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Sarcoma is a rare malignancy with unfavorable prognoses. Accumulating evidence indicates that aberrant alternative splicing (AS) events are generally involved in cancer pathogenesis. The aim of this study was to identify the prognostic value of AS-related survival genes as potential biomarkers, and highlight the functional roles of AS events in sarcoma. RNA-sequencing and AS-event datasets were downloaded from The Cancer Genome Atlas (TCGA) sarcoma cohort and TCGA SpliceSeq, respectively. Survival-related AS events were further assessed using a univariate analysis. A multivariate Cox regression analysis was also performed to establish a survival-gene signature to predict patient survival, and the area-under-the-curve method was used to evaluate prognostic reliability. KOBAS 3.0 and Cytoscape were used to functionally annotate AS-related genes and to assess their network interactions. We detected 9674 AS events in 40,184 genes from 236 sarcoma samples, and the 15 most significant genes were then used to construct a survival regression model. We further validated the involvement of ten potential survival-related genes (TUBB3, TRIM69, ZNFX1, VAV1, KCNN2, VGLL3, AK7, ARMC4, LRRC1, and CRIP1) in the occurrence and development of sarcoma. Multivariate survival model analyses were also performed, and validated that a model using these ten genes provided good classifications for predicting patient outcomes. The present study has increased our understanding of AS events in sarcoma, and the gene-based model using AS-related events may serve as a potential predictor to determine the survival of sarcoma patients.

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy seen in children. In addition to skeletal muscle, DMD also has a significant impact on bone. The pathogenesis of bone abnormalities in DMD is still unknown. Recently, we have identified a novel bone-regulating cytokine, fibroblast growth factor-21 (FGF-21), which is dramatically upregulated in skeletal muscles from DMD animal models. We hypothesize that muscle-derived FGF-21 negatively affects bone homeostasis in DMD. Dystrophin/utrophin double-knockout (dKO) mice were used in this study. We found that the levels of circulating FGF-21 were significantly higher in dKO mice than in age-matched WT controls. Further tests on FGF-21 expressing tissues revealed that both FGF-21 mRNA and protein expression were dramatically upregulated in dystrophic skeletal muscles, whereas FGF-21 mRNA expression was downregulated in liver and white adipose tissue (WAT) compared to WT controls. Neutralization of circulating FGF-21 by i.p. injection of anti-FGF-21 antibody significantly alleviated progressive bone loss in weight-bearing (vertebra, femur, and tibia) and non-weight bearing bones (parietal bones) in dKO mice. We also found that FGF-21 directly promoted RANKL-induced osteoclastogenesis from bone marrow macrophages (BMMs), as well as promoted adipogenesis while concomitantly inhibiting osteogenesis of bone marrow mesenchymal stem cells (BMMSCs). Furthermore, fibroblast growth factor receptors (FGFRs) and co-receptor β-klotho (KLB) were expressed in bone cells (BMM-derived osteoclasts and BMMSCs) and bone tissues. KLB knockdown by small interfering RNAs (siRNAs) significantly inhibited the effects of FGF21 on osteoclast formation of BMMs and on adipogenic differentiation of BMMSCs, indicating that FGF-21 may directly affect dystrophic bone via the FGFRs-β-klotho complex. In conclusion, this study shows that dystrophic skeletal muscles express and secrete significant levels of FGF-21, which negatively regulates bone homeostasis and represents an important pathological factor for the development of bone abnormalities in DMD. The current study highlights the importance of muscle/bone cross-talk via muscle-derived factors (myokines) in the pathogenesis of bone abnormalities in DMD. © 2019 American Society for Bone and Mineral Research.

The ketogenic diet (KD) is a normocaloric diet composed of high-fat, low-carbohydrate, and adequate protein that induces fasting-like effects and results in the production of ketone bodies. Initially used widely for children with refractory epilepsy, the KD gained popularity due to its beneficial effects on weight loss, diabetes, and cancer. In recent years, there has been a resurgence in interest surrounding the KD and exercise performance. This review provides new insights into the adaptation period necessary for enhancement in skeletal muscle fat and ketone oxidation after sustained nutritional ketosis. In addition, this review highlights metabolically active growth factors and cytokines, which may function as important regulators of keto-adaptation in the setting of exercise and the KD.

