Muscle injuries and repair: the role of prostaglandins and inflammation

Skeletal muscle injuries are a common problem in trauma and orthopaedic surgery. Muscle injuries undergo the healing phases of degeneration, inflammation, regeneration, and fibrosis. Current and experimental therapies to improve muscle regeneration and limit muscle fibrosis include conservative and surgical principles with the adjuvant use of non-steroidal anti-inflammatory drugs (NSAIDs) and growth factor manipulation. NSAIDs appear to have a paradoxical effect on the healing of muscle injuries with early signs of improvement and subsequent late impairment in functional capacity and histology. In vitro and in vivo studies have explored the role of the cyclooxygenases and prostaglandins in the biological processes of healing muscle, including precursor cell activation, myoblast proliferation, myoblast fusion, and muscle protein synthesis. Through use of more specific cyclooxygenase inhibitors, we may be able to better understand the role of inflammation in muscle healing.     

Nitric oxide and repair of skeletal muscle injury

The muscle wound healing occurs in three overlapping phases: (1) degeneration and inflammation, (2) muscle regeneration, and (3) fibrosis. Simultaneously to injury cellular infiltration by neutrophils and macrophages occur, as well as cellular ‘respiratory burst’ via activation of the enzyme NADPH oxidase. When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle, divide, differentiate and fuse with muscle fibers to repair damaged regions and to enhance hypertrophy of muscle fibers. This process depends on nitric oxide (NO) production, metalloproteinase (MMP) activation and release of hepatocyte growth factor (HGF) from the extracellular matrix. Generation of a fibrotic scar tissue, with partial loss of function, can also occur, and seems to be dependent, at least in part, on local TGF-β expression, which can be downregulated by NO. Hence, regeneration the muscle depends on the type and severity of the injury, the appropriate inflammatory response and on the balance of the processes of remodeling and fibrosis. It appears that in all these phases NO exerts a significant role. Better comprehension of this role, as well as of the participation of other important mediators, may lead to development of new treatment strategies trying to tip the balance in favor of greater regeneration over fibrosis, resulting in better functional recovery.     

Role of integrin-β3 protein in macrophage polarization and regeneration of injured muscle

Following injury, skeletal muscle achieves repair by a highly coordinated, dynamic process resulting from interplay among numerous inflammatory, growth factors and myogenic regulators. To identify genes involved in muscle regeneration, we used a microarray analysis; there was a significant increase in the expression of a group of integrin genes. To verify these results, we used RT-PCR and Western blotting and found that 12 integrins were up-regulated from 3 h to 15 days following injury. Following muscle injury, integrin-β3 was initially expressed, mainly in macrophages. In integrin-β3 global KO mice, the expression of myogenic genes was decreased and muscle regeneration was impaired, whereas fibrosis was enhanced versus events in wild type (WT) mice. The mechanism for these responses in integrin-β3 KO mice included an infiltration of macrophages that were polarized into the M2 phenotype. These macrophages produced more TGF-β1 and increased TGF-β1/Smad signaling. In vitro, we confirmed that M2 macrophages lacking integrin-β3 produced more TGF-β1. Furthermore, transplantation of bone marrow cells from integrin-β3 KO mice into WT mice led to suppression of the infiltration and accumulation of macrophages into injured muscles. There was also impaired muscle regeneration with an increase in muscle fibrosis. Our results demonstrate that integrin-β3 plays a fundamental role in muscle regeneration through a regulation of macrophage infiltration and polarization leading to suppressed TGF-β1 production. This promotes efficient muscle regeneration. Thus, an improvement in integrin-β3 function could stimulate muscle regeneration.     

Is it possible to predict the outcome of peripheral nerve injuries? A probability model based on prospects for regenerating neurites

The long term functional consequences of peripheral nerve injuries are notoriously unpredictable. We hypothesized that considering the individual regrowth of the elementary components of a nerve (the neurites) rather than the global regeneration of the organ could lead to a better understanding of the mechanisms of nerve repair. Our basic assumptions were that regrowth was random and regrowth of any individual neurite could be defined in terms of its influence on recovery; this influence could be either valid, neutral or invalid. We designed a probability model describing the prospects of regrowth for nerves composed of several types of fibers. Our goal was to propose a general explanation of nerve healing rather than to predict the outcome in individual situations. The results suggested that this model satisfactorily described the events taking place in a healing nerve.     

