Aims. Glucose-insulin-potassium (GIK) is protective following cardiac myocyte ischaemia-reperfusion (IR) injury, however the role of GIK in protecting skeletal muscle from IR injury has not been evaluated. Given the similar mechanisms by which cardiac and skeletal muscle sustain an IR injury, we hypothesized that GIK would similarly protect skeletal muscle viability. Methods. A total of 20 C57BL/6 male mice (10 control, 10 GIK) sustained a hindlimb IR injury using a 2.5-hour rubber band tourniquet. Immediately prior to tourniquet placement, a subcutaneous osmotic pump was placed which infused control mice with saline (0.9% sodium chloride) and treated mice with GIK (40% glucose, 50 U/l insulin, 80 mEq/L KCl, pH 4.5) at a rate of 16 µl/hr for 26.5 hours. At 24 hours following tourniquet removal, bilateral (tourniqueted and non-tourniqueted) gastrocnemius muscles were triphenyltetrazolium chloride (TTC)-stained to quantify percentage muscle viability. Bilateral peroneal muscles were used for gene expression analysis, serum creatinine and creatine kinase activity were measured, and a validated murine ethogram was used to quantify pain before euthanasia. Results. GIK treatment resulted in a significant protection of skeletal muscle with increased viability (GIK 22.07% (SD 15.48%)) compared to saline control (control 3.14% (SD 3.29%)) (p = 0.005). Additionally, GIK led to a statistically significant reduction in gene expression markers of cell death (CASP3, p < 0.001) and inflammation (NOS2, p < 0.001; IGF1, p = 0.007; IL-1β, p = 0.002; TNFα, p = 0.012), and a significant reduction in serum creatine kinase (p = 0.004) and creatinine (p < 0.001). GIK led to a significant reduction in IR-related pain (p = 0.030). Conclusion. Systemic GIK infusion during and after limb ischaemia protects murine skeletal muscle from cell death, kidneys from reperfusion metabolites, and reduces pain by reducing post-ischaemic inflammation. Cite this article: Bone Joint Res 2023;12(3):212–218

Abstract. Introduction. Skeletal muscle wasting is an important clinical issue following acute traumatic injury, and can delay recovery and cause permanent functional disability particularly in the elderly. However, the fundamental mechanisms involved in trauma-induced muscle wasting remain poorly defined and therapeutic interventions are limited. Objectives. To characterise local and systemic mediators of skeletal muscle wasting in elderly patients following acute trauma. Methods. Experiments were approved by a local NHS Research Ethics Committee and all participants provided written informed consent. Vastus lateralis biopsies and serum samples were taken from human male and female patients shortly after acute trauma injury in lower limbs (n=6; mean age 78.7±4.4 y) and compared to age-matched controls (n=6; mean age 72.6±6.3 y). Atrogenes and upstream regulators (MuRF1; MAFbx; IL6, TNFα, PGC-1α) mRNA expression was assessed in muscle samples via RT-qPCR. Serum profiling of inflammatory markers (e.g. IL6, TNFα, IL1β) was further performed via multiplex assays. To determine whether systemic factors induced by trauma directly affect muscle phenotype, differentiated primary human myotubes were treated in vitro with serum from controls or trauma patients (pooled; n=3 each) in the final 24 hours of differentiation. Cells were then fixed, stained for myogenin and imaged to determine minimum ferret diameter. Statistical significance was determined at P<0.05. Results. There was an increase in skeletal muscle mRNA expression for E3 ligase MAFbx and inflammatory cytokine IL-6 (4.6 and 21.5-fold respectively; P<0.05) in trauma patients compared to controls. Expression of myogenic determination factor MyoD and regulator of mitochondrial biogenesis PGC-1α was lower in muscle of trauma patients vs controls (0.5 and 0.39-fold respectively; P<0.05). In serum, trauma patients showed increased concentrations of circulating pro-inflammatory cytokines IL-6 (14.5 vs. 0.3 pg/ml; P<0.05) and IL-16 (182.7 vs. 85.2 pg/ml; P<0.05) compared to controls. Primary myotube experiments revealed serum from trauma patients induced atrophy (32% decrease in diameter) compared to control serum-treated cells (P<0.001). Conclusion. Skeletal muscle from patients following acute trauma injury showed greater expression of atrophy and inflammatory markers. Trauma patient serum exhibited higher circulating pro-inflammatory cytokine concentrations. Primary human myotubes treated with serum from trauma patients showed significant atrophy compared to healthy serum-treated controls. We speculate a mechanism(s) acting via circulating factors may contribute to skeletal muscle pathology following acute trauma. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Osteoarthritis (OA) of the knee joint is a complex peripheral joint disorder with multiple risk factors. We aimed to examine the relationship between the grade of knee OA and anterior thigh length (ATL). A total of 64 geriatric patients who had no total hip or knee replacement with a BMI of ≥30 were evaluated. Patients' OA severity was determined by two independent experts from bilateral standing knee radiographs according to the Kellgren-Lawrence (KL) grade. Joint cartilage structure was assessed using ultrasonography (US). The ATL, the gastrocnemius medialis (GC), the rectus femoris (RF) and the rectus abdominis (RA) skeletal muscle thicknesses as well as the RF cross-sectional area (CSA) were measured with US. Sarcopenia was diagnosed using the handgrip strength (HGS), 5× sit-to-stand test (5xSST) and bioelectrical impedance analysis. The median (IQR) age of participants was 72 (65–88) years. Seventy-one per cent of the patients (n=46) were female. They were divided into the sarcopenic obese (31.3 %) and the non-sarcopenic obese (68.8%) groups. KL grade of all patients correlated negatively with the ATL (mm) and the thickness of GC (mm) (r= -0,517, p<0.001 and r= -0.456, p<0.001, respectively). In the sarcopenic obese and the non-sarcopenic obese groups, KL grade of the all patients was negatively correlated with ATL (mm) and thickness of GC (mm) (r= -0,986, p<0.001; r= -0.456, p=0.05 and r= -0,812, p=0.002; r= −0,427, p=0.006). KL grade negatively correlated with the RF thickness in the sarcopenic obese group (r= -0,928, p=0.008). In conclusion, OA risk may decrease as the lower extremity skeletal muscle mass increases. Acknowledgments: Feza Korkusuz MD is a member of the Turkish Academy of Sciences (TÜBA)

