Stem cells, angiogenesis and muscle healing: a potential role in massage therapies?
- 1Division of Sports Medicine, Department of Family Medicine, Sports Health And Performance Institute, The Ohio State University, Columbus, Ohio, USA
- 2Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
- Correspondence to Dr Burhan Gharaibeh, Department of Orthopaedic Surgery, University of Pittsburgh, Suite 206, Bridgeside Point II, Pittsburgh, PA 15219, USA;
- Received 14 August 2012
- Accepted 30 October 2012
- Published Online First 29 November 2012
Skeletal muscle injuries are among the most common and frequently disabling injuries sustained by athletes.
Repair of injured skeletal muscle is an area that continues to present a challenge for sports medicine clinicians and researchers due, in part, to complete muscle recovery being compromised by development of fibrosis leading to loss of function and susceptibility to re-injury.
Injured skeletal muscle goes through a series of coordinated and interrelated phases of healing including degeneration, inflammation, regeneration and fibrosis. Muscle regeneration initiated shortly after injury can be limited by fibrosis which affects the degree of recovery and predisposes the muscle to reinjury. It has been demonstrated in animal studies that antifibrotic agents that inactivate transforming growth factor (TGF)-β1 have been effective at decreasing scar tissue formation. Several studies have also shown that vascular endothelial growth factor (VEGF) can increase the efficiency of skeletal muscle repair by increasing angiogenesis and, at the same time, reducing the accumulation of fibrosis. We have isolated and thoroughly characterised a population of skeletal muscle-derived stem cells (MDSCs) that enhance repair of damaged skeletal muscle fibres by directly differentiating into myofibres and secreting paracrine factors that promote tissue repair. Indeed, we have found that MDSCs transplanted into skeletal and cardiac muscles have been successful at repair probably because of their ability to secrete VEGF that works in a paracrine fashion. The application of these techniques to the study of sport-related muscle injuries awaits investigation. Other useful strategies to enhance skeletal muscle repair through increased vascularisation may include gene therapy, exercise, neuromuscular electrical stimulation and, potentially, massage therapy. Based on recent studies showing an accelerated recovery of muscle function from intense eccentric exercise through massage-based therapies, we believe that this treatment modality offers a practical and non-invasive form of therapy for skeletal muscle injuries. However, the biological mechanism(s) behind the beneficial effect of massage are still unclear and require further investigation using animal models and potentially randomised, human clinical studies.
- Sports rehabilitation programs
- Muscle damage/injuries
- Sporting injuries
- Muscle injury and inflammation
- Biology of skeletal muscle
Traumatic muscle contusion and strain injuries comprise up to 45% of all sports-related injuries.1–5 Although skeletal muscle has the capacity to regenerate, following injury, the healing process is slow, often with delayed functional recovery and an increased risk for injury recurrence.2 ,6 Despite the considerable need for attention by the sports community, there has been relatively little progress in the development of standardised therapeutic approaches to enhance healing following skeletal muscle injury.7 Moreover, the short-term declines in function and pain associated with intense eccentric exercise often limit individuals from maintaining adherence to exercise programmes. Therefore, innovative approaches that enhance muscle recovery and healing from intense exercise as well as traumatic injury are of critical importance to continued advances in our ability to care for athletes and other active individuals.
Skeletal muscle injury and healing process
Several articles by our group and others have described the process of healing of injured skeletal muscle.8–11 In short, skeletal muscle repair includes four main, interrelated and sequential phases: degeneration, inflammation, regeneration and fibrosis. Initially, in the inflammatory phase, there is an influx of cytokines that promote chemotaxis, removal of necrotic tissue by macrophages and local increases in vascularity. Over time, the regenerative phase follows with the infiltration of reparative cells that consist largely of muscle-derived stem cells (MDSCs).2 ,12 MDSCs are believed to be an earlier progenitor to satellite cells, expressing stem cell markers such as cluster of differentiation 34 (CD34) and stem cell antigen 1 (Sca-1), and have the ability to differentiate into non-muscle lineages and display a high transplantation capacity in both skeletal and cardiac muscles,13–15 owing to their unique ability to survive in harsh microenvironments. Stem cell therapies may represent an exciting venue for enhancing muscle repair and expediting return of athletes to sport.
