Differentiation of the human PAX7-positive myogenic precursors/satellite cell lineage in vitro.

Satellite cells (SC) are muscle stem cells that can regenerate adult muscles upon injury. Most SC originate from PAX7+ myogenic precursors set aside during development. Although myogenesis has been studied in mouse and chicken embryos, little is known about human muscle development. Here, we report the generation of human induced pluripotent stem cell (iPSC) reporter lines in which fluorescent proteins have been introduced into the PAX7 and MYOG loci. We use single cell RNA sequencing to analyze the developmental trajectory of the iPSC-derived PAX7+ myogenic precursors. We show that the PAX7+ cells generated in culture can produce myofibers and self-renew in vitro and in vivo Together, we demonstrate that cells exhibiting characteristics of human fetal satellite cells can be produced in vitro from iPSC, opening interesting avenues for muscular dystrophy cell therapy. This work provides significant insights into the development of the human myogenic lineage.

Lysine methyltransferase 2D regulates muscle fiber size and muscle cell differentiation.

Kabuki syndrome (KS) is a rare genetic disorder caused primarily by mutations in the histone modifier genes KMT2D and KDM6A. The genes have broad temporal and spatial expression in many organs, resulting in complex phenotypes observed in KS patients. Hypotonia is one of the clinical presentations associated with KS, yet detailed examination of skeletal muscle samples from KS patients has not been reported. We studied the consequences of loss of KMT2D function in both mouse and human muscles. In mice, heterozygous loss of Kmt2d resulted in reduced neuromuscular junction (NMJ) perimeter, decreased muscle cell differentiation in vitro and impaired myofiber regeneration in vivo. Muscle samples from KS patients of different ages showed presence of increased fibrotic tissue interspersed between myofiber fascicles, which was not seen in mouse muscles. Importantly, when Kmt2d-deficient muscle stem cells were transplanted in vivo in a physiologic non-Kabuki environment, their differentiation potential is restored to levels undistinguishable from control cells. Thus, the epigenetic changes due to loss of function of KMT2D appear reversible through a change in milieu, opening a potential therapeutic avenue.

Evaluating the role of subacromial impingement in rotator cuff tendinopathy: development and analysis of a novel rat model.

BACKGROUND: Subacromial impingement of the rotator cuff caused by variations in acromial anatomy or altered glenohumeral kinematics leads to inflammation and degeneration of the rotator cuff, ultimately contributing to the development of tendinopathy. However, the underlying cellular and molecular changes in the impinged tendon remain poorly understood. Because the rat is an accepted model for rotator cuff studies, we have developed a rat model to study rotator cuff tendinopathy. METHODS: Forty-four adult male Sprague-Dawley rats were allocated to one of 4 study groups: intact control group (group 1, n = 11); bilateral subacromial surgical clip placement to induce supraspinatus impingement for 2 weeks (group 2, n = 11), 4 weeks (group 3, n = 11), and 8 weeks (group 4, n = 11). Bilateral shoulder specimens were harvested for biomechanical testing, histology, and quantitative real-time polymerase chain reaction (qRT-PCR) analysis. RESULTS: Radiography confirmed that all microvascular clips remained in stable position in the subacromial space. Gross inspection of supraspinatus tendon specimens in the impingement groups revealed changes in tendon morphology at the enthesis and midsubstance. Biomechanical evaluation demonstrated decreased supraspinatus tendon failure force and tissue stiffness at all time points compared with control tendons. Semiquantitative scoring of histologic specimens demonstrated significant, persistent tendinopathic changes over 8 weeks. qRT-PCR analysis of impinged tendon specimens demonstrated upregulation of gene expression for Col3 and Mmp14 in the impingement groups compared with control groups. In muscle samples, significant upregulation was seen in the expression of genes that are commonly associated with muscle atrophy (MuRF1 and Ube2b) and fatty infiltration (Fabp4, Pparg2, and Klf15). CONCLUSION: This new rat subacromial impingement model creates cellular and molecular changes consistent with the development of rotator cuff tendinopathy. The results of this study may serve as a baseline for future investigation.

Current Practices for Rehabilitation After Meniscus Repair: A Survey of Members of the American Orthopaedic Society for Sports Medicine.