Duchenne muscular dystrophy (DMD) is a degenerative muscle disorder characterized by a lack of dystrophin expression in the sarcolemma of muscle fibers. DMD patients acquire bone abnormalities including osteopenia, fragility fractures, and scoliosis indicating a deficiency in skeletal homeostasis. The dKO (dystrophin/Utrophin double knockout) is a more severe mouse model of DMD than the mdx mouse (dystrophin deficient), and display numerous clinically-relevant manifestations, including a spectrum of degenerative changes outside skeletal muscle including bone, articular cartilage, and intervertebral discs. To examine the influence of systemic factors on the bone abnormalities and healing in DMD, parabiotic pairing between dKO mice and mdx mice was established. Notably, heterochronic parabiosis with young mdx mice significantly increased bone mass and improved bone micro-structure in old dKO-hetero mice, which showed progressive bone deterioration. Furthermore, heterochronic parabiosis with WT C56/10J mice significantly improved tibia bone defect healing in dKO-homo mice. These results suggest that systemic blood-borne factor(s) and/or progenitors from WT and young mdx mice can influence the bone deficiencies in dKO mice. Understanding these circulating factors or progenitor cells that are responsible to alleviate the bone abnormalities in dKO mice after heterochronic parabiosis might be useful for the management of poor bone health in DMD.

Introduction Duchenne muscular dystrophy (DMD) has been well characterized as a disease that affects both skeletal muscle and bone. The pathophysiology responsible for the deficits in bone tissue is still unclear. Methods Quantitative reverse-transcription polymerase chain reaction and Western blot analyses of known myokines from skeletal muscle were performed on dystrophic mouse models and wild-type (WT) controls to identify differentially expressed bone-regulating myokines. Results Twenty-four of 43 myokine genes demonstrated significantly different mRNA expression in the skeletal muscles of dystrophic mice when compared with muscles of WT mice. Several differently expressed bone-regulating myokine genes were identified, and their protein levels were also verified by Western blot. Conclusions Dystrophic skeletal muscle demonstrated a significantly altered myokine gene expression profile. mRNA and protein levels of several bone-regulating myokines were significantly altered in dystrophic skeletal muscle, which suggests pathological role of bone-regulating myokines on bone homeostasis in DMD. Muscle Nerve 58: 573-582, 2018.

Background: Patients presenting with cam deformity of the femoral head and neck sustain repeated trauma to the articular cartilage of the superior acetabulum, with chondral delamination injuries found during hip arthroscopy. Two previous studies reveal conflicting chondrocyte viability data in these traumatic cartilage injuries. The full-thickness nature of flaps may suggest that chondrocytes residing in the cartilage flap matrix in the joint environment would remain viable despite shear trauma. Hypothesis/Purpose: The purpose of this study is to determine the in vivo tissue viability of acetabular chondral flaps in patients with femoroacetabular impingement (FAI) when samples are analyzed immediately after biopsy. We hypothesize that the majority of the tissue in acetabular chondral flaps is viable in the joint microenvironment. Study Design: Descriptive laboratory study. Methods: Partially detached cartilage flaps from 10 patients undergoing arthroscopic hip surgery for FAI were biopsied in a minimally traumatic manner before chondroplasty and microfracture. Samples were placed in cold Hank’s Balanced Salt Solution without phenol red solution and immediately transported on ice to our laboratory. The edge of the samples was trimmed and further cut into 3 separate, 1-mm-thick sections. Sections were stained using a live/dead staining kit. Images were obtained with confocal microscopy, and the percentage of live cells was quantified. Results: Patients averaged 36 ± 11 years (range, 18-48 years), and 2 patients were female. The mean body mass index was 28.9 ± 5.6 kg/m2. The total proportion of live cells from all sections analyzed was 85.8%. The proportion of live cells per patient was 87% ± 10%. Conclusion: We determined that acetabular chondral flaps are approximately 87% live cells when analyzed immediately after biopsy, with 6 of 10 patients having greater than 90% live cells. These data point to the importance of laboratory techniques in making viability judgments in biologic systems. Clinical Relevance: Full-thickness cartilage loss is a difficult problem for all active people but particularly in the young population in whom joint preservation is key. We describe the viability of chondrocytes present in full-thickness acetabular-based chondral flaps encountered during hip arthroscopy. Identification of greater than 85% chondrocyte viability supports a foundation for evaluation and creation of novel clinical innovations for repair and replacement techniques using the flap as donor tissue, as alternatives to chondroplasty and microfracture.