MDSCs have proven to be experimentally useful in the improvement of repair processes in a host of musculo-skeletal tissues. MDSCs transplanted without genetic manipulation or transduced to express genes such as VEGF, soluble tyrosine kinase 1 (sFlt1) (VEGF antagonist) and/or BMP-2 and BMP-4 have been shown to be involved and offer promise in repairing full-thickness articular cartilage defects, osteoarthritis, long bone and calvarial bone defects, transected medial collateral ligament (MCL), as well as cardiac injury in several animal models.13 ,15–30
Relationship of MDSCs to blood vessel wall in the skeletal muscle
The vast majority of Sca-1(+) cells found within the skeletal muscle are located outside the muscle fibre's basal lamina, and are located within or closely associated with blood vessels.31 The fact that MDSCs express endothelial markers and promote neo-angiogenesis after implantation in both skeletal and cardiac muscle further strengthens this relationship. These results, when taken together, suggest that murine MDSCs appear to be derived from the blood vessel walls. In addition, recent investigations by our group have demonstrated similarities between MDSCs and blood vessel-derived endothelial cells and perivascular cells.32–34 Accordingly, the regenerative phase in muscle healing is remarkably dependent on angiogenesis. In animal models, the regenerative phase peaks approximately 10 days after injury and shortly thereafter, concentrations of inflammatory cytokines, most notably transforming growth factor (TGF)-β1, peak by 2 weeks following injury and abate the regenerative phase.35 Our studies on TGF-β1 show that this cytokine plays a dominant role in fibrogenesis. Thus, antifibrotic agents that inactivate TGF-β1 such as suramin, IFN-γ and IFN-α, relaxin, decorin and losartan reduce muscle fibrosis and consequently improve skeletal muscle healing in vivo.10 ,36–44 Limited data from a case study show the efficacy of using losartan to treat hamstring injury in humans.11 Clinical trials are proposed to explore the efficacy of some of these agents to improve recovery in humans sustaining muscle injuries.
MDSCS enhance skeletal muscle repair by increased levels of VEGF
Based on our knowledge of the sequential phases of skeletal muscle healing (figure 1), aside from minimising the amount of fibrotic tissue by the pharmacological agents discussed above, an alternative strategy is to maximise muscle regeneration by use of stem cells. Stem cells that are resistant to oxidative stress are able to proliferate and contribute to the healing process by direct differentiation into host tissues. However, it was noted in recent years that direct differentiation is not the only event that happens after stem cell transplantation into the injury site. Indirect effects like production of paracrine molecules that likely enhance repair by recruiting host cells to the damaged area also take place.8 One of the important paracrine molecules produced by MDSCs injected into skeletal and cardiac muscles is vascular endothelial growth factor (VEGF).45–47
VEGF refers to a group of mitogenic proteins (VEGF-A, B, C and D) that can specifically induce the proliferation of endothelial cells and regulate the formation of new blood vessels by interacting with numerous other factors.48 Repetitive exercise of skeletal muscle has been reported to increase angiogenesis via increased expression of VEGF proteins.49 ,50 VEGF transcription is also elevated in response to muscle hypoxia or low oxygen partial pressure after exercise.51
Our group has investigated the role of VEGF after MDSCs transplantation into a mouse model of Duchenne muscular dystrophy (mdx mice) which lack dystrophin.46 Transplantation of MDSCs, transduced to express VEGF, resulted in an increase in angiogenesis and endogenous muscle regeneration as well as a reduction in fibrosis in comparison with control muscles. Muscle injected with cells expressing soluble forms such as sFlt1, a VEGF-specific antagonist, showed a significant decrease in vascularisation and an increase in fibrosis.46 In a different set of experiments, we have found that transplantation of MDSCs after contusion injury in a mouse model was associated with high levels of VEGF expression, significant induction of angiogenesis and consequently improved muscle regeneration and muscle strength.45 The improvement in muscle regeneration was also accompanied by reduced levels of fibrosis after transplantation of MDSCs.45 Results in these studies clearly show the importance of modulation of VEGF expression and neoangiogenesis to optimise repair of injured skeletal muscle.
