nuclei black, was used to compare the amount of scar tissue development among the different groups. Northern
Eclipse software (Empix Imaging, North Tonawanda,
New York) was utilized to quantitate the area of scar
Improvement of Muscle Healing
tissue. Images were collected via the same techniques described above for vimentin, and the software was used to calculate the absolute area of fibrosis. In brief, the images were converted to grayscale, a threshold was set, and the area (m2) was determined.
Physiological
testing for muscle strength was performed 4 weeks after laceration. Four muscles per group were assayed to analyze fast-twitch and tetanus strength. The mice were anesthetized by intramuscular injection of
0.03 ml ketamine (100 mg/ml) and 0.02 ml xylazine
(20 mg/ml). Both GMs were removed and mounted in a 35°C, double-jacketed organ bath of 5 ml Krebs solution (mmol/L: NaCl 113, KCl 4.7, CaCl2 1.25,
MgSO4 1.2, NaHCO3 25, KHPO4 1.2, glucose 11.5) constantly bubbled with a mixture of 95% O2 and
5% CO2. All muscles were subjected to a fixed tension of 20 mN; isometric contractions were measured with strain-gauge transducers coupled with a
TBM4 strain-gauge amplifier (World Precision Instruments, Sarasota, Florida) and recorded on a computer using a data acquisition program
(Windaq, Dataq Instruments, Akron, Ohio). The sampling rate was set to 500 Hz, and the amplitude of stimulation-evoked contractions was computed by a calculation program (Windaq, Dataq Instruments).
After 20-min equilibration, electrical field stimuli were applied through two platinum wire electrodes positioned on the top and bottom of the organ bath
(separated by 4 cm). The muscles were stimulated with 0.25-ms square-wave pulses with a maximal voltage of 50 V. First, 1-Hz stimulations were applied for
6 min and the muscle twitches were recorded, then six tetanic stimulations were applied for 0.5 s. Train duration was measured at 100 Hz every 10 s. The muscles were weighed after testing and the muscle strength was reported as millinewtons per gram
(mN/g).
Characterization of Muscle Strength.
The average and standard deviation of all data were compared among the different groups using repeated measures ANOVA for statistical analysis. Statistical significance was defined as
P Ͻ 0.05.
Statistical Analysis.
RESULTS
Quantitative Histological Analysis of Muscle Regeneration. After counting the number of regenerating
myofibers (i.e., centronucleated myofibers) within the injured site, we observed a significantly higher number of regenerating myofibers in all treated
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FIGURE 1. Muscle healing 4 weeks after laceration injury. Top panels, H&E-stained sections; lower panels, mean number of new regenerating (centronucleated) myofibers per field and mean diameter of these myofibers. Control (C), muscles treated with IGF-1 (IG), muscles treated with decorin (D), and muscles treated with both agents (IGϩD); *P Ͻ 0.05.
muscles (IGF-1, decorin, or both) compared with control muscles injected with PBS alone. We also noted that the muscles treated with IGF-1 and decorin contained a significantly higher number of regenerating fibers compared to muscles treated with only IGF-1 or only decorin (Fig. 1).
Measurement of the diameter of the regenerating myofibers revealed similar results. The diameters of the regenerating myofibers in all treated muscles
(IGF-1, decorin, or both) were statistically larger than the diameters of the regenerating myofibers in the control muscles. Moreover, the muscles treated with IGF-1 and decorin contained regenerating myofibers with diameters significantly larger than those observed in the muscles treated with IGF-1 alone or decorin alone (Fig. 1). These results indicate that combination treatment with IGF-1 and decorin is more effective in increasing both the number and the diameter of regenerating myofibers than is treatment with either agent alone.
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Quantitative Histological Analysis of Muscle Fibrosis.
By using vimentin and trichrome staining to characterize the fibrotic area, we found that scar tissue formation was significantly decreased in the muscles treated with decorin alone or with IGF-1 and decorin when compared to the control muscles or muscles treated with IGF-1 alone. There was no significant difference in fibrotic area in muscles treated with decorin alone and those injected with IGF-1 and decorin (Figs. 2, 3). These results suggest that IGF-1 treatment does not significantly reduce fibrosis, and that combination treatment with IGF-1 and decorin is no better than treatment with decorin alone in terms of reducing muscle fibrosis.