Muscle fatigue is common among humans and also a crucial indicator of many muscular diseases such as muscular dystrophy and disorders. Timely evaluation of muscle fatigue, hence, is important to track disease progress and avoid disease exacerbations. However, convenient tools for evaluating muscle fatigue out of clinic are still missing. In this paper, we present a new technique that uses commodity smartphones to evaluate muscle fatigue through simple and daily muscle exercises. The basic idea of our technique is to mimic an active sonar system with the smartphone’s built-in microphone and speaker, and use this sonar system to evaluate muscle fatigue from the muscle’s surface characteristics that can be measured from the transmitted acoustic signal. More specifically, our technique first measures the acoustic channel disturbances caused by fatigue-induced muscle tremor via channel estimation, and then derives quantitative fatigue levels from the variation of acoustic channel estimation. By using the arm bicep muscle as our primary target, we designed the exercise protocol and implemented a smartphone app for fatigue evaluation. Experiment results verified that our technique can precisely evaluate the speed of muscle fatigue accumulation, as well as identifying the actual fatigue occurrence. This technique, hence, could be used in practical home settings for effective fatigue evaluation on a daily basis.

Biological research in the areas of skeletal, cartilaginous, tendinous, and muscular tissues has led to the advancement of various products designed to augment healing. Platelet-rich plasma (PRP) has been in clinical use and researched since the 1970s because of its regenerative properties. In this chapter, we will first define PRP and its components and discuss various methods of preparation and isolation. The second section will focus on the clinical applications of PRP on tissue specific pathologies including tendinopathy, ligamentous injuries, osteoarthritis, and muscle injuries in the field of orthopedic surgery and sports medicine. Lastly, the latest progress in PRP research will be briefly listed and some promising future directions will be discussed.

Objectives: Individuals who participate in sports have an increased risk of osteoarthritis (OA), characterized by articular cartilage degeneration. Currently, there is no cure for OA with treatment aimed at symptom relief and improved function. Muscle-derived stem cells (MDSCs) have been shown to exhibit long-term proliferation, high self-renewal, and multipotent differentiation capabilities in vitro. Previously, we have demonstrated that murine MDSCs retrovirally transduced to express chondrogenic proteins (BMPs) differentiate into chondrocytes and enhance cartilage repair in vivo. Direct injection of therapeutic proteins can promote cartilage healing; however, they have relatively short half-lives requiring muitiple injections of high dosages. This presents a challenge in terms of maintaining adequate local BMP levels and could negatively affect both injured and normal structures and lead to side effects such as osteophyte formation. Gene therapy is a promising approach that addresses this problem; however, its utilization in clinical applications is much further down the road. In order to circumvent viral transduction of cells for cartilage regeneration, we developed a unique growth factor delivery platform comprised of native heparin and a synthetic polycation, poly(ethylene argininylaspartate diglyceride) (PEAD) incorporated with BMP2 (BMP2 coacervate). In this study, we show that sustained delivery of BMP2 via a BMP2 coacervate can induce the differentiation of MDSCs to a chondrocyte lineage for in vivo cartilage regeneration and healing in a Monoiodoacetate (MIA)-induced osteoarthritis model. Methods: mMDSCs were isolated from muscle biopsies via a modified pre-plated technique. The BMP2 coacervates were prepared as previously described. The release profiles of BMP2 coacervate were tested by ELISA. The chondrogenic effects that delivery of BMP2 had on MDSCs were evaluated by RT-PCR. The efficacy of MDSC with BMP2 coacervate were evaluated in vivo in a MIA-induced OA model in C57B6 mice. Mice received an intraarticular injection of 20μl of MIA to induce osteoarthritic lesions. Two weeks after MIA injection, mice were injected with PBS (control), MDSC + free BMP2, or MDSC + BMP2 coacervate. Histologic evaluations of cartilage regeneration were conducted at week 4. Institutional Animal Care and Use Committee approval was obtained prior to all animal studies. Results: Release profiles of BMP2 showed sustained release for more than 28 days demonstrating sustained release. The chondrogenic differentiation of MDSCs following BMP2 delivery was assayed using cell culture. The mRNA expression of Aggrecan and Col2A were significantly higher in each BMP2 group compared to control or vehicle only (PConclusion: This study demonstrates that sustained growth factor delivery (BMP2) is a potential therapeutic option for muscle-derived stem cell based cartilage regeneration for the treatment of osteoarthritis. Our results demonstrate an effective method for prolonged exposure to BMP2 via a non-gene therapy approach which is preferred and clinically translatable.