Increased VEGF expression in MDSCs by preconditioning with mechanical stimulation prior to transplantation
We have investigated whether in vitro mechanical stimulation has a beneficial effect on improving the transplantation capacity of MDSCs in cardiac muscle.47 ,52 We cultured MDSCs on flexible collagen-coated culture plates (Flexcell Intl Corp, Hillsborough, NC, USA). Twelve hours later, the cells were subjected to 10% equibiaxial strain, 0.5 Hz, sine wave for 4 or 24 h. Controls were cultured in the same manner but not subjected to strain. Stimulated MDSCs showed higher levels of VEGF expression than non-stimulated cells. After mechanical stimulation, the cells were injected into the left ventricular myocardium of mdx mice. At 2 weeks after cell implantation, the mice were sacrificed, and their hearts were harvested and stained for dystrophin expression. Infarcted hearts transplanted with stimulated MDSCs showed improved cardiac contractility, increased angiogenesis and decreased fibrosis compared with the hearts treated with non-stimulated MDSCs.52 Dystrophin-positive cells were counted for the largest engraftment area. An increased number of dystrophin-positive fibres were detected for cells mechanically stimulated for both 4 and 24 h, when compared with non-stimulated cells (0 h; figure 2). For cells mechanically stimulated for 24 h, there was a significant increase in the number of dystrophin-expressing myocytes within the heart, when compared with non-stimulated and 4 h-stimulated cells. These results suggest that mechanical stimulation prior to injection improves the transplantation capacity of MDSCs into cardiac muscle. The important implication of these findings is that stimulated MDSCs can also be used in skeletal muscle repair. Furthermore, mechanical stimulation of muscle tissue probably activates in situ stem and satellite cells which would accelerate remodelling of damaged muscle.
Effect of exercise and electrical stimulation of the skeletal muscle on stem cells and VEGF expression
Sca-1 positive, non-haematopoietic (CD45−) stem cells53 responded to eccentric (lengthening) exercise by doubling their number in wild-type skeletal muscle in the presence of α7 integrin. Cells isolated from α7Tg muscle following exercise were characterised as mesenchymal-like stem cells, with a majority being pericytes. Hypoxia-inducible factor HIF-1α and HIF-2α regulate VEGF levels. HIF-1α and HIF-2α levels increased in microvessels isolated from rat muscle after stretch by overloading.54 Ambrosio et al55 have investigated the effect of treadmill exercise on MSDC transplantation success and reported an increased ability for healing in a mouse skeletal muscle contusion injury model. MDSC-injected muscles have shown significantly higher number of regenerated myofibres, lower collagen I-positive (fibrotic) and increased CD31 positive (neoangiogenesis) levels indicating a synergistic effect between stem cells and exercise.55
Several studies have shown that electrical stimulation of rat hindlimb skeletal muscles resulted in a significant increase in expression of VEGF gene56 and blood vessel density poststimulation.57 ,58 Direct effect of the electrical field on the tissue, chronic muscle contractions, hypoxic conditions and activating host and donor stem cells are all likely causes that trigger the expression of VEGF.55 ,59 Ambrosio et al55 administered an electrical current via the peroneal nerve to the anterior crural muscles of dystrophic mice after MDSCs injection. Harvested muscles show a nearly threefold increase in the number of dystrophin-positive fibres in MDSC-injected muscles that were subjected to an electrical stimulation as compared with stimulated controls. Stimulation could increase the vascular supply and hence increase the transplantation capacity of the injected MDSCs. These results suggest to us that electrical stimulation of skeletal muscle following injury may offer a promising avenue for enhancing angiogenesis and potentially upregulating the events of muscle regeneration through stem cell activation and triggering of VEGF. Further work is needed to understand in a better manner the mechanisms by which electrical stimulation increases angiogenesis as well as clinical trials in humans to test its application and efficacy to sports-related muscle problems.
Massage-based therapies and modulation of skeletal muscle recovery and healing
Muscle can perceive mechanical stimuli and respond by generation of corresponding intracellular signals. Muscle cells respond to biomechanical signals in a magnitude-dependent manner, and lead to qualitative and quantitative changes in gene expression that initiate muscle damage or muscle repair. Trauma induced by both excessive mechanical forces and repetitive lengthening contractions, even those associated with intense eccentric exercise, can lead to the induction of proinflammatory cytokines and subsequent expression of mediators that initiate muscle damage.60 ,61 For example, 20–50% mechanical stretch applied to muscle ex vivo results in activation of nuclear factor kappa B (NF-κ)-mediated transcription factors and proinflammatory gene induction62 suggesting that critical levels of mechanical forces are necessary for tissue homeostasis. Activation of the NF-κ pathway by exercise is associated with regulation of distinct gene clusters and has been shown to be a major stimulator of inflammation and muscle protein turnover.63 ,64 Thus, regulation of NF-κ activity may present an exciting intervention target aimed at preventing or attenuating muscle weakness and wasting.
