FOXO-regulated DEAF1 controls muscle regeneration through autophagy.

The commonality between various muscle diseases is the loss of muscle mass, function, and regeneration, which severely restricts mobility and impairs the quality of life. With muscle stem cells (MuSCs) playing a key role in facilitating muscle repair, targeting regulators of muscle regeneration has been shown to be a promising therapeutic approach to repair muscles. However, the underlying molecular mechanisms driving muscle regeneration are complex and poorly understood. Here, we identified a new regulator of muscle regeneration, Deaf1 (Deformed epidermal autoregulatory factor-1) - a transcriptional factor downstream of foxo signaling. We showed that Deaf1 is transcriptionally repressed by FOXOs and that DEAF1 targets to Pik3c3 and Atg16l1 promoter regions and suppresses their expression. Deaf1 depletion therefore induces macroautophagy/autophagy, which in turn blocks MuSC survival and differentiation. In contrast, Deaf1 overexpression inactivates autophagy in MuSCs, leading to increased protein aggregation and cell death. The fact that Deaf1 depletion and its overexpression both lead to defects in muscle regeneration highlights the importance of fine tuning DEAF1-regulated autophagy during muscle regeneration. We further showed that Deaf1 expression is altered in aging and cachectic MuSCs. Manipulation of Deaf1 expression can attenuate muscle atrophy and restore muscle regeneration in aged mice or mice with cachectic cancers. Together, our findings unveil an evolutionarily conserved role for DEAF1 in muscle regeneration, providing insights into the development of new therapeutic strategies against muscle atrophy.Abbreviations: DEAF1: Deformed epidermal autoregulatory factor-1; FOXO: Forkhead box O; MuSC: Muscle Stem Cell; PAX7: Paired box 7; PIK3C3: Phosphatidylinositol 3-kinase catalytic subunit type 3.

ETC-159, an Upstream Wnt inhibitor, Induces Tumour Necrosis via Modulation of Angiogenesis in Osteosarcoma.

There is an increasing urgency in the search for new drugs to target high-grade cancers such as osteosarcomas (OS), as these have limited therapeutic options and poor prognostic outlook. Even though key molecular events leading to tumorigenesis are not well understood, it is widely agreed that OS tumours are Wnt-driven. ETC-159, a PORCN inhibitor that inhibits the extracellular secretion of Wnt, has recently progressed on to clinical trials. In vitro and in vivo murine and chick chorioallantoic membrane xenograft models were established to examine the effect of ETC-159 on OS. Consistent with our hypothesis, we noted that ETC-159 treatment not only resulted in markedly decreased ß-catenin staining in xenografts, but also increased tumour necrosis and a significant reduction in vascularity-a hereby yet undescribed phenotype following ETC-159 treatment. Through further understanding the mechanism of this new window of vulnerability, therapies can be developed to potentiate and maximize the effectiveness of ETC-159, further increasing its clinical utility for the treatment of OS.

Hematopoietic Wnts Modulate Endochondral Ossification During Fracture Healing.

Wnt signaling plays a critical role in bone formation, homeostasis, and injury repair. Multiple cell types in bone have been proposed to produce the Wnts required for these processes. The specific role of Wnts produced from cells of hematopoietic origin has not been previously characterized. Here, we examined if hematopoietic Wnts play a role in physiological musculoskeletal development and in fracture healing. Wnt secretion from hematopoietic cells was blocked by genetic knockout of the essential Wnt modifying enzyme PORCN, achieved by crossing Vav-Cre transgenic mice with Porcnflox mice. Knockout mice were compared with their wild-type littermates for musculoskeletal development including bone quantity and quality at maturation. Fracture healing including callus quality and quantity was assessed in a diaphyseal fracture model using quantitative micro computer-assisted tomographic scans, histological analysis, as well as biomechanical torsional and 4-point bending stress tests. The hematopoietic Porcn knockout mice had normal musculoskeletal development, with normal bone quantity and quality on micro-CT scans of the vertebrae. They also had normal gross skeletal dimensions and normal bone strength. Hematopoietic Wnt depletion in the healing fracture resulted in fewer osteoclasts in the fracture callus, with a resultant delay in callus remodeling. All calluses eventually progressed to full maturation. Hematopoietic Wnts, while not essential, modulate osteoclast numbers during fracture healing. These osteoclasts participate in callus maturation and remodeling. This demonstrates the importance of diverse Wnt sources in bone repair.

Anterior Cruciate Ligament Surgery Performed Less Than 3 Weeks After Injury Is Not Inferior to Delayed Surgery.