Abstract. Objectives. Acute compartment syndrome (ACS) is a progressive form of muscle ischaemia that is a surgical emergency and can have detrimental outcomes for patients if not treated optimally. The current problem is that there is no clear diagnostic threshold for ACS or guidance as to when fasciotomies should be performed. A new diagnostic method(s) is necessary to provide real-time information about the extent of muscle ischaemia in ACS. Given that lactic acid is produced by cells through anaerobic respiration, it may be possible to measure H+ ion concentration and to use this as a measure of ischaemia within muscle. Although we are familiar with the key biochemical metabolites involved in ischaemia; and the use of viability dyes in cell culture to distinguish between living or dead cells is well recognised; research has not been undertaken to correlate the biochemical and histological findings of ischaemia in skeletal muscle biopsies. Our primary aim was to investigate the potential for viability dyes to be used on live skeletal muscle biopsies (explants). Our secondary aim was to correlate the intramuscular pH readings with muscle biopsy viability. Methods. Nine euthanised Wistar rats were used. A pH catheter was inserted into one exposed gluteus medius muscles to record real-time pH levels and muscle biopsies were taken from the contralateral gluteus medius at the start of experiment and subsequently at every 0.1 of pH unit drop. Prior to muscle biopsy, the surface of the gluteus medius was painted with a layer of 50µmol/l Brilliant blue FCF solution to facilitate biopsy orientation. A 4mm punch biopsy tool was used to take biopsies. Each muscle biopsy was placed in a base mould filled with 4% ultra-low melting point agarose. The agarose embedded tissue block was sectioned to generate 400 micron thick tissue slices with a vibratome. The tissue slices were then placed in the staining solution with Hoechst 33342, Ethidium homodimer-1 and Calcein am. The tissue slices were imaged with Zeiss LSM880 confocal microscope's Z stack function. A dead muscle control was created by adding TritonX-100 to other tissue slices. For quantitative analyses, the images were analysed in Image J using the selection tool. This permitted individual cells to be identified and the mean grey value of each channel to be defined. Using the dead control, we were able to identify the threshold value for living cells using the Calcein AM channel. Results. Viability dyes, used primarily for cell cultures, can be used with skeletal muscle explants. Our study also showed that despite a significant reduction in tissue pH concentration over time, that almost 100% of muscle cells were still viable at pH 6.0, suggesting that skeletal muscle cells are robust to hypoxic insult in the absence of reperfusion. Conclusions. Viability dyes can be used on skeletal muscle biopsies. Further research investigating the likely associations between direct measured pH using a pH catheter, the concentrations of key cellular metabolic markers, and muscle tissue histology using vitality dyes in response to ischaemia, rather than hypoxia, is warranted. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Scar tissue formation secondary to acute muscle injury, surgical wounding and compartment syndrome can result in significant functional impairment and predispose to further injury. The source of fibroblasts, and the molecular mechanisms driving their activation and persistence in skeletal muscle fibrosis are not known. We hypothesized that cells expressing PDGFRβ become fibroblasts in response to injury and that targeting αv integrins in these cells reduces skeletal muscle fibrosis. We used double-fluorescent reporter mice to demonstrate that cells expressing PDGFRβ become activated myofibroblasts in response to cardiotoxin (CTX) induced skeletal muscle injury. Following injury, PDGFRβ+ cells moved from perivascular locations into the interstitium in a distribution characteristic of fibroblasts, and showed marked induction of fibroblastic genes including αSMA and collagen1 (all p<0.0001). To confirm that αv integrins present on PDGFRβ cells critically regulate skeletal muscle fibrosis we used Itgavflox/flox;PDGFRβ-Cre mice (transgenic mice in which αv integrins are ‘knocked-down’ in PDGFRβ+ cells). These mice were significantly protected from CTX induced fibrosis (p<0.01). To demonstrate potential clinical utility of targeting αv integrins, we used a small molecule inhibitor of αv integrins (CWHM12). Treatment with CWHM12 significantly reduced fibrosis when delivered from the time of injury (p<0.01) and when delivered after the fibrotic response had become established (p<0.01). We have identified a core pathway regulating fibrosis in skeletal muscle. Pharmacologic inhibition of αv integrins has potential clinical utility in the treatment and prevention of skeletal muscle fibrosis