The strength of the lacerated muscles was determined by physiological testing to evaluate fast-twitch and tetanic strength, and was compared among the different treatment groups.
The fast-twitch muscle strength was 370.13 Ϯ 83.77
Physiological Analysis.
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FIGURE 2. Quantitative histological analysis of the laceration site by vimentin immunohistochemical staining 4 weeks after injury. Top panels (A), vimentin immunohistochemical stain; lower panel (B), area of scar tissue per field. Control (C), muscles treated with IGF-1
(IG), muscles treated with decorin (D), and muscles treated with both agents (IGϩD); *P Ͻ 0.05.
FIGURE 3. Quantitative analysis of scar tissue by trichrome staining. Scar tissue containing collagen (blue) was analyzed. Top panels, trichrome stain; lower panel, area of scar tissue per field (original magnification ϫ100). Control (C), muscles treated with IGF-1 (IG), muscles treated with decorin (D), and muscles treated with both agents (IGϩD); *P Ͻ 0.05.
Improvement of Muscle Healing
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FIGURE 4. Histogram of physiological evaluation. The strength of the muscle measured ex vivo: (A) tetanus strength; (B) fast-twitch strength. Control (C), normal, nonlacerated muscle (N), muscles treated with IGF-1 (IG), muscles treated with decorin (D), and muscles treated with both agents (IGϩD); *P Ͻ
0.05.
mN/g in normal muscle, 135.62 Ϯ 34.72 mN/g in the control (20 l PBS) group, 196.12 Ϯ 126.82 in the group receiving IGF-1, 300.48 Ϯ 26.63 mN/g in the group given decorin, and 202.50 Ϯ 27.65 mN/g in the group receiving combination therapy (Fig.
4B). Similarly, the corresponding values for tetanic muscle strength were 140.15 Ϯ 29.79 mN/g, 72.09 Ϯ
2.87 mN/g, 81.76 Ϯ 17.47 mN/g, 129.23 Ϯ 18.67 mN/g, and 93.84 Ϯ 13.40 mN/g, respectively (Fig.
4A).
These results indicate a significant improvement in both fast-twitch and tetanic strength for every treated group (IGF-1, decorin, or both) when compared to the lacerated, nontreated control group. Of the treated groups, the group treated with decorin alone showed the best overall improvement. The strength of muscles in this group returned to nearly the same level as muscles in the normal, nonlacerated group. Muscles in the decorin-treated group also displayed a significant improvement in fasttwitch and tetanic strength compared to the muscles treated with only IGF-1 and those treated with both
IGF-1 and decorin. This finding indicates that treatment with decorin alone is the most effective method by which to restore the physiological strength of injured muscle. The addition of IGF-1 to the treatment actually decreased the resultant physiological strength of the injured muscle. This finding was not supported by the histological analysis, which indicated a beneficial increase in the number and diameter of regenerating myofibers when IGF-1 was used in combination with decorin. These seemingly contradictory results suggest that the histological evaluation of injured muscle does not always correlate with the physiological performance of the muscle.
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DISCUSSION
Optimal treatment methods for muscle injuries have not yet been determined.2,3,12,24 Treatments traditionally have focused on rather conservative techniques such as RICE (rest, ice, compression, and elevation), heat, water pool, or immobilization, though treatment options such as continuous passive motion machines, drugs, and hospitalization also have been used.9,15,18 In previous studies, we have observed that the immobilization of lacerated muscle does not limit the development of fibrosis, whereas suture repair can limit fibrosis development deep in the injured muscle but is incapable of eliminating superficial muscle fibrosis.25 These traditional treatments usually do not promote a rapid enough recovery to satisfy patients and the recurrence of injury is common, indicating that such treatments also fail to provide full functional recovery and are likely ineffective at preventing the formation of permanent scar tissue at an injured site.2,3,12,24 These factors have prompted us to seek novel biological approaches that may enable faster, more complete muscle recovery. For the present study, we hypothesized that the injection of IGF-1 at
1, 3, and 5 days postinjury would increase the proliferation and differentiation of mononucleated satellite cells—thus improving regeneration—and that the injection of decorin at 14 days postinjury would inhibit the effects of TGF-1, thereby reducing fibrosis.