Performing anterior cruciate ligament (ACL) surgery in the immediate period after injury is controversial. However, there may be instances where the opportunity cost of delayed surgery for the patient may be unacceptable. Concomitant meniscus injuries may also prevent the patient from regaining range of motion in the preoperative period. Every week that surgery is delayed may increase pain and impair mobility for this group of patients. We investigate the functional and clinical outcomes in patients with ACL surgery in the immediate 3-week period following ACL injury. A cohort study was performed to compare the outcome of early ACL (less than 3 weeks after injury) and late ACL surgery (more than 3 weeks after surgery). A total of 58 patients were followed up at fixed time points over a 2-year period. Clinical measurements (range of motion and knee laxity scores) and functional outcome scores (International Knee Documentation Committee, Lysholm's Knee and Tegner's scores) were used to document outcomes over time. The mean time to surgery from the time of injury in the early ACL surgery group was 2 weeks (standard deviation [SD] = 0.45) and the 20 weeks (SD = 9.64) in the late ACL surgery group. The absence or presence of meniscal injuries had no significant effect on the improvement over time for both groups of patients (p > 0.05). Patients in the early ACL group had faster rates of improvement for clinical measurements and functional outcome scores but had started out initially with poorer outcomes at baseline. However, both groups had comparable outcomes (p > 0.05) at the 1- and 2-year mark postsurgery. Patients in both groups had no adverse outcomes. ACL Surgery within 3 weeks of injury (2 weeks SD = 0.45) is safe and has comparable outcomes compared to patients with delayed surgery (20 weeks SD = 9.64).

Subchondroplasty for Bone Marrow Lesions in the Arthritic Knee Results in Pain Relief and Improvement in Function.

Subchondroplasty is a relatively new joint preserving procedure, which involves the localized injection of calcium pyrophosphate bone substitute into the bone marrow lesion. The advent of magnetic resonance imaging (MRI) has greatly facilitated the identification of these bone marrow lesions. We investigated the clinical efficacy of subchondroplasty in the treatment of symptomatic bone marrow lesions in the knee, including knees with preexisting osteoarthritis. This study comprised of 12 patients whose knees were evaluated with standard radiographs and MRI to identify and localize the bone marrow lesions. They then underwent subchondroplasty under intraoperative radiographic guidance. Preoperative and postoperative visual analog scale (VAS) pain scores, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores, and Knee Injury and Arthritis Outcome Scores (KOOS) were obtained. VAS scores improved significantly from 7.5 ± 1.8 before surgery to 5.2 ± 2.7 after surgery. This further improved to 2.1 ± 2.4 at the one-year follow-up. KOOS scores improved significantly from 38.5 ± 17.0 before surgery to 73.2 ± 19.0 at the one-year follow-up. WOMAC scores improved significantly from 47.8 ± 20.5 before surgery to 14.3 ± 13.2 at the one-year follow-up. Subchondroplasty offers an effective way to treat subchondral bone marrow lesions in the arthritic knee, resulting in improvement in symptoms and early return to activity. Long-term studies are required to evaluate if these benefits can last. This is a Level II study.

YJ5 as an immunohistochemical marker of osteogenic lineage.

Overexpression of WLS, an upstream protein in the Wnt pathway, has been implicated in several non-osteogenic tumours. This study represents the first attempt at evaluating WLS expression in various bone and soft tissue tumours using YJ5, a monoclonal antibody specific to WLS, with the aim of elucidating its utility in discerning tumours with aberrant Wnt signalling and as a marker of osteogenic lineage in challenging cases. Tumour tissue sections of 144 bone mass lesions and 63 soft tissue mass lesions were immunostained with the YJ5 antibody following standardised protocols. Subsequent assessment of immunoreactivity segregated cases into one of three groups: absent/weak, moderate, or strong YJ5 immunoreactivity. For the bone tumours, strong YJ5 immunoreactivity was seen in almost all osteosarcomas and chondroblastomas, all osteoblastomas and osteoid osteomas. In contrast, all other cartilaginous tumours, chordomas, aneurysmal bone cysts, chondromyxoid fibromas, most fibrous dysplasias and most giant cell tumours exhibited absent/weak YJ5 immunostaining. For the soft tissue tumours, a more heterogeneous pattern of YJ5 immunoreactivity was observed. Because diffuse and strong YJ5 expression is identified in almost all benign and malignant bone tumours with osteoblastic activity, it can be potentially utilised as an immunohistochemical marker to support osteogenic lineage. If interpreted in the appropriate context, this marker is useful in determining whether a malignant bone tumour is an osteosarcoma, particularly in those subtypes with no or minimal osteoid or unusual morphological features. This marker can also complement SATB2 to denote osteogenic lineage.

Cord Lining Mesenchymal Stem Cells Have a Modest Positive Effect on Angiogenesis in Hindlimb Ischemia.

