Skeletal muscle injuries often occur during sports activities and trauma. Several reports recommend RICE (Rest, Icing, Compression, and Elevation) therapy to control bleeding and inflammation during the acute stage after injury.1 Recently, some reports have described that skeletal muscle exhibits a high capacity to repair and regenerate after injury. However, when skeletal muscle suffers extensive injury or a wide defect, the tissue heals with fibrous scarring and occasionally with ectopic calcification. To establish sound treatment modalities, many investigators have focused on minimizing fibrosis after muscle injury using drugs including cytokine inhibitors with antifibrotic properties.2, 3 Although cell transplantation has become popular in regenerative medicine, treatment of skeletal muscle injuries using cell-based therapy has not been established yet. Delivery of the transplanted cells to the injury site is of particular importance in cell-based treatment strategies. We developed a magnetic cell delivery system to efficiently localize cells to injury sites and reported that bone and spinal cord were efficiency regenerated using this system.4–7 In these reports, cell kinetics was only observed at the endpoint and not over time. There are currently no reports on skeletal muscle regeneration using mesenchymal stem cells (MSCs) with external magnetic targeting. In this study, we describe the repair of skeletal muscle injury by MSCs under an external magnetic force, where the kinetics and localization of the MSCs were observed using an in vivo bioluminescence imaging system.
The purpose of this study is to clarify the kinetics of transplanted mesenchymal stem cells (MSCs) in rat skeletal muscle injury model and the contribution of the magnetic cell delivery system to muscle injury repair. A magnetic field generator was used to apply an external magnetic force to the injury site of the tibia anterior muscle, and 1 × 106 MSCs labeled with ferucarbotran–protamine complexes, which were isolated from luciferase transgenic rats, were injected into the injury site. MSCs were injected with and without an external magnetic force (MSC M+ and MSC M− groups, respectively), and phosphate-buffered saline was injected into injury sites as a control. In vivo bioluminescence imaging was performed immediately after the transplantation and, at 12, 24, and 72 h, and 1 and 4 weeks post-transplantation. Also, muscle regeneration and function were histologically and electromechanically evaluated. In vivo bioluminescence imaging showed that the photon of the MSC M+ group was significantly higher than that of the MSC M− group throughout the observation period. In addition, muscle regeneration and function in the MSC M+ group was histologically and functionally better than that of the MSC M− group. The results of our study indicated that magnetic cell delivery system may be of use in directing the transplanted MSCs to the injury site to promote skeletal muscle regeneration. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 754–759, 2013
MATERIALS AND METHODS
Preparation of MSCs from Luciferase Transgenic Rats and Magnetic Labeling of MSCs
MSCs were isolated from luciferase transgenic rats (4–8 weeks old, male) which were provided by Kyoto University (Kyoto, Japan). MSCs at passages 2–4 were used for experimentation. In addition, cells were labeled with fercarbotran and protamine as transfection agent8–11 (Supplementary S1).
Rat Model of Skeletal Muscle Injury and Cell Transplantation
Muscle injury in rats was created based on previously published reports from our department.12, 13 Lewis rats (LEW/CrlCrl, 9 weeks old, female N = 6/group) were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg). An anterolateral skin incision was made in the left leg to expose the tibialis anterior muscle. The fascia was longitudinally incised and carefully released from the muscle belly. The muscle belly was transversely lacerated at the mid-portion using a scalpel. The defect of the muscle was wedged-shaped at approximately 6 mm long, 4 mm wide, and 5 mm deep. The fascia was then tightly sutured using 5-0 nylon.12, 13 The MSCs (1.0 × 106 in PBS 30 µl) were injected into the injury site with external magnetic force [3.0 T (Supplementary S2), exposure time: 10 min] as MSC M+ group and without external magnetic force as MSC M− group. The same volume of PBS without MSCs was administered to injury sites as a control (PBS group).
In Vivo and Ex Vivo Bioluminescence Imaging
In vivo bioluminescence imaging of luciferase was performed using a Night Owl LB 981 system controlled by Indigo 2 software (Berthold Technologies, Bad Wildbad, Germany). Imaging was performed at 15 min after intraperitoneal injection of luciferin. Images were obtained during the 5 min immediately after the operation, and at 12, 24, and 72 h, 1 and 4 weeks post-operatively. In addition, skeletal muscles in MCS M+ group and MSC M− group at 4 weeks were removed from legs. Muscles were placed on 100 mm culture dishes in the imaging chamber. Ex vivo bioluminescence imaging of luciferase was performed after luciferin addition. Images were obtained over 5 min.