Agent-based model provides insight into the mechanisms behind failed regeneration following volumetric muscle loss injury

Skeletal muscle possesses a remarkable capacity for repair and regeneration following a variety of injuries. When successful, this highly orchestrated regenerative process requires the contribution of several muscle resident cell populations including satellite stem cells (SSCs), fibroblasts, macrophages and vascular cells. However, volumetric muscle loss injuries (VML) involve simultaneous destruction of multiple tissue components (e.g., as a result of battlefield injuries or vehicular accidents) and are so extensive that they exceed the intrinsic capability for scarless wound healing and result in permanent cosmetic and functional deficits. In this scenario, the regenerative process fails and is dominated by an unproductive inflammatory response and accompanying fibrosis. The failure of current regenerative therapeutics to completely restore functional muscle tissue is not surprising considering the incomplete understanding of the cellular mechanisms that drive the regeneration response in the setting of VML injury. To begin to address this profound knowledge gap, we developed an agent-based model to predict the tissue remodeling response following surgical creation of a VML injury. Once the model was able to recapitulate key aspects of the tissue remodeling response in the absence of repair, we validated the model by simulating the tissue remodeling response to VML injury following implantation of either a decellularized extracellular matrix scaffold or a minced muscle graft. The model suggested that the SSC microenvironment and absence of pro-differentiation SSC signals were the most important aspects of failed muscle regeneration in VML injuries. The major implication of this work is that agent-based models may provide a much-needed predictive tool to optimize the design of new therapies, and thereby, accelerate the clinical translation of regenerative therapeutics for VML injuries.     

Functional analysis of limb recovery following autograft treatment of volumetric muscle loss in the quadriceps femoris

Severe injuries to the extremities often result in muscle trauma and, in some cases, significant volumetric muscle loss (VML). These injuries continue to be challenging to treat, with few available clinical options, a high rate of complications, and often persistent loss of limb function. To facilitate the testing of regenerative strategies for skeletal muscle, we developed a novel quadriceps VML model in the rat, specifically addressing functional recovery of the limb. Our outcome measures included muscle contractility measurements to assess muscle function and gait analysis for evaluation of overall limb function. We also investigated treatment with muscle autografts, whole or minced, to promote regeneration of the defect area. Our defect model resulted in a loss of muscle function, with injured legs generating less than 55% of muscle strength from the contralateral uninjured control legs, even at 4 weeks post-injury. The autograft treatments did not result in significant recovery of muscle function. Measures of static and dynamic gait were significantly decreased in the untreated, empty defect group, indicating a decrease in limb function. Histological sections of the affected muscles showed extensive fibrosis, suggesting that this scarring of the muscle may be in part the cause of the loss of muscle function in this VML model. Taken together, these data are consistent with clinical findings of reduced muscle function in large VML injuries. This new model with quantitative functional outcome measures offers a platform on which to evaluate treatment strategies designed to regenerate muscle tissue volume and restore limb function.     

Lentiviral-mediated transfer of CDNF promotes nerve regeneration and functional recovery after sciatic nerve injury in adult rats

Peripheral nerve injury is often followed by incomplete and unsatisfactory functional recovery and may be associated with sensory and motor impairment of the affected limb. Therefore, a novel method is needed to improve the speed of recovery and the final functional outcome after peripheral nerve injuries. This report investigates the effect of lentiviral-mediated transfer of conserved dopamine neurotrophic factor (CDNF) on regeneration of the rat peripheral nerve in a transection model in vivo. We observed notable overexpression of CDNF protein in the distal sciatic nerve after recombinant CDNF lentiviral vector application. We evaluated sciatic nerve regeneration after surgery using light and electron microscopy and the functional recovery using the sciatic functional index and target muscle weight. HE staining revealed better ordered structured in the CDNF-treated group at 8 weeks post-surgery. Quantitative analysis of immunohistochemistry of NF200 and S-100 in the CDNF group revealed significant improvement of axonal and Schwann cell regeneration compared with the control groups at 4 weeks and 8 weeks after injury. The thickness of the myelination around the axons in the CDNF group was significantly higher than in the control groups at 8 weeks post-surgery. The CDNF group displayed higher muscle weights and significantly increased sciatic nerve index values. Our findings suggest that CDNF gene therapy could provide durable and stable CDNF protein concentration and has the potential to enhance peripheral nerve regeneration, morphological and functional recovery following nerve injury, which suggests a promising strategy for peripheral nerve repair.     