Skeletal muscle tissue engineering has made progress towards production of functional tissues in line with the development in materials science and fabrication techniques. In particular, combining the specificity of 3D printing with smart materials has introduced a new concept called the 4D printing. Inspired by the unique properties of smart/responsive materials, we designed a bioink made of gelatin, a polymer with well-known cell compatibility, to be 3D printed on a magnetically responsive substrate. Gelatin was made photocrosslinkable by the methacrylate reaction (GELMA), and its viscosity was finetuned by blending with alginate which was later removed by alginate lyase treatment, so that the printability of the bioink as well as the cell viability can be finetuned. C2C12 mouse myoblasts-laden bioink was then 3D printed on a magnetic substrate for 4D shape-shifting. The magnetic substrate was produced using silicon rubber (EcoFlex) and carbonyl iron powders. After 3D printing, the bioink was crosslinked on the substrate, and the substrate was rolled with the help of a permanent magnet. Unrolled (Open) samples were used as the control group. The stiffness of the bioink matrix was found to be in the range of 13–45 kPa, which is the appropriate value for the adhesion of C2C12 cells. In the cell viability analysis, it was observed that the cells survived and could proliferate within the 7-day duration of the experiment. As a result of the immunofluorescence test, compared to the Open Group, more cell nuclei were observed overlapping MyoD1 expression in the Rolled Group; this indicated that the cells in these samples had more cell-cell interactions and therefore tended to form more myotubes. Acknowledgements: This research was supported by the TÜBİTAK 2211-A and YÖK 100/2000 scholarship programs

Background. There are currently no effective treatments for skeletal muscle fibrosis. Myofibroblasts are the major cellular effectors of fibrosis but their origin in muscle is unknown. We report that PDGFRβ (platelet derived growth factor receptor beta) Cre inactivates genes in murine PDGFRβ+ cells and myofibroblasts in muscle with high efficiency. We used this system to delete the integrin αv subunit because of the suggested role of multiple αv integrins as central mediators of fibrosis in multiple organs. Methods. Muscle fibrosis was induced by intramuscular cardiotoxin (CTX) injection. The contribution of PDGFRβ+ cells to fibrosis was assessed in double-flourescent reporter (mTmG) mice under PDGFRβ-Cre control. Itgavflox/flox;PDGFRβ-Cre mice were used to investigate whether loss of αv integrins on PDGFRβ+ cells influences fibrosis development. A small-molecule inhibitor of αv integrins (CWHM12) was used to determine whether pharmacological blockade of αv integrins could attenuate fibrosis. Results. At 21 days following injury PDGFRβ+ cells in mTmG;PDGFRβ-Cre mice were distributed in a manner characteristic of myofibroblasts. PDGFRβ+ cells sorted from injured muscles of mTmG;PDGFRβ-Cre mice showed induction of genes associated with myofibroblastic transition. Itgavflox/flox;PDGFRβ-Cre mice were protected from CTX induced fibrosis, as determined by picrosirius red staining for collagen (p<0.01). Sorted and culture activated αv-null PDGFRβ+ cells demonstrated significant reduction in collagen1 over controls (p<0.05). CWHM12 significantly reduced muscle fibrosis when delivered from the time of injury (prophylactic model: p<0.01) and from day 10 post injury (therapeutic model: p<0.01). Furthermore, CWHM12 inhibited collagen1 expression by PDGFRβ+ cells ex-vivo (p<0.05). Conclusions. PDGFRβ-Cre labels profibrotic cells in skeletal muscle and depletion of αv integrins in these cells reduces muscle fibrosis. Most importantly from a treatment standpoint, pharmacologic inhibition of αv integrins using a small molecule inhibitor may have utility in the prevention and treatment of skeletal muscle fibrosis. Level of Evidence. Basic Science

Objectives. Ischaemic preconditioning (IPC) is a phenomenon whereby tissues develop an increased tolerance to ischaemia and subsequent reperfusion if first subjected to sublethal periods of ischaemia. Despite extensive investigation of IPC, the molecular mechanism remains largely unknown. Our aim was to show genetic changes that occur in skeletal muscle cells in response to IPC. Methods. We established an in-vitro model of IPC using a human skeletal muscle cell line. Gene expression of both control and preconditioned cells at various time points was determined. The genes examined were HIF-1?, EGR1, JUN, FOS, and DUSP1. HIF-1? is a marker of hypoxia. EGR1, JUN, FOS and DUSP1 are early response genes and may play a role in the protective responses induced by IPC. Secondly, the expression of HSP22 was examined in a cohort of preconditioned total knee arthroplasty patients. Results. HIF-1? was upregulated following 1 and 2 hours of simulated ischaemia (p = 0.076 and 0.841 respectively) verifying that hypoxic conditions were met. Expression of EGR1, FOS and DUSP1 were upregulated and peaked after 1 hour of hypoxia (p = 0.001, < 0.00, and 0.038 respectively). cFOS was upregulated at 2 and 3 hours of hypoxia. IPC prior to simulated hypoxia resulted in a greater level of upregulation of EGR1, JUN and FOS genes (p = < 0.00, 0.047, and < 0.00 respectively). HSP22 was not significantly upregulated following IPC using the hypoxic model. It was, however, upregulated on an mRNA level in total knee arthroplasty patients (p = 0.15). Conclusion. This study has validated the use of our hypoxic model for studying IPC in-vitro. IPC results in a greater upregulation of protective genes in skeletal muscle cells exposed to hypoxia than in control cells. We have demonstrated hitherto unknown molecular mechanisms of IPC in cell culture and in patients undergoing TKA