Background: There is no consensus among sports medicine surgeons in North America on postoperative rehabilitation strategy after meniscus repair. Various meniscal tear types may necessitate a unique range of motion (ROM) and weightbearing rehabilitation protocol. Purpose: To assess the current landscape of how sports medicine practitioners in the American Orthopedic Society for Sports Medicine (AOSSM) rehabilitate patients after the repair of varying meniscal tears. Study Design: Cross-sectional study. Methods: A survey was distributed to 2973 AOSSM members by email. Participants reviewed arthroscopic images and brief patient history from 6 deidentified cases of meniscus repair-in cases 1 to 3, the tears retained hoop integrity (more stable repair), and in cases 4 to 6, the tear patterns represented a loss of hoop integrity. Cases were shuffled before the presentation. For each case, providers were asked at what postoperative time point they would permit (1) partial weightbearing (PWB), (2) full weightbearing (FWB), (3) full ROM, and (4) ROM allowed immediately after surgery. Results: In total, 451 surveys were completed (15.2% response). The times to PWB and FWB in cases 1 to 3 (median, 0 and 4 weeks, respectively) were significantly lower than those in cases 4 to 6 (median, 4 and 6 weeks, respectively) (P < .001). In tears with retained hoop integrity, the median time to PWB was immediately after surgery, whereas in tears without hoop integrity, the median time to PWB was at 4 weeks postoperatively. Similarly, the median time to FWB in each tear with retained hoop integrity was 4 weeks after surgery, while it was 6 weeks in each tear without hoop integrity. However, regardless of tear type, most providers (67.1%) allowed 0° to 90° of ROM immediately after surgery and allowed full ROM at 6 weeks. Most providers (83.3%) braced the knee after repair regardless of hoop integrity and utilized synovial rasping/trephination with notch microfracture-a much lower proportion of providers utilized biologic augmentation (9%). Conclusion: Sports medicine practitioners in the AOSSM rehabilitated meniscal tears differently based on hoop integrity, with loss of hoop stresses triggering a more conservative approach. A majority braced and utilized in situ adjuncts for biological healing, while a minority added extrinsic biologics.

Open-Source Ultrasound Trainer for Healthcare Professionals: A Pilot Randomized Control Trial.

INTRODUCTION: This technical report describes the development of a high-fidelity, open-source ultrasound trainer and showcases its abilities through a proof-of-concept, pilot randomized control trial. The open-source ultrasound trainer (OSUT) aims to enhance anatomical visualization during ultrasound education. The OSUT can attach to any ultrasound transducer, uses minimal hardware, and is able to be used during live patient ultrasound examinations. METHODS: After viewing a standardized training video lecture, 24 incoming first-year medical students with no prior ultrasound experience were randomized into a control group given an ultrasound system or an intervention group given the OSUT in addition to an ultrasound system. Both groups were tasked with localizing the thyroid, abdominal aorta, and right kidney on a patient. Performance outcomes were structure localization time, ultrasound image accuracy, and preactivity and postactivity participant confidence. RESULTS: The OSUT decreased right kidney localization time (Kruskal-Wallis, P < 0.001), increased sonographer right kidney accuracy ratings (Mann-Whitney U , U = 10.5, P < 0.05), and increased confidence in structure identification (Mann-Whitney U , U = 37, P = 0.045) and overall ultrasound ability (Wilcoxon signed-rank test, P = 0.007). There was no significant change in localization time, accuracy ratings, or participant confidence for locating the thyroid and abdominal aorta. CONCLUSIONS: A high-fidelity, open-source ultrasound trainer was developed to aid healthcare professionals in learning diagnostic ultrasound. The study demonstrated the potential beneficial effects of the OSUT in localizing the right kidney, showcasing its adaptability and accessibility for ultrasound education for certain anatomical structures.

Tetraspanin CD82 Associates with Trafficking Vesicle in Muscle Cells and Binds to Dysferlin and Myoferlin.

Tetraspanins organize protein complexes at the cell membrane and are responsible for assembling diverse binding partners in changing cellular states. Tetraspanin CD82 is a useful cell surface marker for prospective isolation of human myogenic progenitors and its expression is decreased in Duchenne muscular dystrophy (DMD) cell lines. The function of CD82 in skeletal muscle remains elusive, partly because the binding partners of this tetraspanin in muscle cells have not been identified. CD82-associated proteins are sought to be identified in human myotubes via mass spectrometry proteomics, which identifies dysferlin and myoferlin as CD82-binding partners. In human dysferlinopathy (Limb girdle muscular dystrophy R2, LGMDR2) myogenic cell lines, expression of CD82 protein is near absent in two of four patient samples. In the cell lines where CD82 protein levels are unaffected, increased expression of the ≈72 kDa mini-dysferlin product is identified using an antibody recognizing the dysferlin C-terminus. These data demonstrate that CD82 binds dysferlin/myoferlin in differentiating muscle cells and its expression can be affected by loss of dysferlin in human myogenic cells.

Anatomical Considerations for Gallbladder Sonography: A Cross-Sectional Study of CT Imaging by BMI Group.