Collagen scaffolds modified with collagen-binding bFGF promotes the neural regeneration in a rat hemisected spinal cord injury model

Nerve conduit is one of strategies for spine cord injury(SCI)treatment.Recently,studies showed that biomaterials could guide the neurite growth and promote axon regeneration at the injury site.However,the scaffold by itself was difficult to meet the need of SCI functional recovery.The basic fibroblast growth factor(bFGF)administration significantly promotes functional recovery after organ injuries.Here,using a rat model of T9 hemisected SCI,we aimed at assessing the repair capacity of implantation of collagen scaffold(CS)modified by collagen binding bFGF(CBD-bFGF).The results showed that CS combined with CBD-bFGF treatment improved survival rates after the lateral hemisection SCI.The CS/CBD-bFGF group showed more significant improvements in motor than the simply CS-implanted and untreated control group,when evaluated by the 21-point Basso-Beattie-Bresnahan(BBB)score and footprint analysis.Both hematoxylin and eosin(H&E)and immunohistochemical staining of neurofilament(NF)and glial fibrillary acidic protein(GFAP)demonstrated that fibers were guided to grow through the implants.These findings indicated that administration of CS modified with CBD-bFGF could promote spinal cord regeneration and functional recovery.     

Biological approaches to improve skeletal muscle healing after injury and disease

Skeletal muscle injury and repair are complex processes, including well‐coordinated steps of degeneration, inflammation, regeneration, and fibrosis. We have reviewed the recent literature including studies by our group that describe how to modulate the processes of skeletal muscle repair and regeneration. Antiinflammatory drugs that target cyclooxygenase‐2 were found to hamper the skeletal muscle repair process. Muscle regeneration phase can be aided by growth factors, including insulin‐like growth factor‐1 and nerve growth factor, but these factors are typically short‐lived, and thus more effective methods of delivery are needed. Skeletal muscle damage caused by traumatic injury or genetic diseases can benefit from cell therapy; however, the majority of transplanted muscle cells (myoblasts) are unable to survive the immune response and hypoxic conditions. Our group has isolated neonatal skeletal muscle derived stem cells (MDSCs) that appear to repair muscle tissue in a more effective manner than myoblasts, most likely due to their better resistance to oxidative stress. Enhancing antioxidant levels of MDSCs led to improved regenerative potential. It is becoming increasingly clear that stem cells tissue repair by direct differentiation and paracrine effects leading to neovascularization of injured site and chemoattraction of host cells. The factors invoked in paracrine action are still under investigation. Our group has found that angiotensin II receptor blocker (losartan) significantly reduces fibrotic tissue formation and improves repair of murine injured muscle. Based on these data, we have conducted a case study on two hamstring injury patients and found that losartan treatment was well tolerated and possibly improved recovery time. We believe this medication holds great promise to optimize muscle repair in humans. (Part C) 96:82–94, 2012. © 2012 Wiley Periodicals, Inc.     

MALDI imaging mass spectrometry: Discrimination of pathophysiological regions in traumatized skeletal muscle by characteristic peptide signatures

Due to formation of fibrosis and the loss of contractile muscle tissue, severe muscle injuries often result in insufficient healing marked by a significant reduction of muscle force and motor activity. Our previous studies demonstrated that the local transplantation of mesenchymal stromal cells into an injured skeletal muscle of the rat improves the functional outcome of the healing process. Since, due to the lack of sufficient markers, the accurate discrimination of pathophysiological regions in injured skeletal muscle is inadequate, underlying mechanisms of the beneficial effects of mesenchymal stromal cell transplantation on primary trauma and trauma adjacent muscle area remain elusive. For discrimination of these pathophysiological regions, formalin‐fixed injured skeletal muscle tissue was analyzed by MALDI imaging MS. By using two computational evaluation strategies, a supervised approach (ClinProTools) and unsupervised segmentation (SCiLS Lab), characteristic m/z species could be assigned to primary trauma and trauma adjacent muscle regions. Using “bottom‐up” MS for protein identification and validation of results by immunohistochemistry, we could identify two proteins, skeletal muscle alpha actin and carbonic anhydrase III, which discriminate between the secondary damage on adjacent tissue and the primary traumatized muscle area. Our results underscore the high potential of MALDI imaging MS to describe the spatial characteristics of pathophysiological changes in muscle.     