Muscle soreness and weakness accompany intense or prolonged physical activity. The science of sports massage interests athletes, athletic trainers and sports physiologists. While evidence to support or reject its effect on sports performance is insufficient at this time, new reports help formulate an understanding of massage and its role in exercise-related muscle pain.65 It does appear that under certain conditions, massage can reduce muscle soreness associated with eccentric muscle contractions, although whether force output recovers more quickly is still unclear. Zainuddin et al66 found that massage treatment applied after eccentric exercise in 10 subjects had significant effects on lowering plasma creatine kinase and reducing arm circumference 3–4 days postexercise but no effect on the recovery of maximal isometric and isokinetic voluntary strength. In another clinical study, Hart et al67 found no evidence that sports massage reduced symptoms of DOS in subjects exposed to eccentric exercise. Despite the lack of conclusive scientific evidence, providers spend a large portion of their time providing massage.68 The contrast between current scientific understanding of sports massage and its practice is notable, and scientific evidence to corroborate or refute the effect of massage on muscle recovery remains an important area of investigation.65
According to the American Massage Therapy Association (http://www.amtamassage.org), the physical benefits of therapeutic massage on muscle include; relief from muscle tension and stiffness, faster healing of strained muscles and sprained ligaments, reduced muscle pain, swelling and spasm, greater joint flexibility and range of motion and even enhanced athletic performance. Using our well-established model of eccentric exercise-induced muscle inflammation and damage, we have begun to examine the efficacy of massage therapies in mitigating the strength loss associated with repeated eccentric contractions. In our early studies, we demonstrated that 4 days of massage-like loading at a predetermined magnitude, duration and frequency of tissue loading led to an accelerated recovery of muscle function, as measured by isometric torque at 21 joint angles.69
We have subsequently designed a new device for applying massage-like compressive loads that allows for the simultaneous recording of shear forces.70 Our device can be accommodated to both rabbit and mouse muscle permitting a variety of applications and investigations. In a recent study, we showed that magnitude and frequency of massage-like loading effected recovery of active muscle properties (isometric torque) following a controlled bout of eccentric exercise.71 Interestingly, loading duration did not affect recovery of muscle properties (isometric joint torque). Massage also reversed the typical peak torque-joint angle shift that occurs following eccentric exercise, a further indication of muscle recovery when this modality was utilised. Furthermore, four consecutive days of massage resulted in decreased muscle wet weight and decreased tissue damage (torn muscle fibres) compared with the contralateral leg that was exercised but received no massage. We have also shown that the same 4-day massage-like loading protocol applied following eccentric exercise restores normal tissue stiffness.72 The majority of this decrease in tissue stiffness towards the pre-exercise values occurs on the first 2 days, suggesting a time limit for the beneficial and cumulative effects of repeated bouts of massage. These findings need further validation in human studies to make definitive recommendations about massage and its utilisation in athletes and other exercising individuals. Taken together, we have shown for the first time to our knowledge that magnitude, duration and frequency of tissue loading should be considered in the prescription of massage-based therapies and that there may be a finite limit to the benefits of repeated sessions of massage. Moreover, these findings are consistent with in vitro work demonstrating the sensitivity of muscle cells to various levels of perturbation. An unknown but important question that remains unanswered in humans receiving manual therapies such as massage is the ability of the therapist to know how much force, for how long and how often to utilise this therapy to achieve optimal results. Clinical trials could be based on findings from animal studies to potentially address these important questions.