The outcome, though promising, is somewhat different than anticipated. We expected the dual treatment with IGF-1 and decorin to promote better muscle recovery than treatment with either agent alone. Our histological studies supported this hypothesis. Treatment with IGF-1 and decorin had an additive effect on muscle regeneration, increasing
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both the number and diameter of the regenerating muscle fibers. In contrast, the histological analysis of muscle fibrosis did not indicate any benefit associated with the use of both decorin and IGF-1. Decorin alone was capable of reducing fibrosis, and the addition of IGF-1 neither added nor detracted significantly from this effect.
Surprisingly the physiological testing suggested that the histological appearance of muscle does not always correspond directly with its physiological strength. The injured muscle treated with decorin alone actually displayed greater fast-twitch and tetanic strength than the muscle treated with both
IGF-1 and decorin. The reason for this finding is unclear. It may be that in reconstructed muscle there is an optimum ratio between the extracellular matrix (ECM) and myofibers, which maximizes physiological strength.17,33 Perhaps the fibrotic tissues inhibited by decorin are required to ensure proper realignment of the myofibers and the myofibrils in order to maximize muscle mechanics. Disrupting this ratio with too high a proportion of either ECM or myofibers thus would not prove beneficial for tissue reconstruction in injured skeletal muscle. Furthermore, molecular studies have indicated an interaction between decorin and IGF-1 in muscle, a finding that has not yet been elaborated upon. Decorin has been shown to bind to other growth factors— and even growth factor receptors—to alter their function.30,32 IGF-1 itself could play a role in accelerating the growth of the ECM, and such an interaction could result in an unpredicted change in the physiological strength of the regenerated muscle.29
As mentioned above, we recently have determined that myogenic cells can differentiate into fibrotic cells after laceration injury.23 It is not yet clear, however, how the application of decorin and IGF-1 in combination might affect this process. In future research, we plan to further explore the relationship between decorin and IGF-1.
The use of IGF-1 to increase muscle regeneration in combination with decorin to inhibit fibrosis promoted good histological recovery of lacerated muscle, but this high level of recovery was not supported by physiological testing at the time points tested in this study. Even so, these approaches need to be studied both for longer time points to delineate possible side effects and in other animal models of muscle injury, such as strain and contusion models.
It is also necessary to more fully evaluate potential intramuscular and intracellular interactions between antifibrotic agents, such as decorin and growth factors, that appear to aid muscle regeneration.
Improvement of Muscle Healing
The authors thank Ryan Pruchnic and Marcelle Pellerin for technical assistance, Victor Prisk, Yi-Sheng Chan, and Takashi Horaguchi for their critical comments about the manuscript, and Ryan Sauder for his assistance in editing the manuscript. This work was supported by a grant awarded to Johnny Huard from the National Institutes of
Health (NIH 1, RO1 AR47973-01) and by the Orris C. Hirtzel and
Beatrice Dewey Hirtzel Memorial Foundation, the William F. and
Jean W. Donaldson Chair at Children’s Hospital of Pittsburgh, and the Henry J. Mankin Chair at the University of Pittsburgh.
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However, an injured muscle often does not fully recover its strength because complete muscle regeneration is hindered by the development of fibrosis.
Biological approaches to improve muscle healing by enhancing muscle regeneration and reducing the formation of fibrosis are being investigated.
Previously, we have determined that insulin-like growth factor–1 (IGF-1) can improve muscle regeneration in injured muscle. We also have investigated the use of an antifibrotic agent, decorin, to reduce muscle fibrosis following injury. The aim of this study was to combine these two therapeutic methods in an attempt to develop a new biological approach to promote efficient
healing …show more content…
and recovery of strength after muscle injuries. Our findings indicate that further improvement in the healing of muscle lacerations is attained histologically by the combined administration of IGF-1 to enhance muscle regeneration and decorin to reduce the formation of fibrosis. This improvement was not associated with improved responses to physiological testing, at least at the time-points tested in this study.