OBJECTIVES. Ischaemic preconditioning (IPC) is a phenomenon whereby tissues develop an increased tolerance to ischaemia and subsequent reperfusion if first subjected to sublethal periods of ischaemia. Despite extensive investigation of IPC, the molecular mechanism remains largely unknown. Our aim was to show genetic changes that occur in skeletal muscle cells in response to IPC. METHODS. Firstly, we established an in-vitro model of IPC using a human skeletal muscle cell line. Gene expression of both control and preconditioned cells at various time points was determined. The genes examined were HIF-1 alpha, EGR1, JUN, FOS, and DUSP1. HIF-1 alpha is a marker of hypoxia. EGR1, JUN, FOS and DUSP1 are early response genes and may play a role in the protective responses induced by IPC. Secondly, the expression of HSPB8 was examined in a cohort of preconditioned total knee arthroplasty patients. RESULTS. HIF-1 alpha was upregulated following 1 and 2 hours of simulated ischaemia (p = 0.076 and 0.841 respectively) verifying that hypoxic conditions were met using our model. Expression of EGR1, FOS and DUSP1 were upregulated and peaked after 1 hour of hypoxia (p = 0.001, <0.00, and 0.038 respectively). cFOS was upregulated at 2 and 3 hours of hypoxia. IPC prior to simulated hypoxia resulted in a greater level of upregulation of EGR1, JUN and FOS genes (p = <0.00, 0.047, and <0.00 respectively). HSPB8 was not significantly upregulated following IPC using the hypoxic model. It was, however, upregulated on an mRNA level in total knee arthroplasty patients (p = 0.15). CONCLUSION. This study has validated the use of our hypoxic model for studying IPC in-vitro. IPC results in a greater upregulation of protective genes in skeletal muscle cells exposed to hypoxia than in control cells. We have demonstrated hitherto unknown molecular mechanisms of IPC in cell culture and in patients undergoing TKA

Aims: Pharmacological modulation of skeletal muscle reperfusion injury after trauma associated ischaemia may improve limb salvage rates and prevent the associated systemic sequelae. Resuscitation with hypertonic saline restores the circulating volume and has favourable effects on tissue perfusion and blood pressure. The purpose of our study was to evaluate the effects of hypertonic saline on skeletal muscle ischaemia reperfusion (I/R) injury and the associated endorgan injury. Methods: Adult male Sprague Dawley rats (n=24) were randomised into three groups: control group, I/R group treated with normal saline and I/R group treated with hypertonic saline. Bilateral hind-limb ischaemia was induced by rubber band application proximal to the level of the greater trochanters for 2.5 hours. Treatment groups received either normal saline or hypertonic saline prior to tourniquet release. Following twelve hours reperfusion, the tibialis anterior muscle was dissected and muscle function assessed electrophysiologically by electrical field stimulation. The animals were then sacrificed and skeletal muscle harvested for evaluation. Lung tissue was also harvested for measurement of wet-to-dry ratio, myeloperoxidase content and histological analysis. Results: Hypertonic saline significantly attenuated skeletal muscle reperfusion injury as shown by reduced twitch and tetanic contractions of the skeletal muscle (Table). There was also a significant reduction in lung injury as demonstrated by differences in wet-to-dry ratio, myeloperoxidase content and histological analysis. Conclusion: Resuscitation with hypertonic saline may have a protective role in attenuating skeletal muscle ischaemia reperfusion injury and its associated systemic sequelae