Objective The purpose of this study was to establish an association between the body mass index (BMI) group and anatomical gallbladder position to aid novices in gallbladder sonography. Methods This was a cross-sectional, Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)-compliant study that examined the association between gender and the BMI group with quantitative gallbladder position measurements from computed tomography (CT) scans. Results A quantitative analysis determined that the gallbladder was positioned relatively higher and oriented more horizontally within the abdomen of individuals with obese BMI than those with normal BMI (p < 0.001), irrespective of gender. Additionally, the gallbladder was more obstructed by the rib cage in individuals with obese BMI than those with normal BMI (p = 0.007 for females and p < 0.001 for males). The gallbladder was significantly more horizontal in overweight males than females (p < 0.001) and more obstructed by the rib cage in obese males than females (p = 0.013). Conclusion This association provides ultrasound novices knowledge for a more targeted approach in localizing the gallbladder and evidence to recommend an intercostal approach for gallbladder sonography in obese patients.

Conceptualizing Biological Aging and Frailty in Orthopaedics: A Framework for Clinical Practice.

➤: Biological aging can best be conceptualized clinically as a combination of 3 components: frailty, comorbidity, and disability. ➤: Despite advancements in the understanding of senescence, chronological age remains the best estimate of biological age. However, a useful exercise for practitioners is to look beyond chronological age in clinical and surgical decision-making. ➤: A chronologically aging person does not age biologically at the same rate. ➤: The best way to understand frailty is to consider it as a physical phenotype. ➤: Physical optimization should parallel medical optimization before elective surgery. ➤: The poorer the host (both in terms of bone quality and propensity for healing), the more robust the implant construct must be to minimize reliance on host biology.

3-D modeling applications in ultrasound education: a systematic review.

Ultrasound offers a real-time 2-D view of structures within the human body. While many medical education programs have already dedicated a portion of their curriculum to ultrasound, others are concerned about cost, accessibility and limits to student practice. Student benefit may be affected by cognitive errors, which are in part owing to the mental heuristics required to visualize a 3-D structure by interpreting a 2-D image. A possible solution to eliminating subjectivity in ultrasound interpretation is the use of 3-D models to augment the traditional 2-D ultrasound experience. PubMed, Embase and Web of Science were searched for primary literature exploring relationships between 3-D modeling applications and their use in ultrasound education. The search and review process was guided by the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) checklist. Overall, 14 of the included 16 studies indicated a significant improvement in medical education of ultrasound with the intervention of 3-D modeling applications. This systematic review confirms that 3-D modeling applications benefit student learning in ultrasound education while illuminating the need for more research in this field.

The Role of Indian Hedgehog Signaling in Tendon Response to Subacromial Impingement: Evaluation Using a Mouse Model.

BACKGROUND: The underlying cellular and molecular mechanisms involved in the development of tendinopathy due to subacromial supraspinatus tendon (SST) impingement and the response to subsequent removal of impingement remain unknown. PURPOSE: To investigate the involvement of Indian hedgehog (IHH) signaling in the development of SST tendinopathy and the subsequent healing process after the relief of subacromial impingement in a novel mouse shoulder impingement model. STUDY DESIGN: Controlled laboratory study. METHODS: A total of 48 male wild-type C57BL/6 mice were used in this study. Supraspinatus tendinopathy was induced by inserting a microsurgical clip into the subacromial space bilaterally. Eleven mice were sacrificed at 4 weeks after surgery to establish impingement baseline; 24 mice underwent clip removal at 4 weeks after surgery and then were euthanized at 2 or 4 weeks after clip removal. Thirteen mice without surgical intervention were utilized as the control group. All SSTs were evaluated with biomechanical testing; quantitative histomorphometry after staining with hematoxylin and eosin, Alcian blue, and picrosirius red; and immunohistochemical staining (factor VIII, IHH, Patched1 [PTCH1], and glioma-associated oncogene homolog 1 [GLI1]). RESULTS: The mean failure force and stiffness in the 4-week impingement group decreased significantly compared with the control group (P < .001) and gradually increased at 2 and 4 weeks after clip removal. Histological analysis demonstrated increased cellularity and disorganized collagen fibers in the SST, with higher modified Bonar scores at 4 weeks, followed by gradual improvement after clip removal. The IHH-positive area and PTCH1- and GLI1-positive cell percentages significantly increased after 4 weeks of clip impingement (20.64% vs 2.06%, P < .001; 53.9% vs 28.03%, P = .016; and 30% vs 12.19%, P = .036, respectively) and continuously increased after clip removal. CONCLUSION: The authors' findings suggest that the hedgehog signaling pathway and its downstream signaling mediator and target GLI1 may play a role in the development and healing process of rotator cuff tendinopathy due to extrinsic rotator cuff impingement. CLINICAL RELEVANCE: This study suggests the potential for the hedgehog pathway, together with its downstream targets, as candidates for further study as potential therapeutic targets in the treatment of supraspinatus tendinopathy.