Biological mechanisms for massage-based therapies to improve recovery following eccentric exercise
Sports massage has been suggested as a means to help prepare an athlete for competition, as a tool to enhance athletic performance, as a treatment approach to help the athlete recover after exercise or competition and as a manual therapy intervention for sports-related musculoskeletal injuries. In addition to a lack of conclusive evidence for efficacy, the exact mechanisms by which massage may aid in recovery of muscle function following intense exercise remain unknown. However, recent studies have begun to shed light on this important topic. Crane et al73 have documented the changes in gene expression that occur within the vastus lateralis following intense eccentric exercise and the ability of a single 10 min bout of massage to alter expression of key transcription factors regulating inflammation such as NF-κβ. In addition, massage led to an increase in mitochondrial biogenesis and a decrease in heat shock protein 27 phosphorylation, thereby decreasing cellular stress caused by muscle fibre injury.73
Our laboratory has similarly begun to investigate the potential mechanisms for massage-based therapies to mitigate muscle damage and enhance recovery from intense eccentric exercise. Using our animal model and the methods described above, we have recently completed studies comparing the effects of immediate versus massage delayed by 48 h following eccentric exercise.62 ,74 Both strategies led to an accelerated recovery of muscle function (isometric torque) compared with a control, exercised and non-massaged muscle. However, the immediate massage condition also showed an additional benefit over the delayed massage condition for recovery of muscle function, as well as decreasing tissue wet weight, myofibre damage and inflammatory cell infiltration.
The importance of manual therapies, including massage-based therapies, and their use for improving the time course and extent of muscle recovery following intense exercise is a topic of relevance to athletes, coaches and all medical personnel. Using massage, it is possible to increase the duration of the muscle repair phase and decrease the duration of the fibrotic phase by increasing circulatory clearance of locally expressed TGF-β, while antifibrotic agents decrease the duration of the fibrotic phase. However, further work is necessary to translate the findings from animal-based laboratory research into clinical practice. Additional research on massage, muscle recovery and healing and the conditions necessary (applied forces, duration of therapy, etc) is needed to fully understand the clinical utility of our research.
Muscle injury and repair is an area in sports medicine research that has not been extensively investigated and, consequently, therapies have virtually not changed significantly in the past few decades. Skeletal muscle goes through several well-coordinated and interrelated phases of healing that are well described. Research into how to optimise muscle healing by upregulating the regeneration phase, minimising fibrosis or scar tissue formation and modulating the effects of inflammation are all potential venues that can be investigated to improve recovery and minimise risk of reinjury. Several studies have shown that VEGF can enhance skeletal muscle repair by effectively increasing angiogenesis. Angiogenesis has also been found to be inversely related to levels of fibrosis in injured muscle. Strategies to modulate angiogenesis include direct injection of growth factors, gene therapy, exercise, neuromuscular electrical stimulation and potentially massage therapy. The latter in particular seems to be a very attractive and the least invasive form of therapy that warrants continued investigation both in animal studies exploring the mechanism of action and human trials evaluating efficacy and best practice (figure 3).
The interrelated phases of skeletal muscle healing include degeneration, inflammation, regeneration and fibrosis.
Vascular endothelial growth factor (VEGF) can promote skeletal muscle repair by increasing angiogenesis and reducing fibrosis.
Skeletal muscle-derived stem cells (MDSCs) enhance repair of damaged skeletal muscle fibres by directly differentiating into myofibres and secreting VEGF.
Mechanical stimulation of MDSCs in vitro increases their expression of VEGF, while neuromuscular electrical stimulation and exercise in vivo improves their regenerative index.
Animal studies demonstrated the potential for massage-based therapies to improve muscle function and recovery from intense exercise.
One possible mechanism for this therapy is enhanced tissue revascularisation through increased expression of VEGF.
Research reported in this publication was supported by the National Center for Complementary and Alternative Medicine of the National Institutes of Health under Award Number R01AT004922. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We also acknowledge funding provided to Johnny Huard through the Henry J Mankin endowed chair at the University of Pittsburgh, the William F. and Jean W Donaldson endowed chair at the Children's Hospital of Pittsburgh and the Department of Defense (W81XWH-06-1-0406).
Contributors JH and TMB conceived the idea of the article. JH prepared the part on muscle injury, VEGF and muscle-derived stem cells. TMB prepared part of the review regarding massage therapy. BG prepared the first draft and did the literature search to update the citations. TMB, BG and JH prepared the final draft in tandem. BG formatted the manuscript and submitted to BJSM.
Funding Government Agency. National Institutes of Health.
Competing interest JH receives remuneration as a consultant and royalties from Cook MyoSite, Inc. The other authors declare no conflict of interest.
Provenance and peer review Not commissioned; externally peer reviewed.