Muscle Nerve 28: 365–372, 2003
IMPROVEMENT OF MUSCLE HEALING THROUGH
ENHANCEMENT OF MUSCLE REGENERATION
AND PREVENTION OF FIBROSIS
KENJI SATO, MD,1,2 YONG LI, MD, PhD,1 WILLIAM FOSTER, BS,1
KAZUMASA FUKUSHIMA, MD,1,2 NEIL BADLANI, MS,1 NOBUO ADACHI, MD,1,2
ARVYDAS USAS, MD,1 FREDDIE H. FU, MD,2 and JOHNNY HUARD, PhD1,2
1
Growth and Development Laboratory, Department of Orthopaedic Surgery,
Children’s Hospital and University of Pittsburgh, Pittsburgh, Pennsylvania, USA
2
Department of Orthopaedic Surgery, Division of Sports Medicine, University of Pittsburgh,
Pittsburgh, Pennsylvania, USA
Accepted 24 April 2003
There are various types of muscle injury, including those that occur through direct trauma (e.g., laceration and contusion) and those attributable to indirect damage (e.g., ischemia, denervation, and strain), but the general process of muscle damage and repair is similar in most cases.2,10,12,19 –21,24 –26
Muscle fibers have the ability to regenerate following injury through the activation of satellite cells,13 but the healing process is very slow and often results in incomplete recovery of strength due to the development of scar tissue.12,24,25 The muscle healing …show more content…
process comprises several phases, including degeneration and
Abbreviations: ECM, extracellular matrix; bFGF, basic-fibroblast growth factor; GM, gastrocnemius muscle; H&E, hematoxylin and eosin; IGF-1, insulin-like growth factor–1; NGF, nerve growth factor; PBS, phosphate-buffered saline; TGF-1, transforming growth factor-1
Key words: decorin; fibrosis; IGF-1; muscle healing; regeneration.
Correspondence to: J. Huard, Growth and Development Laboratory, 4151
Rangos Research Center, Children’s Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213-2583; e-mail: jhuardϩ@pitt.edu
© 2003 Wiley Periodicals, Inc.
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inflammation, regeneration, and scar formation (after severe muscle injury).12,24 Muscle regeneration occurs early in this process; it begins about 1 week after injury, peaks during the 2nd week, and then rapidly declines.3,12,16,20,24,25 We have shown previously that growth factors, including insulin-like growth factor–1
(IGF-1), basic-fibroblast growth factor (bFGF), and nerve growth factor (NGF), can improve muscle regeneration during this preliminary phase of healing.19 –21,26
Of these growth factors, IGF-1 was found to have the greatest effect on the healing of the injured muscle by increasing both the efficiency of muscle regeneration and muscle strength. However, none of the growth factors that have been investigated appears able to completely heal injured muscle, possibly due to the development of muscle fibrosis.
The development of fibrosis begins 2 weeks after muscle injury and continues over time. This process hinders muscle regeneration and prevents full strength recovery in the injured skeletal muscle.2,3,12,24,25 We have reported that myogenic cells can differentiate into myofibroblasts upon muscle
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laceration injury.23 We also have observed high levels of transforming growth factor-1 (TGF-1) expression in injured skeletal muscle.23 TGF-1 stimulates the deposition of collagens and overgrowth of the extracellular matrix, thereby leading to the accumulation of fibrotic tissue.4,31 Many studies have shown that upregulation and increased expression of the cytokine TGF- at the injured site constitute central events during fibrosis development through the activation of myofibroblasts.1,4,,6,7,22,23,27,28,31 Accordingly, we believe that TGF-1 plays a pivotal role in skeletal muscle fibrosis and have targeted TGF- inhibition via an anti–TGF- human proteoglycan, decorin,11,14 as an approach to preventing muscle fibrosis.8 We determined that direct injection of human recombinant decorin at 2 weeks after laceration effectively prevents muscle fibrosis and, in turn, enhances muscle regeneration.8
In this study, we investigated the combined use of
IGF-1 and decorin to enhance muscle regeneration, reduce fibrosis, and thereby facilitate improved healing of muscle injuries in vivo. To investigate this hypothesis, we developed a reproducible muscle laceration injury model in mice.8,12,23,25 After treatment of the injured muscles with IGF-1 and decorin, muscle healing and fibrosis were analyzed both histologically and physiologically to assess muscle regeneration and strength. Our primary goal was to improve muscle regeneration through the use of IGF-1 and reduce muscle fibrosis through the use of decorin, in the hope that these two substances would have an additive or synergistic effect on muscle healing and thus facilitate more complete recovery of muscle strength. MATERIALS AND METHODS
Twenty-four mice (C57BL10Jϩ/ϩ, 8 weeks old, 20 –30 g) were used in this experiment. The policies and procedures of the animal laboratory are in accordance with those detailed by the U.S. Department of
Health and Human Services. The Animal Research and Care Committee of the authors’ institutions approved the research protocol used for these experiments.