Duchenne muscular dystrophy (DMD) is a degenerative muscle disorder characterized by the lack of dystrophin expression at the sarcolemma of muscle fibers. In addition, DMD patients acquire osteopenia, fragility fractures, and scoliosis indicating that a deficiency in skeletal homeostasis coexists but little is known about the effects of DMD on bone and other connective tissues within the musculoskeletal system. Recent evidence has emerged implicating adult stem cell dysfunction in DMD myopathogenesis. Given the common mesenchymal origin of muscle and bone, we sought to investigate bone and other musculoskeletal tissues in a DMD mouse model. Here, we report that dystrophin-utrophin double knockout (dko) mice exhibit a spectrum of degenerative changes, outside skeletal muscle, in bone, articular cartilage, and intervertebral discs, in addition to reduced lifespan, muscle degeneration, spinal deformity, and cardiomyopathy previously reported. We also report these mice to have a reduced capacity for bone healing and exhibit spontaneous heterotopic ossification in the hind limb muscles. Therefore, we propose the dko mouse as a model for premature musculoskeletal aging and posit that a similar phenomenon may occur in patients with DMD.

The formation of fibrous tissue during the healing of skeletal muscle injuries leads to incomplete recovery of the injured muscle. Platelet-rich-plasma (PRP) contains beneficial growth factors for skeletal muscle repair; however, it also contains deleterious cytokines and growth factors, such as TGF-β1, that can cause fibrosis and inhibit optimal muscle healing. Objective To test if neutralizing TGF-β1's action within PRP, through neutralization antibodies, could improve PRP's beneficial effect on skeletal muscle repair. Methods PRP was isolated from in-bred Fisher rats. TGF-β1 neutralization antibody (Ab) was used to block the TGF-β1 within the PRP prior to injection. The effects of customized PRP (TGF-β1 neutralized PRP) on muscle healing was tested on a cardiotoxin (CTX) induced muscle injury model. Results A significant increase in the numbers of regenerative myofibers was observed in the PRP and customized PRP groups compared to the untreated control. A significant decrease in collagen deposition was observed in customized PRP groups when compared to the control and PRP groups. Significantly enhanced angiogenesis and more Pax-7 positive satellite cells were found in the PRP and customized PRP groups compared to the control group. Macrophage infiltration was increased in the customized PRP groups when compared with the PRP group. More M2 macrophages were recruited to the injury site in the customized PRP groups when compared with the PRP and control groups. Conclusion Neutralizing TGF-β1 within PRP significantly promotes muscle regeneration while significantly reducing fibrosis. Not only did the neutralization reduce fibrosis, it enhanced angiogenesis, prolonged satellite cell activation, and recruited a greater number of M2 macrophages to the injury site which also contributed to the efficacy that the customized PRP had on muscle healing.