Introduction: Ischaemic preconditioning (IPC) is a well recognised and powerful phenomenon where a tissue becomes more tolerant to prolonged ischaemia when it is first subjected to short bursts of ischaemia/reperfusion. IPC has been most comprehensively studied in cardiothoracic surgery, to date there has been little use of this powerful phenomenon in orthopaedic surgery. In this study, we report on the first clinical trial of IPC on human skeletal muscle, and show the potential of IPC in orthopaedics using global gene expression analysis. Methods: After local ethics committee approval and informed consent, patients undergoing primary knee arthroplasty were randomly assigned into an IPC group and a control group. Diabetic patients or patients with an ankle/brachial index of less than 1 were excluded. The IPC consisted of three five-minute periods of tourniquet insufflation on the operative limb, interrupted by five minute periods of reperfusion. The tourniquet was again insufflated and the operation started. The control group simply had tourniquet insufflation as normal prior to the start of surgery. Muscle samples were taken from the operative knee of all patients at the immediate onset of surgery (t=0), and again, at one hour into the surgery (t=1). Total RNA was extracted from the muscle samples, and the gene expression profiles were determined using microarray technology. Results: Comparison of IPC and control samples identified 702 transcripts with differences of ≥1.5-fold in their expression. Of these, 137 were altered at t=0 while 565 were altered at t=1. Amongst these changes was an up-regulation in the expression of a number of heat shock proteins (HSPs) in the IPC group as compared to the control group. Notably, there was up-regulation of the well known cytoprotective/anti-apoptotic gene, HSP72, at one hour post IPC (1.5-fold, p=0.039). There was also up-regulation of important oxidative stress defense genes, such as glutathione-S-transferase (1.6-fold, p = 0.021) and superoxide dismutase 2 (3.6-fold, p= 0.048). Microarray analysis also revealed a down-regulation in the expression of genes involved in metabolism, down-regulation of pro-apoptotic genes and up-regulation of genes necessary for transformation to a hypoxia-tolerant state. Discussion: We present convincing evidence that IPC is beneficial to human skeletal muscle and for the first time show that IPC of human skeletal muscle works in the clinical setting. In this study, the protective effect of IPC involved a down-regulation in the expression of genes associated with metabolism, and an up-regulation in the expression of genes that provide protection from cell stress, oxidative stress and apoptosis. HSPs, and especially HSP72, have well documented roles in cell stress protection. Their presence has been cited by other studies as an indicator of cell adaptation to stress

Introduction: Reactive oxygen species (ROS) have important roles in the pathogenesis of ischemia reperfusion injury (I/R) of skeletal muscles Melatonin was proved to be an antioxidant agent and many experimental models showed that it reduces I/R injury in many tissues. The objective of present study was to detect protective antioxidant effect of melatonin on I/R injury of skeletal muscles. Material and Methods: Albimino wistar rats were randomly allocated into 3 groups. There were 8, 10, 10 rats in sham, I/R and I/R + melatonin (Mel) groups respectively. Right hind limb ischemia was achieved by clamping femoral arteries in all groups except for control group. Melatonin (10 mg/kg) was administered intraperitoneally in I/R + Mel group 48, 24, 1 hour before reperfusion. After a period of 2 hour ischemia followed by 1.5 hour reperfusion, muscles and venous blood samples were collected for biochemical analysis and histopathological examination. Plasma antioksidant enzyme activities of süperoxide dismutase (SOD), glutathion peroxidase (GSH-Px), and levels of MDA and NO. were investigated. Enzyme activities of catalase (CAT), protein carbonyl (PC), SOD, GSH-Px and levels of MDA and NO. were analysed in muscle tissues. Results: Antioxidant enzyme activities and levels of MDA and NO. in plasma were significantly higher in I/R group compared to control group (p< 0,001). Muscle tissues of I/R groups revealed significant higher antioxidant enzyme activity and MDA, NO. levels with respect to control group (p< 0,001). Levels of these parameters in muscle and plasma revealed significant reduction in I/R + Mel group with respect to I/R group (p< 0.001). Histopathological examination of ischemic muscles in I/R group showed significant degeneration and inflammation compared to control group whereas melatonin administered ischemic muscles showed significant reduction of degeneration and inflammation with respect to I/R group (p< 0.001). Conclusions: Levels of NO. and MDA and antioxidant enzyme activity were significantly higher and also revealed significant degeneration and inflammation in I/R group. These results support the opinion that ROS is an important factor in the pathogenesis of I/R injury in skeletal muscles. We attribute the increasing enzyme activities in I/R group to a compensatory mechanism against ROS. Levels of NO. and MDA and antioxidant enzyme activity in tissue and plasma of I/R + Mel group were significantly lower and additionally revealed significant improvement in inflammation and degenaration. This proves the potential ROS scavenging effect of melatonin in reduction of I/R injury. In conclusion we suggest that melatonin may be used in the treatment of I/R injury due extremity injuries with vascular compromise, extremity surgery with prolonged tourniquet time and compartment syndrome

Aims: Pharmocological modulation of skeletal muscle reperfusion injury after an ischaemic insult may improve limb salvage rates and prevent the associated systemic sequelae. Activated Protein c (APC) is an endogenous anti-coagulant with anti-inflammatory properties. The purpose of our study was to evaluate the effects of APC on skeletal muscle ischaemia reperfusion injury and to examine the direct effects of APC on neutrophil activation. Methods: Adult male Sprague Dawley rats (n=30) were randomised into three groups: control group, I/R group treated with normal saline and I/R treated with APC. Bilateral hind-limb ischaemia was induced by rubber ban application proximal to the level of the greater trochanters for two hours. Treatment groups received either normal saline or APC prior to tourniquet release. Following twelve hours reperfusion, the tibialis anterior was dissected and muscle function assessed electrophysiologically by electrical field stimulation. The animals were then sacrificed and skeletal muscle harvested for evaluation. Skeletal muscle injury was assessed based on myeloperoxidase content, wet-to-dry ratio and histological analysis. The effect of APC on TNF-α stimulated human peripheral blood neutrophils was also examined by measuring CD 18 expression and reactive oxygen species (ROS) generation. Results: APC significantly attenuated skeletal muscle reperfusion injury as shown by reduced myeloperoxidase content, wet-to-dry ratio and electrical properties of skeletal muscle. These findings were supported by our histological findings. Our in-vitro work demonstrated a reduction in CD 18 expression and ROS generation. Conclusion: Activated Protein C may have a protective role in the setting of skeletal muscle ischaemia reperfusion injury and this is in part mediated by a direct inhibitory effect on neutrophil activation