To evaluate functional regeneration, intrinsic tensile strength produced by stimulating the common peroneal nerve was measured with transducer load cells (LVS-1KA; Kyowa Electronic Instruments, Tokyo, Japan) and recorded with a sensor interface (PCD-300A; Kyowa Electronic Instruments) and software (PCD-30A; Kyowa Electronic Instruments) as described elsewhere12, 13 (Supplementary S3).
To evaluate fibrosis and muscle regeneration, Masson trichrome staining was performed on frozen sections from each sample. To evaluate muscle regeneration and angiogenesis in regenerated tissues, frozen sections from each sample at 1 week were immunostained with an anti-desmin antibody (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) and an Alexa Fluor 568-conjugated goat anti-rabbit IgG (1:500; Molecular Probes Inc., Eugene, OR). Endothelial-specific chemical staining was performed using a fluorescein isothiocyanate conjugated anti-isolectin B4 antibody (1:100; Vector Laboratories, Burlingame, CA) to assess vascularization of injured muscles. The regenerated muscle area and capillaries were evaluated by histological examination of six randomly selected fields around the fibrotic area in axial sections at 200× magnification. Capillaries were identified as tubular structures positive for isolectin B4. For histological assessment, each sample was digitally imaged by a BIOREVO BZ-9000 (Keyence Japan, Osaka, Japan) at 400× magnification. Images were analyzed using the BZ II analyzer image analysis software package (Keyence Japan) (Supplementary S4).
For in vivo bioluminescence imaging data, statistical analysis was performed with repeated measures analysis of variance (ANOVA) and the Mann–Whitney U-test with Bonferroni correction. Data were represented as the mean ± standard error (SE). Results of electromechanical and histological evaluations were analyzed using one-way ANOVA followed by Fisher's protected least significant difference post hoc comparisons. A value of p < 0.05 was considered statistically significant.
Immediately after the operation, photon in MSC M+ and MSC M− groups increased up to 24 h and then decreased in both groups. The photon in the MSC M+ group was significantly higher compared with that of the MSC M− group over time (Fig. 1A). Although the photon was almost undetectable both in MSC M+ and MSC M− groups in vivo at 4 weeks, ex vivo bioluminescence imaging showed that the photon of resected muscles at 4 weeks in the MSC M+ group was significantly higher than that of the MSC M− group (Fig. 1B).
We compared fast-twitch and tetanus strength ratios among the three groups. At 1 and 4 weeks, the fast-twitch ratio was significantly higher in MSC M+ and MSC M− groups compared with that of PBS group. At 1 week, the fast-twitch ratio of the MSC M+ group was higher than that of the MSC M− group, but there was no significant difference. However, at 4 weeks, the fast-twitch ratio of the MSC M+ group was significantly higher among the three groups. Similarly, the tetanus strength ratios in MSC M+ and MSC M− groups were significantly higher compared with that of the PBS group at 1 and 4 weeks. However, there was no significant difference between MSC M+ and MSC M− groups at 1 week. At 4 weeks, the tetanus strength ratio of the MSC M+ group was significantly higher than that of the MSC M− group (Fig. 2). Masson trichrome staining was used to evaluate fibrosis and demonstrated that there was almost complete healing with a very small fibrotic area at 4 weeks in the MSC M+ group. At 1 week, the fibrotic area in the MSC M+ group was smaller than that in the MSC M− group, but there was no significant difference between MSC M+ and MSC M− groups. At 4 weeks, the fibrotic area in the MSC M+ group was significantly the smallest among the three groups (Fig. 3). The diameter of myofibers near perifibrous sites in all groups was evaluated in axial sections. The diameter of myofibers in the MSC M+ group was significantly the largest compared with the other two groups at 1 and 4 weeks. In addition, the diameter of myofibers in the MSC M− group was significantly larger than those in the PBS group at 1 and 4 weeks (Fig. 4).
Immunohistological Assessment of Regeneration
Regenerated muscle by MSC transplantation was assessed by the desmin-positive area at 1 week. Desmin-positive areas in MSC M+ and MSC M− group were significantly larger compared with those in the PBS group. The desmin-positive area in the MSC M+ group was significantly the largest among the three groups. Enhanced angiogenesis by MSC transplantation was assessed by the number of capillaries at 1 week. Vascular staining of isolectin B4 in tissue samples demonstrated the significantly highest degree of neovascularization around the fibrotic area in the MSC M+ group among the three groups. Although the average number of Pax7 (marker for satellite cell) positive in MSC M+ group was greater than that in the other groups, there was no significant deference in the number of Pax7-positive cells among the three groups. In addition, there was no significant deference in the number of CD11b (marker for leukocyte) positive cells was no significant deference among the three groups (Fig. 5A–F).