Purpose: We investigated the use of human Cord Lining Mesenchymal Stem Cells (CL-MSCs) (US Patent number 9,737,568), in a rabbit hindlimb ischemia model, and evaluated their potential in stimulating neovascularization. Allogenic human CL- MSCs could potentially be used to treat patients with lower limb ischemia and non-healing wounds. Methods: Twenty rabbits were divided into two separate groups. We created a hindlimb ischemia model surgically. At 21 and 49 days post-operatively, animals in the treatment group were injected with CL-MSCs (500,000 cells per 0.2 ml on each site) at 10 different sites (Quadriceps- 4 sites, Hamstrings- 4 sites and Calf--2 sites) in the hindlimb muscles. The control group received only saline injection to the corresponding sites at the same time point as the treatment group. We then evaluated the effects of treatment on neovascularization by angiography, laser doppler perfusion imaging, as well as by histology. We evaluated the tissue samples for any signs of local immune reaction to the cell implantation. We also observed the rabbit clinically for any adverse effects after treatment. Results: We found a higher number of CD31 positive cells in the treatment group, with a greater number of capillaries found in the treated muscles. The Rectus Femoris demonstrated a median vessel count/muscle fiber of 0.121 for the treatment group, compared to 0.076 in the control group (median difference 0.04; 95% CI 0.001-0.11; p = 0.041). The Gastrocnemius demonstrated a median vessel count/muscle fiber of 0.175 for the treatment group, compared to 0.089 in the control group (median difference 0.087; 95% CI -0.006 to 0.234; p = 0.07). Blood perfusion quantification through Laser Doppler Perfusion Imaging (LDPI) also demonstrated a non-statistically significant increase in perfusion in favor of the treatment group. CL-MSCs demonstrated no toxicity associated morbidity and minimal local immune reaction to implantation. Conclusion: CL-MSCs have a positive effect on angiogenesis in a rabbit hindlimb ischemia model. This preliminary data is encouraging and paves the way for future large animal studies or for clinical trials.

Non-prosthetic peri-implant fractures: classification, management and outcomes.

INTRODUCTION: Non-prosthetic peri-implant fractures (NPPIFs) are an under-reported entity. Management is challenging because of alterations in anatomy, the presence of orthopaedic implants and phenomena such as stress shielding, disuse osteopenia and fracture remodeling. The aims of this paper were to review patterns of injury, management and outcomes and to propose a classification system to aid further research. MATERIALS AND METHODS: This study is a multi-centered retrospective case series. Patients were identified from the orthopaedic department trauma databases of public hospitals in Singapore and individual surgeon case series of members of the Singapore Orthopaedic Research Collaborative (SORCE) group. RESULTS: We collected a series of 60 NPPIFs in 53 patients. 38 fractures involved the femur, 12 the radius/ulna, 5 humeri, 3 tibia/fibula and 1 clavicle. 39 patients had fractures around plates and screws, 12 around nails, and 3 around screws. Fractures were managed with a variety of surgical techniques. Six patients had surgical complications with refracture in four and non-union in two cases. Two patients had multiple refractures (total 12 additional fractures). All surgical complications required further surgery. Three patients had deep vein thrombosis and one patient died of post-operative pneumonia. Fractures were classified according to the initial implant (plate or nail), the position of the new fracture relative to the original implant (at the tip or distant) and the status of the original fracture (healed, not healed or failing). Surgical strategies for common subtypes were reviewed. CONCLUSIONS: This study represents the largest series in the literature. NPPIFs are a challenging clinical problem with a high rate of post-operative complications. They are distinct from peri-prosthetic fractures and should be understood as a separate entity. We, therefore, propose a novel classification system. Further research is needed to determine the optimal treatment for the various subtypes. LEVEL OF EVIDENCE: Therapeutic Level IV-case series.

Long-term Follow-up of Pulmonary Function and Scoliosis in Patients With Duchenne's Muscular Dystrophy and Spinal Muscular Atrophy.

BACKGROUND: Spine surgery for neuromuscular scoliosis in patients with Duchenne's Muscular Dystrophy (DMD) and Spinal Muscular Atrophy (SMA) remained controversial. This study aimed to review the long-term results of spine surgery and its effect on pulmonary function in these patients. METHODS: A retrospective review was conducted for the above patients who had undergone surgery from 1990 to 2006 in a tertiary hospital. Their yearly lung function tests, clinical records, and x-ray films before and after surgery were reviewed. All patients had at least 2 lung function tests performed before surgery and at least 3 lung function tests performed after surgery. Records of perioperative pulmonary infections that resulted in hospital admissions were also retrieved from the hospital computer system. RESULTS: Forty patients were reviewed: 29 with DMD, 11 with SMA. The mean follow-up period was 11.6 years. For patients with DMD, the mean correction of Cobb's angle from surgery was 34.1 degrees. The rate of decline of the predicted forced vital capacity preoperatively was 7.80% per year, and was reduced to 4.26% per year postoperatively (P<0.001). For patients with SMA, the mean correction of Cobb's angle from surgery was 44.1 degrees. The rate of decline of the predicted forced vital capacity preoperatively was 5.31% per year, and was reduced to 1.77% per year postoperatively (P<0.001). For both DMD and SMA patients, the difference between the rate of preoperative and postoperative pulmonary infections that resulted in hospital admission were, however, not significant (P=0.433 and 0.452, respectively). CONCLUSIONS: Scoliosis surgery in patients with DMD and SMA results in a long-term decreased rate of decline in pulmonary function over a follow-up period of more than 10 years. The level of the apical vertebrae of the scoliosis did not demonstrate a significant trend on the pulmonary function. The frequency of chest infections did not improve by scoliosis surgery. LEVEL OF SIGNIFICANCE: Level III­Retrospective study.