Injured skeletal muscle repairs spontaneously via regeneration, however, this process is often incomplete because of fibrotic tissue formation. In our study we wanted to show improved efficiency of regeneration process induced by antifibrotic agent decorin in a combination with Platelet Rich Plasma (PRP)-derived growth factors. A novel human myoblast cell (hMC) culture, defined as CD56 (NCAM)+ developed in our laboratory, was used for evaluation of potential bioactivity of PRP and decorin. To determine the their effect on the viability of hMC we performed a MTT assay. To perform the cell proliferation assay, hMCs were separately seeded on plates at a concentration of 30 viable cells per well. Cell growth medium prepared with different concentrations of PRP exudates (5%, 10%, and 20%) and decorin (10 ng/mL, 25 ng/mL, and 50 ng/mL) were added and incubated for 7 days. After incubation we stained the cells with crystal-violet and measured the absorbance. To study the expression of Transforming Growth Factor Beta (TGF-β) and myostatin (MSTN), two main fibrotic factors in the process of muscle regeneration we performed several ELISA assays in groups treated with all therapeutic agents (PRP, decorin and their combination). Further, we have studied the ability of these agents to influence the differential cascade of dormant myoblasts towards fully differentiated myotubes by monitoring step wise activation of single nuclear factors like MyoD and Myogenin via multicolor flow cytometry. We stained the cells simultaneously with antibodies against CD56, MyoD and myogenin. We acquired cell images of 5,000 events per sample at 40 x magnification using 488 nm and 658 nm lasers and fluorescence was collected using three spectral detection channels. We analysed the cells populations according to expression of single or multiple markers and their ratios. Finally, we examined the treated cell populations using a multicolour laser microscope after staining for desmin (a key marker of myogenic differentiation of hMC), α-tubulin, and nuclei. Optical images were acquired at the center of chamber slides where the cell density is at its highest using a Leica TCS SP5 II confocal microscope and analysed using Photoshop CS6, where a “Color Range” tool was used in combination with a histogram palette to count the pixels that correspond to desmin-positive areas in an image. The mitochondrial activity of cells, as determined by the MTT assay, was significantly increased (p < 0 .001) after exposure to tested concentrations of PRP exudate. Similarly, viability was elevated in all tested concentrations of decorin. PRP exudate enhanced the viability of cells to more than 400% when compared to the control (p < 0 .001). The viability of cells treated with PRP exudates was also significantly higher when compared to decorin (p < 0 .001). Decorin did not show a significant effect on cell proliferation compared to the control, however, cultivation with PRP exudate leads to a 5-fold increase in cell proliferation (p < 0 .001). Decorin was shown to down-regulate the expression of TGF-β when compared to the control by more than 15% (p < 0 .001) but significantly less than PRP exudate p < 0 .005). PRP significantly down-regulated TGF-β expression by more than 30% (p < 0 .001). Similarly, the MSTN expression levels were significantly down-regulated by decorin and PRP. MSTN levels of cells treated with decorin were decreased by 28.4% (p < 0 .001) and 23.1% by PRP (p < 0 .001) when compared to the control group. Using flow cytometry we detected a 39.1% increase in count of myogenin positive cells in the PRP-treated group compared to the control. Moreover, there was a 3.09% increase in cells positive only for myogenin, whereas no such cells were found in the control cell population. The population of cells positive only for myogenin is considered as fully differentiated and capable of fusion into myotubes as well as future mucle fibers and is thus of great importance for muscle regeneration. At the same time 20.6% fewer cells remained quiescent (positive only for CD56). Cells positive for both MyoD and myogenin represent the population that shifted significantly towards mature myocites during myogenesis but are not yet fully committed. Finally, a statistically significant up-regulation of desmin expression (p < 0 .01 for the PRP treated group, p < 0 .005 for the decorin and PRP + decorin treated groups) was present in all therapeutic groups when compared to the control. While no significant difference was found between the PRP and decorin-treated groups, their combination led to a more than 3-fold increase (p < 0 .005) of desmin expression when compared to single bioactives. PRP can be a highly potential therapeutic agent for skeletal muscle regeneration and repair, especially if in combination with a TGF-β antagonis decorin. Achieving better healing could likely result in faster return to play and lower reinjury rate