The results of the present study demonstrates that the transplantation of MSCs under external magnetic control enhances the localization of transplanted cells at the muscle injury site and promotes the regeneration of injured skeletal muscle, as indicated by the reduction of fibrosis and the acceleration of angiogenesis and myogenesis. The localization of transplanted MSCs at the muscle injury site was confirmed by in vivo bioluminescence imaging of luciferase.
In this study, the localization of transplanted MSCs at the muscle injury site was enhanced by the magnetic force from outside the body. Previous studies reported that cultured MCSs labeled with magnetic particles could be guided and localized at a desired region by magnetic force.5, 14 Furthermore, some tissues, including bone, and spinal cord, are regenerated by localization of MSCs using magnetic force in our department.4, 6, 7, 15
Additionally, in this study, the localization of transplanted MSCs at the muscle injury by magnetic force was evaluated using an in vivo bioluminescence imaging system. This is the first report of regenerated muscle that was promoted by MSCs under an external magnetic force and assessed in vivo over time in rats.
To understand profound biological processes as they occur in living animals, imaging strategies have been developed and refined to reveal cellular and molecular biological events in real-time. Recently, kinetics of various cells, infection, bacterium, and inflammation were observed using in vivo bioluminescence imaging.16–19 However, there is no previous report on the kinetics of transplanted MSCs under an external magnetic force using in vivo bioluminescence imaging system. In previous reports, evaluation of cell localization was only performed by histological or immunohistological assessment at one time-point after animal sacrifice.
We expected that the peak of photon would be observed immediately after cell transplantation. However, the peak photon was observed from 12 to 24 h post-transplantation. This observation may be due to neovascularization at the injury site not being initiated immediately after transplantation, resulting in the inability of luciferin to access the injury site and transplanted MSCs. At 4 weeks, the photon of the MSC M+ group was diminished as determined by in vivo bioluminescence imaging. However, the photon of the MSC M+ group was consistent at the injury site as determined by ex vivo bioluminescence imaging. This result demonstrated that transplanted MSCs were engrafted at the injury site after transplantation under an external magnetic force.
Various cells participate in the repair of injured skeletal muscles. Initially neutrophils, and subsequently macrophages, both inflammatory cells, migrate to phagocytosis necrotic tissue. Simultaneously, muscle satellite cells are activated, and regeneration of skeletal muscle tissues is initiated. Stem cells are generally <1% of tissue, but muscle satellite cells are reported to represent 1–5% of skeletal muscle tissue.20 In this study, Pax7-positive cells in MSC M+ group do not proliferate significantly, but satellite cells might promote to differentiated progenitor cells. On the other hand, the infiltration of inflammatory cells was neither enhanced nor inhibited by magnetic cell targeting.
In severe or recurring injuries, the amount of stem cells becomes insufficient for repair because rapid replacement of stem cells is difficult. Hence, defects are filled with fibroblasts that simultaneously proliferate and generate fibrotic tissues. Tobias and colleagues reported a dose-dependent relationship between MSCs and functional regeneration after muscle injury in rats. In their report, the transplantation of 10 × 106 MSCs resulted in the most pronounced improvement to muscle force.21 In this study, we demonstrated that transplanted MSCs were efficiently localized at the injury site over long term using a magnetic delivery system. Localization MSCs over a long term resulted in promotion of muscle regeneration as demonstrated functionally and histologically. Maturation of myofibers was histologically accelerated, normal muscle strength was functionally regained earlier post-transplantation, and early repair of skeletal muscle was promoted by transplanting MSCs into injured skeletal muscle under an external magnetic force.
In conclusion, present study reports the effects of bone marrow-derived MSCs on muscle tissue regeneration. We observed that a magnetic targeting system enables efficient MSC localization to the injury site using an in vivo bioluminescence imaging system without the need to sacrifice the animals. The results of the current study have demonstrated a positive correlation between effective localization of transplanted cells at the injury site and tissue regeneration. Our device is smaller than conventional device, so it easy to use in wide clinical application. Further research is needed to introduce magnetically targeted bone marrow-derived mesenchymal stem cell therapy as a valid and safe cell-based treatment modality in regeneration of various tissues.
This research was partly supported by a Grant-in-Aid to Prof. Ochi M. for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology, Japan (No. 21249079).