Asynchronous remodeling is a driver of failed regeneration in Duchenne muscular dystrophy

We sought to determine the mechanisms underlying failure of muscle regeneration that is observed in dystrophic muscle through hypothesis generation using muscle profiling data (human dystrophy and murine regeneration). We found that transforming growth factor β–centered networks strongly associated with pathological fibrosis and failed regeneration were also induced during normal regeneration but at distinct time points. We hypothesized that asynchronously regenerating microenvironments are an underlying driver of fibrosis and failed regeneration. We validated this hypothesis using an experimental model of focal asynchronous bouts of muscle regeneration in wild-type (WT) mice. A chronic inflammatory state and reduced mitochondrial oxidative capacity are observed in bouts separated by 4 d, whereas a chronic profibrotic state was seen in bouts separated by 10 d. Treatment of asynchronously remodeling WT muscle with either prednisone or VBP15 mitigated the molecular phenotype. Our asynchronous regeneration model for pathological fibrosis and muscle wasting in the muscular dystrophies is likely generalizable to tissue failure in chronic inflammatory states in other regenerative tissues.     

TGF-beta1 is a negative regulator of lymphatic regeneration during wound repair

Although clinical studies have identified scarring/fibrosis as significant risk factors for lymphedema, the mechanisms by which lymphatic repair is impaired remain unknown. Transforming growth factor -beta1 (TGF-beta1) is a critical regulator of tissue fibrosis/scarring and may therefore also play a role in the regulation of lymphatic regeneration. The purpose of this study was therefore to assess the role of TGF-beta1 on scarring/fibrosis and lymphatic regeneration in a mouse tail model. Acute lymphedema was induced in mouse tails by full-thickness skin excision and lymphatic ligation. TGF-beta1 expression and scarring were modulated by repairing the wounds with or without a topical collagen gel. Lymphatic function and histological analyses were performed at various time points. Finally, the effects of TGF-beta1 on lymphatic endothelial cells (LECs) in vitro were evaluated. As a result, the wound repair with collagen gel significantly reduced the expression of TGF-beta1, decreased scarring/fibrosis, and significantly accelerated lymphatic regeneration. The addition of recombinant TGF-beta1 to the collagen gel negated these effects. The improved lymphatic regeneration secondary to TGF-beta1 inhibition was associated with increased infiltration and proliferation of LECs and macrophages. TGF-beta1 caused a dose-dependent significant decrease in cellular proliferation and tubule formation of isolated LECs without changes in the expression of VEGF-C/D. Finally, the increased expression of TGF-beta1 during wound repair resulted in lymphatic fibrosis and the coexpression of alpha-smooth muscle actin and lymphatic vessel endothelial receptor-1 in regenerated lymphatics. In conclusion, the inhibition of TGF-beta1 expression significantly accelerates lymphatic regeneration during wound healing. An increased TGF-beta1 expression inhibits LEC proliferation and function and promotes lymphatic fibrosis. These findings imply that the clinical interventions that diminish TGF-beta1 expression may be useful in promoting more rapid lymphatic regeneration.     

Antagonistic regulation of transmembrane 4 L6 family member 5 attenuates fibrotic phenotypes in CCl(4) -treated mice

The development of liver fibrosis from chronic inflammation can involve epithelial-mesenchymal transition (EMT). Severe liver fibrosis can progress to cirrhosis, and further to hepatocellular carcinoma. Because the tetraspanin transmembrane 4 L6 family member 5 (TM4SF5) induces EMT and is highly expressed in hepatocellular carcinoma, it is of interest to investigate whether TM4SF5 expression is correlated with EMT processes during the development of fibrotic liver features. Using hepatic cells in vitro and a CCl(4) -mediated mouse liver in?vivo model, we examined whether TM4SF5 is expressed during liver fibrosis mediated by CCl(4) administration and whether treatment with anti-TM4SF5 reagent blocks the fibrotic liver features. Here, we found that TM4SF5 expression was induced by the transforming growth factor (TGF)β1 and epidermal growth factor signaling pathways in hepatocytes in vitro. In the CCl(4) -mediated mouse liver model, TM4SF5 was expressed during the liver fibrosis mediated by CCl(4) administration and correlated with α-smooth muscle actin expression, collagen I deposition, and TGFβ1 and epidermal growth factor receptor signaling activation in fibrotic septa regions. Interestingly, treatment with anti-TM4SF5 reagent blocked the TM4SF5-mediated liver fibrotic features: the formation of fibrotic septa with α-smooth muscle actin expression and collagen I deposition was attenuated by treatment with anti-TM4SF5 reagent. These results suggest that TM4SF5 expression mediated by TGFβ1 and growth factor can facilitate fibrotic processes during chronic liver injuries. TM4SF5 is thus a candidate target for prevention of liver fibrosis following chronic liver injury.     