Summary Statement. Ischaemic preconditioning protected skeletal myotubes against the effects of ischaemia-reperfusion in vitro. This protection was associated with increased Nrf2 signalling. Introduction. Ischaemic preconditioning (IPC) is a well recognised and powerful phenomenon where a tissue becomes more tolerant to a period of prolonged ischaemia when it is first subjected to short bursts of ischaemia/reperfusion. While much is known about the ability of ischaemic preconditioning to protect myocardial tissue against ischaemia-reperfusion injury, its potential to confer benefit in an orthopaedic setting by protecting skeletal muscle remains relatively unexplored to date. One mechanism by which ischaemic preconditioning may induce protection is through a reduction in oxidative stress. Reactive oxygen species (ROS) are generated both during prolonged ischaemia and also upon reperfusion by infiltrating neutrophils, thereby leading to an increase in oxidative stress. The transcription factor, NF-E2-related factor 2 (Nrf2), is a key regulator of the cells response to oxidative stress as it regulates the expression of a network of anti-oxidant/detoxifying enzymes. Nrf2 signalling has recently been shown to protect against ischaemia-reperfusion injury in both a kidney cell line and in liver biopsies, indicating that this transcription factor may play a key role in the protection provided by ischaemic preconditioning. To date, the involvement of Nrf2 in the response of skeletal muscle to ischaemia-reperfusion has not been investigated. Thus, the aims of this study were to investigate the ability of ischaemic preconditioning to protect skeletal myotubes against ischaemia-reperfusion and to determine the role of Nrf2 signalling in this protection. Materials & Methods. C2C12 mouse myoblasts were maintained at 37. o. C in a humidified atmosphere of 95% air and 5% CO. 2. in DMEM containing 20% FBS. When cultures were approximately 90% confluent, myoblasts were differentiated to myotubes by changing to DMEM supplemented with 2% horse serum and culturing for 7–10 days. Differentiated myotubes were then exposed to simulated ischaemia for 4h (1% O. 2. ) followed by 2h reoxygenation (21% O. 2. ). To precondition myotubes, cells were subjected to 30 min of simulated ischaemia followed by 1 hour reoxygenation prior to the prolonged ischaemic event. Cell survival was assessed by lactate dehydrogenase release. Changes in Nrf2 expression were assessed using real-time PCR, Western blotting and immunofluorescence. Changes in sequestosome-1 (SQSTM1), catalase (CAT), glutathione S-transferase theta-1 (GSTT1), heme oxygenase-1 (HO-1) expression were assessed using a combination of real-time PCR and Western blotting. Results. Preconditioned myotubes showed greater viability both after 4h of ischaemia, and after 4h ischaemia followed by 2h of reoxygenation. This increase in cell viability was associated with increased Nrf2 expression. In addition, increased expression of SQSTM1, and the antioxidant enzymes, CAT, GSTT1 and HO-1 was observed in preconditioned myotubes. Discussion. Our findings indicate that ischaemic preconditioning can protect skeletal myotubes against the effects of ischaemia-reperfusion in vitro. This protection is associated with increased Nrf2 signalling indicating that this transcription factor may play a role in mediating the protection induced by ischaemic preconditioning. By modulating the response of skeletal muscle to ischaemia, ischaemic preconditioning has the potential to limit reperfusion injury, which in turn, may lead to improvements in outcome following orthopaedic surgery

Objectives: Skeletal muscle trauma leads to severe functional deficits. Present therapeutic treatments are unsatisfying and insufficient posttraumatic regeneration is a problem in trauma and orthopaedic surgery. Mesenchymal stem cell (MSC) therapy is a promising tool in the regeneration of muscle function after severe trauma. Our group showed increased contraction forces compared to a non-treated control group 3 weeks after MSC transplantation (TX) into a skeletal muscle trauma. In addition we demonstrated a dose-response relationship of the amount of MSC and force enhancement. We furthermore investigated the fate of the transplanted MSC labelled with very small iron oxide particles using 7 Tesla-MRI. Histological analysis revealed fusion events between existing myofibers but only to a low amount. The increase of muscle force can not be explained by these events only. Before further steps are taken the impact of paracrine effects and the homing to the site of trauma of the MSC has to be evaluated. Experimental studies about the functional regeneration of traumatized skeletal muscule after systemic MSC-TX do not exist. Methods: 36 female SD-rats received open crush trauma of the left soleus muscle. One week after trauma 2.5 x 106 autologous MSC, harvested from tibial biopsies, were transplanted intraarterially (i.a., femoral arte-ria, group 1) or intravenously (i.v., tail vein, group 2) (n=18). Control animals received saline (i.a.: group 3; i.v.: group 4) (n=18). Histological analysis and biomechanical evaluation by in vivo muscle force measurement was performed 3 weeks after TX. Results: Twitch stimulation of the healthy right soleus muscles resulted in a contraction force of 0.52±0.14 N. Forces of tetanic contraction in the uninjured muscles reached 0.98±0.27 N. The i.a. MSC-TX improved the muscle force of the injured soleus significantly compared to control (twitch: 82,4%, p=0.02, tetany: 61.6%, p=0.02). Contraction forces of muscles treated i.v. (MSC vs. saline) showed no significant difference. The histological analysis showed no differences in the amount of fibrotic tissue. Conclusions: The presented study demonstrates the effect of systemic MSC-TX in the treatment of severe skeletal muscle injuries. Interestingly, the functional regeneration could only be increased by i.a. application. The entrapment of MSC in the lungs and the dilution effect in the circulation, when injecting the MSC i.v. could be the reason. For possible future therapeutic approaches a systemic application is considered to be favourable compared to local injections due to the better distribution of the cells in the target muscle