Tetraspanin CD82 is necessary for muscle stem cell activation and supports dystrophic muscle function.

BACKGROUND: Tetraspanins are a family of proteins known to assemble protein complexes at the cell membrane. They are thought to play diverse cellular functions in tissues by modifying protein-binding partners, thus bringing complexity and diversity in their regulatory networks. Previously, we identified the tetraspanin KAI/CD82 as a prospective marker for human muscle stem cells. CD82 expression appeared decreased in human Duchenne muscular dystrophy (DMD) muscle, suggesting a functional link to muscular dystrophy, yet whether this decrease is a consequence of dystrophic pathology or a compensatory mechanism in an attempt to rescue muscle from degeneration is currently unknown. METHODS: We studied the consequences of loss of CD82 expression in normal and dystrophic skeletal muscle and examined the dysregulation of downstream functions in mice aged up to 1 year. RESULTS: Expression of CD82 is important to sustain satellite cell activation, as in its absence there is decreased cell proliferation and less efficient repair of injured muscle. Loss of CD82 in dystrophic muscle leads to a worsened phenotype compared to control dystrophic mice, with decreased pulmonary function, myofiber size, and muscle strength. Mechanistically, decreased myofiber size in CD82-/- dystrophic mice is not due to altered PTEN/AKT signaling, although increased phosphorylation of mTOR at Ser2448 was observed. CONCLUSION: Basal CD82 expression is important to dystrophic muscle, as its loss leads to significantly weakened myofibers and impaired muscle function, accompanied by decreased satellite cell activity that is unable to protect and repair myofiber damage.

Postoperative Tendon Loading With Treadmill Running Delays Tendon-to-Bone Healing: Immunohistochemical Evaluation in a Murine Rotator Cuff Repair Model.

Mechanical stress has an important effect on tendon-to-bone healing. The purpose of the present study was to compare tendon-to-bone healing in animals exposed to either tendon unloading (botulinum toxin injection) or excessive loading (treadmill running) in a murine rotator cuff repair model. Forty-eight C57BL/6 mice underwent unilateral supraspinatus tendon detachment and repair. Mice in the unloaded group were injected with botulinum toxin to the supraspinatus muscle. The contralateral shoulder of the unloaded group was used as a control. Mice were euthanized at 1, 2, and 4 weeks after surgery and evaluated with hematoxylin-eosin and immunohistochemical (IHC) staining for Ihh, Gli1, Wnt3a, and ß-catenin. The positive staining area on IHC and the Modified Tendon Maturing Score were measured. The score of the unloaded group was significantly higher (better healing) than that of the treadmill group at 4 weeks. Ihh and the glioma-associated oncogene homolog 1 (Gli1) positive area in the unloaded group were significantly higher than those of the control group at 1 week. The peak time-points of the Ihh and Gli1 positive area was 1 week for the unloaded group and 2 weeks for the treadmill group. The Wnt3a positive area in the unloaded group was significantly higher than that of the control group at 2 weeks. The ß-catenin positive area in the unloaded group was significantly higher than that of the treadmill group and the control group at 1 week. Our data indicated that the unloaded group has superior tendon maturation compared to the treadmill running group. Excessive tendon loading may delay the tendon healing process by affecting the activity of Ihh and Wnt/ß-Catenin pathways. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1628-1637, 2019.

Biomechanics and Microstructural Analysis of the Mouse Knee and Ligaments.

The purpose of this study is to determine the feasibility of using murine models for translational study of knee ligament injury, repair, and reconstruction. To achieve this aim, we provide objective, quantitative data detailing the gross anatomy, biomechanical characteristics, and microscopic structure of knee ligaments of 44 male mice (C57BL6, 12 weeks of age). Biomechanical testing determined the load-to-failure force, stiffness, and the site of ligament failure for the anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), and the medial and lateral collateral ligaments (MCL and LCL). These data are complemented by histological characterization of each of the knee ligaments. In addition, the osseous morphology of the mouse knee was examined using high-resolution nanofocus computed tomography (CT), while standard micro-CT was employed to measure bone morphometrics of the distal femur and proximal tibia. Collectively, our findings suggest that the gross anatomy of the mouse knee is similar to the human knee despite some minor differences and features unique to the murine knee. The ACL had the highest load to failure (5.60 ± 0.75 N), the MCL (3.33 ± 1.45 N), and the PCL (3.45 ± 0.84 N) were similar, and the LCL (1.44 ± 0.37 N) had the lowest load to failure and stiffness. Murine models provide a unique opportunity to focus on biological processes that impact ligament pathology and healing due to the availability of transgenic strains. Our data support their use as a translational platform for the in vivo study of ligament injury, repair, and reconstruction.