Macrophage-specific expression of urokinase-type plasminogen activator promotes skeletal muscle regeneration

Macrophages (Mp) and the plasminogen system play important roles in tissue repair following injury. We hypothesized that Mp-specific expression of urokinase-type plasminogen activator (uPA) is sufficient for Mp to migrate into damaged muscle and for efficient muscle regeneration. We generated transgenic mice expressing uPA only in Mp, and we assessed the ability of these mice to repair muscle injury. Mp-only uPA expression was sufficient to induce wild-type levels of Mp accumulation, angiogenesis, and new muscle fiber formation. In mice with wild-type uPA expression, Mp-specific overexpression further increased Mp accumulation and enhanced muscle fiber regeneration. Furthermore, Mp expression of uPA regulated the level of active hepatocyte growth factor, which is required for muscle fiber regeneration, in damaged muscle. In vitro studies demonstrated that uPA promotes Mp migration through proteolytic and nonproteolytic mechanisms, including proteolytic activation of hepatocyte growth factor. In summary, Mp-derived uPA promotes muscle regeneration by inducing Mp migration, angiogenesis, and myogenesis.     

Platelet-Rich Plasma,Especially When Combined with a TGF-β Inhibitor Promotes Proliferation,Viability and Myogenic Differentiation of Myoblasts In Vitro

Regeneration of skeletal muscle after injury is limited by scar formation, slow healing time and a high recurrence rate. A therapy based on platelet-rich plasma (PRP) has become a promising lead for tendon and ligament injuries in recent years, however concerns have been raised that PRP-derived TGF-β could contribute to fibrotic remodelling in skeletal muscle after injury. Due to the lack of scientific grounds for a PRP -based muscle regeneration therapy, we have designed a study using human myogenic progenitors and evaluated the potential of PRP alone and in combination with decorin (a TGF-β inhibitor), to alter myoblast proliferation, metabolic activity, cytokine profile and expression of myogenic regulatory factors (MRFs). Advanced imaging multicolor single-cell analysis enabled us to create a valuable picture on the ratio of quiescent, activated and terminally committed myoblasts in treated versus control cell populations. Finally high-resolution confocal microscopy validated the potential of PRP and decorin to stimulate the formation of polynucleated myotubules. PRP was shown to down-regulate fibrotic cytokines, increase cell viability and proliferation, enhance the expression of MRFs, and contribute to a significant myogenic shift during differentiation. When combined with decorin further synergistc effects were identified. These results suggest that PRP could not only prevent fibrosis but could also stimulate muscle commitment, especially when combined with a TGF-β inhibitor.     

Regulation of matrix metalloproteinase-1 and alpha-smooth muscle actin expression by interleukin-1 alpha and tumour necrosis factor alpha in hepatic stellate cells

Hepatic stellate cells (HSCs) are key players in liver fibrosis and regeneration via collagen degradation and synthesis. These phenomena involve inflammatory cytokines released from non-parenchymal liver cells such as Kupffer cells. Although the effects of individual cytokines on many cell types have been investigated in various conditions, such as inflammation and tissue fibrosis, investigating the effect of combined cytokines would further our understanding of the regulatory mechanisms in tissue fibrosis. Here, we report the effect of multiple cytokine combinations on primary HSCs. We first examined the effect of individual cytokines and then the simultaneous exposure of different cytokines, including interleukin-6 (IL-6), IL-1 alpha (IL-1α), platelet-derived growth factor (PDGF), tumour necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β), on matrix metalloproteinase-1 (MMP1) gene expression in primary HSCs. We observed that the combination of all five cytokines induced higher levels of MMP1 gene expression. Of these cytokines, TNF-α and IL-1α were found to be the key cytokines for not only inducing MMP1 expression, but also increasing α-smooth muscle actin gene expression. In conclusion, the combined treatment of TNF-α and IL-1α on HSCs had an enhanced effect on the expression of the fibrotic genes, MMP1 and α-smooth muscle actin, so appears to be an important regulator for tissue regeneration. This finding suggests that stimulation with combined anti-fibrotic cytokines is a potential approach in the development of a novel therapy for the recovery of liver fibrosis.