Summary Statement. Bio-impedance analysis (BIA) provides a convenient method for the estimation of whole body and segmental measurement of skeletal muscle mass (SMM). BIA-measured SMM parameters may be effectively used for the normalisation of muscle strength and removing body-size dependence. Introduction. Despite an increasing interest in using bio-impedance analysis (BIA) for the estimation of segmental skeletal muscle mass (SMM); existing data is sparse. On the other hand, there is a need for better understanding of the influence of SMM on gender-related differences in muscle strength. Using BIA technique, this study aimed to measure the SMM, determine its correlation with muscle strength, and examine its relation with gender-related differences in muscle strength. Patients and Methods. Segmental and whole body SMM (3-segment electrode configuration) and maximum voluntary contraction in five distinct shoulder planes (forward flexion, abduction in scapular plane, abduction in coronal plane, and internal- and external rotation) were measured in 45 healthy participants (22 males, 23 females) with a mean age of 30.3 years. Independent t-tests and Pearson Correlation test were applied for comparative and correlational analysis, respectively. Results. All muscle-related parameters including muscle volume, SMM, and SMM index were significantly different between men and women. There was a significant gender-related difference in the absolute shoulder strength but not after normalisation to SMM. A strong correlation was found between strength and SMM and in-between strength measurements. Conclusion. BIA provided a convenient method for SMM estimation. SMM parameters may be effectively used for strength normalisation allowing comparisons of individuals with differing body masses. Strong correlations between SMM and muscle strength supported the use of BIA in assessing muscle size-strength relations and its applicability in muscle function assessments

Purpose: It is well established that skeletal muscle ischemia followed by reperfusion induces oxidative damage, metabolic stress, and an inflammatory response. This ischemia-reperfusion injury has been studied extensively in experimental models and, importantly, in the clinical setting where it is associated with tourniquet (TQ) inflation during orthopedic trauma surgery. Of particular clinical concern is the notion that reperfusion upon TQ release is central to oxidative injury, since release necessarily follows surgery. Consequently, the effects of ischemia alone, without reperfusion, is poorly documented. That is, it remains unknown what are the effects of muscle ischemia, per se, on muscle properties that could influence functional recovery postoperatively or what preventative measures might be taken to minimize the potentially deleterious effects of the ischemic period alone. Hence the purpose of this study was to investigate changes in myofibrillar contractile protein oxidation over the course of TQ-induced leg muscle ischemia during orthopedic trauma surgery. Method: Among patients with unilateral ankle fractures requiring surgery at our institution, 24 subjects gave informed consent to participate. All subjects underwent standard general anesthesia. PRE surgical biopsies were collected from the peroneus tertius muscle (PT) immediately after TQ inflation and incision of the skin and underlying connective tissue. POST surgical biopsies were collected from the same muscle immediately before TQ release. Oxidation of PT myosin, actin, and total protein was quantified using Western blot analysis for 4-hydroxynonenal (4-HNE) modified proteins. Results are reported as mean ± standard deviation. Results: Total TQ time ranged from about 21 to 84 min (50.5±16). As anticipated, in PRE biopsies compared to POST biopsies there were large increases in the PT content of 4-NE modified myosin (174.4±128%; P< 1×10-6), actin (223.7±182%; P< 5×10-9), and total protein (567.5±378%; P< 5×10-7). Intriguingly, there was a much greater increase in PT protein oxidation in males than in females (43.3% difference; P< 0.05), although there was no relationship observed between PT protein oxidation and subject age. Surprisingly, there was no significant relationship between muscle protein oxidation and duration of the TQ-induced ischemia. Conclusion: TQ-induced skeletal muscle ischemia for 21 to 84 min during orthopedic trauma surgery leads to considerable oxidative muscle injury as measured by muscle protein oxidation, including of the functionally relevant contractile proteins myosin and actin. This injury occurs even without reperfusion. Interestingly, the extent of oxidative muscle injury appears to be influenced by gender, but is not dependent upon the duration of ischemia. FUNDING: MSFHR, COF, BCLA

Introduction and Aims: To determine whether taurine influences skeletal muscle ischaemia-reperfusion injury in a rat hindlimb model. Method: Twenty-three rats in five groups were subjected to right hindlimb ischaemia-reperfusion injury. The right femoral vein was exposed and cannulated, and a tourniquet applied to the right hindlimb. The left hindlimb acted as a control in each animal. In group 1 there was no treatment, group 2 had normal saline injected into the femoral vein distal to the tourniquet, group 3 had taurine 200mg/Kg injected distal to the tourniquet, group 4 had taurine 200mg/Kg injected proximal to the tourniquet, and group 5 had taurine 100mg/Kg distally and 100mg/Kg proximally. After ischaemia (four hours) and reperfusion (30 minutes), right and left gastrocnemius biopsies were taken and their contraction strength in response to a standardised electrical impulse was measured. Results: Histology of all right-sided biopsies confirmed inflammatory changes consistent with ischaemia-reperfusion injury. In groups 1, 2, 4, and 5, the mean contraction strength of the right (ischaemia-reperfusion injury) biopsies was significantly less than that of the left (control) biopsies. In group 3 (taurine distal to the tourniquet) the difference in strength between right and left was an order of magnitude less than in the other groups, and was not statistically significant. Conclusion: These data suggest that taurine during ischaemia confers some protection against ischaemia-reperfusion injury mediated functional impairment in rat skeletal muscle