Muscle-derived cells have been successfully isolated using a variety of different methods and have been shown to possess multilineage differentiation capacities, including an ability to differentiate into articular cartilage and bone in vivo; however, the characterization of human muscle-derived stem cells (hMDSCs) and their bone regenerative capacities have not been fully investigated. Genetic modification of these cells may enhance their osteogenic capacity, which could potentially be applied to bone regenerative therapies. We found that hMDSCs, isolated by the preplate technique, consistently expressed the myogenic marker CD56, the pericyte/endothelial cell marker CD146, and the mesenchymal stem cell markers CD73, CD90, CD105, and CD44 but did not express the hematopoietic stem cell marker CD45, and they could undergo osteogenic, chondrogenic, adipogenic, and myogenic differentiation in vitro. In order to investigate the osteoinductive potential of hMDSCs, we constructed a retroviral vector expressing BMP4 and GFP and a lentiviral vector expressing BMP2. The BMP4-expressing hMDSCs were able to undergo osteogenic differentiation in vitro and exhibited enhanced mineralization compared to nontransduced cells; however, when transplanted into a calvarial defect, they failed to regenerate bone. Local administration of BMP4 protein and cell pretreatment with N-acetylcysteine (NAC), which improves cell survival, did not enhance the osteogenic capacity of the retro-BMP4-transduced cells. In contrast, lenti-BMP2-transduced hMDSCs not only exhibited enhanced in vitro osteogenic differentiation but also induced robust bone formation and nearly completely healed a critical-sized calvarial defect in CD-1 nude mice 6 weeks following transplantation. Herovici's staining of the regenerated bone demonstrated that the bone matrix contained a large amount of type I collagen. Our findings indicated that the hMDSCs are likely mesenchymal stem cells of muscle origin and that BMP2 is more efficient than BMP4 in promoting the bone regenerative capacity of the hMDSCs in vivo.

Objectives: Skeletal muscle injuries are among the most common sports-related trauma. Current treatment strategies result in formation of fibrous tissue that hinders the healing process before complete recovery. Incomplete recovery impairs muscle function and predisposes to re-injury. Platelet-Rich-Plasma (PRP) contains a multitude of growth factors and is an autologous source of growth factors for various tissue repairs. It is well established that PRP contains beneficial growth factors for muscle repair; however, it also contains high concentrations of deleterious growth factors for optimal muscle healing, such as transforming growth factor-beta 1 (TGF-β1). TGF-β1 leads to increased fibrosis impeding muscle healing. We therefore hypothesized that neutralization of TGF-β1’s action within PRP could improve PRP’s beneficial effect on skeletal muscle repair. Methods: Sixteen week old in-bred Fisher rats were used. Three rats were used for PRP isolation. 10 ml of blood were extracted from abdominal aorta and mixed with citrate phosphate dextrose solution. PRP were isolated by twice centrifugation. 24 rats were randomly assigned to four groups. A small incision was made along the tibialis anterior (TA) muscle; 50 µl cardiotoxin (CTX) (0.15ug/ul) was injected intramuscularly to the TA. One day after CTX injection, the animals were treated with PBS (control), plain PRP (PRP group), customized PRP+Ab-1x, and PRP+Ab-5x. Animals were sacrificed, and TA muscles were dissected on week 1 and 2 for assessment of muscle regeneration, fibrosis, macrophage infiltration, and satellite cell activation. Results: We observed significantly more regenerative myofibers in the PRP and customized PRP groups compared to control (Fig 1A-C). Collagen deposition (fibrosis) was detected in all groups at week 1 and week 2 after injury; while customized PRP group showed significantly decreased collagen deposition at week 1 and week 2 when compare to control and PRP groups (Fig. 1D-F). PRP was also shown to activate satellite cells at day 7 while customized PRP extended satellite cell activation through day 14 (Fig. 1G, H). Macrophage infiltration was significantly decreased in PRP group when compared to control at week 1 after injury; however the macrophage infiltration was increased in customized PRP group when compare with PRP group, specifically, we observed an increased in the percentage of the M2 macrophage subtype in the customized PRP group at week 1 but not week 2(Fig. 1I, J). Conclusion: This study demonstrates that substantial fibrosis occurs in skeletal muscle following CTX injury. PRP treatment significantly increases muscle regeneration by promoting angiogenesis, satellite cell activation, and reducing the infiltration of inflammatory macrophages. However, augmented fibrosis was also observed. Neutralizing TGF-β1 within PRP also significantly promotes muscle regeneration while significantly reducing fibrosis. It has been suggested that M2 macrophages are involved in the suppression, resolution and reversal of fibrosis. Hence, the increase in M2 macrophages in the customized PRP group may have contributed to the observed decrease in fibrosis. Our results demonstrate that neutralization of TGF-β1 within PRP is a promising approach to improve PRP’s beneficial effect on skeletal muscle repair while decreasing deleterious fibrosis.