The muscle laceration model developed in mice entailed laceration of the gastrocnemius muscle
(GM) of both legs based on previously described studies.8,12,19,25,26 The mice were anesthetized by intramuscular injection of 0.03 ml ketamine (100 mg/ ml) and 0.02 ml xylazine (20 mg/ml). A surgical blade (#11 SteriSharps, Mansfield, Massachusetts) was used to lacerate each GM at 60% of its length
Animal Model of Muscle Laceration.
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from its distal insertion through the lateral 50% of muscle width and 100% of muscle thickness. PDSII
5-0 suture (Ethicon, Somerville, New Jersey) was placed at the medial edge of the lacerated site, with
4 mm of the suture remaining visible on the posterior of each leg as a marker for the lacerated site.
After laceration, the skin was closed with black silk
4-0 suture (Ethicon).
Injection of IGF-1 and Decorin following Laceration.
The mice were divided into three groups (eight mice per group). After laceration, IGF-1 (human recombinant IGF-1; Gibco BRL, Carlsbad, California) and decorin (from bovine articular cartilage; Sigma, St.
Louis, Missouri) were injected along the suture material using a micro-syringe (Hamilton, Reno, Nevada).
In group 1, repeated injection of IGF-1 (100 ng/injection) was performed in the right GM at different time points (1, 3, and 5 days postlaceration), while the left
GM was injected with phosphate-buffered saline (PBS) to serve as the control. In group 2, decorin (50 g) was injected into the right GM at 2 weeks postlaceration, while the left GM was injected with PBS as the control.
In group 3, the right GM was injected three times with
IGF-1 (100 ng/injection at days 1, 3, and 5 postlaceration) and once with decorin (50 g at 2 weeks postlaceration), and the left GM served as the lacerated control. Four weeks after laceration, all animals were sacrificed to evaluate muscle healing and regeneration.
The GMs were isolated and frozen in 2-methylbutane precooled in liquid nitrogen.
The harvested muscles were divided into five groups: group N contained the normal, nonlacerated muscle; group C contained the lacerated, shaminjected muscle; group IG contained the lacerated,
IGF-1–injected muscle; group D contained the lacerated, decorin-injected muscle; and group IGϩD contained the lacerated muscle injected with IGF-1 and decorin. Four muscles from each group were evaluated by quantitative histological staining and quantitative physiological analysis.
Characterization of Muscle Regeneration following Injection. Hematoxylin and eosin (H&E) staining was
used to evaluate histologically the degree of muscle regeneration following laceration. We monitored the number and diameter of the centronucleated, regenerating myofibers within the injured injection sites and control groups. As previously described,8,19,20,23,25 five random fields within the injured area were selected in each sample. In brief, low-power images (10ϫ) were obtained with a Nikon
Eclipse E800 microscope (Nikon, Melville, New
York) connected to the Spot Image capture system
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(Spot Image, Chantilly, Virginia). Using the Spot software calibrated with a micrometer, the number and diameter (m) of the centronucleated myofibers (i.e., the regenerating myofibers) were measured directly from the images of the injured area.
The number and diameter of the centronucleated myofibers were expressed as number of centronucleated myofibers per field and diameter of myofibers, respectively. Characterization of Muscle Fibrosis following Injection.
As previously described,8,19,20,23,25 immunohistochemical techniques that measure the expression of vimentin under a fluorescence microscope were used to evaluate scar tissue formation within the injured muscle. The intermediate filament vimentin was used as a marker for fibrosis in this study because it is expressed mainly in monocytes and fibrocytes and is expressed at high levels within the scar tissue of injured muscle.5,8,19,20,23,25 Vimentin is expressed by myofibers early in development and shortly after injury. However, mature myofibers—as well as regenerating myofibers present in injured muscle 2– 4 weeks after injury— do not express vimentin.5,19,20,23,25 Cryosectioned muscles were used for the vimentin staining. In brief, slides were blocked in
2% horse serum for 1 h. After washing, anti-vimentin conjugated Cy3 (Sigma C-9080) antibody then was applied for 1 h at a dilution of 1:250. The total vimentin-positive area was measured in 10 random fields from each sample (n ϭ 4) of each group.
Images were collected using an Olympus Provis epifluorescence microscope (Olympus Optical, Tokyo,
Japan) and a Sony 970 3 chip CCD camera (Sony,
Tokyo, Japan). These images then were digitized using a Coreco (San Jose, California) frame grabber board and were rendered to monochrome. To evaluate the number of vimentin-positive pixels, the images were collected, converted to grayscale, and a threshold was set. The National Institutes of Health
(NIH) image software package expressed the vimentin-positive area as the number of square pixels. The area of fibrosis (i.e., the vimentin-positive area) was compared among the groups.
We also performed Masson modified trichrome staining (IMEB Inc., San Marcos, California) according to the manufacturer’s protocol to further confirm that the vimentin-positive area was indeed collagenous scar tissue. This staining procedure, which stains collagen blue, muscle red, and nuclei black, was used to compare the amount of scar tissue development among the different groups. Northern
Eclipse software (Empix Imaging, North Tonawanda,
New York) was utilized to quantitate the area of scar
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tissue. Images were collected via the same techniques described above for vimentin, and the software was used to calculate the absolute area of fibrosis. In brief, the images were converted to grayscale, a threshold was set, and the area (m2) was determined.
Physiological
testing for muscle strength was performed 4 weeks after laceration. Four muscles per group were assayed to analyze fast-twitch and tetanus strength. The mice were anesthetized by intramuscular injection of
0.03 ml ketamine (100 mg/ml) and 0.02 ml xylazine
(20 mg/ml). Both GMs were removed and mounted in a 35°C, double-jacketed organ bath of 5 ml Krebs solution (mmol/L: NaCl 113, KCl 4.7, CaCl2 1.25,
MgSO4 1.2, NaHCO3 25, KHPO4 1.2, glucose 11.5) constantly bubbled with a mixture of 95% O2 and
5% CO2. All muscles were subjected to a fixed tension of 20 mN; isometric contractions were measured with strain-gauge transducers coupled with a
TBM4 strain-gauge amplifier (World Precision Instruments, Sarasota, Florida) and recorded on a computer using a data acquisition program
(Windaq, Dataq Instruments, Akron, Ohio). The sampling rate was set to 500 Hz, and the amplitude of stimulation-evoked contractions was computed by a calculation program (Windaq, Dataq Instruments).
After 20-min equilibration, electrical field stimuli were applied through two platinum wire electrodes positioned on the top and bottom of the organ bath
(separated by 4 cm). The muscles were stimulated with 0.25-ms square-wave pulses with a maximal voltage of 50 V. First, 1-Hz stimulations were applied for
6 min and the muscle twitches were recorded, then six tetanic stimulations were applied for 0.5 s. Train duration was measured at 100 Hz every 10 s. The muscles were weighed after testing and the muscle strength was reported as millinewtons per gram
(mN/g).
Characterization of Muscle Strength.
The average and standard deviation of all data were compared among the different groups using repeated measures ANOVA for statistical analysis. Statistical significance was defined as
P Ͻ 0.05.
Statistical Analysis.
RESULTS
Quantitative Histological Analysis of Muscle Regeneration. After counting the number of regenerating
myofibers (i.e., centronucleated myofibers) within the injured site, we observed a significantly higher number of regenerating myofibers in all treated
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367
FIGURE 1. Muscle healing 4 weeks after laceration injury. Top panels, H&E-stained sections; lower panels, mean number of new regenerating (centronucleated) myofibers per field and mean diameter of these myofibers. Control (C), muscles treated with IGF-1 (IG), muscles treated with decorin (D), and muscles treated with both agents (IGϩD); *P Ͻ 0.05.
muscles (IGF-1, decorin, or both) compared with control muscles injected with PBS alone. We also noted that the muscles treated with IGF-1 and decorin contained a significantly higher number of regenerating fibers compared to muscles treated with only IGF-1 or only decorin (Fig. 1).
Measurement of the diameter of the regenerating myofibers revealed similar results. The diameters of the regenerating myofibers in all treated muscles
(IGF-1, decorin, or both) were statistically larger than the diameters of the regenerating myofibers in the control muscles. Moreover, the muscles treated with IGF-1 and decorin contained regenerating myofibers with diameters significantly larger than those observed in the muscles treated with IGF-1 alone or decorin alone (Fig. 1). These results indicate that combination treatment with IGF-1 and decorin is more effective in increasing both the number and the diameter of regenerating myofibers than is treatment with either agent alone.
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Quantitative Histological Analysis of Muscle Fibrosis.
By using vimentin and trichrome staining to characterize the fibrotic area, we found that scar tissue formation was significantly decreased in the muscles treated with decorin alone or with IGF-1 and decorin when compared to the control muscles or muscles treated with IGF-1 alone. There was no significant difference in fibrotic area in muscles treated with decorin alone and those injected with IGF-1 and decorin (Figs. 2, 3). These results suggest that IGF-1 treatment does not significantly reduce fibrosis, and that combination treatment with IGF-1 and decorin is no better than treatment with decorin alone in terms of reducing muscle fibrosis.
The strength of the lacerated muscles was determined by physiological testing to evaluate fast-twitch and tetanic strength, and was compared among the different treatment groups.
The fast-twitch muscle strength was 370.13 Ϯ 83.77
Physiological Analysis.
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FIGURE 2. Quantitative histological analysis of the laceration site by vimentin immunohistochemical staining 4 weeks after injury. Top panels (A), vimentin immunohistochemical stain; lower panel (B), area of scar tissue per field. Control (C), muscles treated with IGF-1
(IG), muscles treated with decorin (D), and muscles treated with both agents (IGϩD); *P Ͻ 0.05.
FIGURE 3. Quantitative analysis of scar tissue by trichrome staining. Scar tissue containing collagen (blue) was analyzed. Top panels, trichrome stain; lower panel, area of scar tissue per field (original magnification ϫ100). Control (C), muscles treated with IGF-1 (IG), muscles treated with decorin (D), and muscles treated with both agents (IGϩD); *P Ͻ 0.05.
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FIGURE 4. Histogram of physiological evaluation. The strength of the muscle measured ex vivo: (A) tetanus strength; (B) fast-twitch strength. Control (C), normal, nonlacerated muscle (N), muscles treated with IGF-1 (IG), muscles treated with decorin (D), and muscles treated with both agents (IGϩD); *P Ͻ
0.05.
mN/g in normal muscle, 135.62 Ϯ 34.72 mN/g in the control (20 l PBS) group, 196.12 Ϯ 126.82 in the group receiving IGF-1, 300.48 Ϯ 26.63 mN/g in the group given decorin, and 202.50 Ϯ 27.65 mN/g in the group receiving combination therapy (Fig.
4B). Similarly, the corresponding values for tetanic muscle strength were 140.15 Ϯ 29.79 mN/g, 72.09 Ϯ
2.87 mN/g, 81.76 Ϯ 17.47 mN/g, 129.23 Ϯ 18.67 mN/g, and 93.84 Ϯ 13.40 mN/g, respectively (Fig.
4A).
These results indicate a significant improvement in both fast-twitch and tetanic strength for every treated group (IGF-1, decorin, or both) when compared to the lacerated, nontreated control group. Of the treated groups, the group treated with decorin alone showed the best overall improvement. The strength of muscles in this group returned to nearly the same level as muscles in the normal, nonlacerated group. Muscles in the decorin-treated group also displayed a significant improvement in fasttwitch and tetanic strength compared to the muscles treated with only IGF-1 and those treated with both
IGF-1 and decorin. This finding indicates that treatment with decorin alone is the most effective method by which to restore the physiological strength of injured muscle. The addition of IGF-1 to the treatment actually decreased the resultant physiological strength of the injured muscle. This finding was not supported by the histological analysis, which indicated a beneficial increase in the number and diameter of regenerating myofibers when IGF-1 was used in combination with decorin. These seemingly contradictory results suggest that the histological evaluation of injured muscle does not always correlate with the physiological performance of the muscle.
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DISCUSSION
Optimal treatment methods for muscle injuries have not yet been determined.2,3,12,24 Treatments traditionally have focused on rather conservative techniques such as RICE (rest, ice, compression, and elevation), heat, water pool, or immobilization, though treatment options such as continuous passive motion machines, drugs, and hospitalization also have been used.9,15,18 In previous studies, we have observed that the immobilization of lacerated muscle does not limit the development of fibrosis, whereas suture repair can limit fibrosis development deep in the injured muscle but is incapable of eliminating superficial muscle fibrosis.25 These traditional treatments usually do not promote a rapid enough recovery to satisfy patients and the recurrence of injury is common, indicating that such treatments also fail to provide full functional recovery and are likely ineffective at preventing the formation of permanent scar tissue at an injured site.2,3,12,24 These factors have prompted us to seek novel biological approaches that may enable faster, more complete muscle recovery. For the present study, we hypothesized that the injection of IGF-1 at
1, 3, and 5 days postinjury would increase the proliferation and differentiation of mononucleated satellite cells—thus improving regeneration—and that the injection of decorin at 14 days postinjury would inhibit the effects of TGF-1, thereby reducing fibrosis.
The outcome, though promising, is somewhat different than anticipated. We expected the dual treatment with IGF-1 and decorin to promote better muscle recovery than treatment with either agent alone. Our histological studies supported this hypothesis. Treatment with IGF-1 and decorin had an additive effect on muscle regeneration, increasing
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both the number and diameter of the regenerating muscle fibers. In contrast, the histological analysis of muscle fibrosis did not indicate any benefit associated with the use of both decorin and IGF-1. Decorin alone was capable of reducing fibrosis, and the addition of IGF-1 neither added nor detracted significantly from this effect.
Surprisingly the physiological testing suggested that the histological appearance of muscle does not always correspond directly with its physiological strength. The injured muscle treated with decorin alone actually displayed greater fast-twitch and tetanic strength than the muscle treated with both
IGF-1 and decorin. The reason for this finding is unclear. It may be that in reconstructed muscle there is an optimum ratio between the extracellular matrix (ECM) and myofibers, which maximizes physiological strength.17,33 Perhaps the fibrotic tissues inhibited by decorin are required to ensure proper realignment of the myofibers and the myofibrils in order to maximize muscle mechanics. Disrupting this ratio with too high a proportion of either ECM or myofibers thus would not prove beneficial for tissue reconstruction in injured skeletal muscle. Furthermore, molecular studies have indicated an interaction between decorin and IGF-1 in muscle, a finding that has not yet been elaborated upon. Decorin has been shown to bind to other growth factors— and even growth factor receptors—to alter their function.30,32 IGF-1 itself could play a role in accelerating the growth of the ECM, and such an interaction could result in an unpredicted change in the physiological strength of the regenerated muscle.29
As mentioned above, we recently have determined that myogenic cells can differentiate into fibrotic cells after laceration injury.23 It is not yet clear, however, how the application of decorin and IGF-1 in combination might affect this process. In future research, we plan to further explore the relationship between decorin and IGF-1.
The use of IGF-1 to increase muscle regeneration in combination with decorin to inhibit fibrosis promoted good histological recovery of lacerated muscle, but this high level of recovery was not supported by physiological testing at the time points tested in this study. Even so, these approaches need to be studied both for longer time points to delineate possible side effects and in other animal models of muscle injury, such as strain and contusion models.
It is also necessary to more fully evaluate potential intramuscular and intracellular interactions between antifibrotic agents, such as decorin and growth factors, that appear to aid muscle regeneration.
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The authors thank Ryan Pruchnic and Marcelle Pellerin for technical assistance, Victor Prisk, Yi-Sheng Chan, and Takashi Horaguchi for their critical comments about the manuscript, and Ryan Sauder for his assistance in editing the manuscript. This work was supported by a grant awarded to Johnny Huard from the National Institutes of
Health (NIH 1, RO1 AR47973-01) and by the Orris C. Hirtzel and
Beatrice Dewey Hirtzel Memorial Foundation, the William F. and
Jean W. Donaldson Chair at Children’s Hospital of Pittsburgh, and the Henry J. Mankin Chair at the University of Pittsburgh.
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