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Background: Muscle atrophy, fibrosis, and fatty infiltration are common to a variety of sports-related and degenerative conditions and are thought to be irreversible. Fibroadipogenic progenitors (FAPs) are multipotent resident muscle stem cells with the capacity to differentiate into fibrogenic as well as white and beige adipose tissue (BAT). FAPs that have assumed a BAT differentiation state (FAP-BAT) have proven efficacious in treating muscle degeneration in numerous injury models. Purpose: To characterize the subpopulation of murine FAPs with FAP-BAT activity, determine whether their promyogenic effect is mediated via exosomes, and analyze human FAPs for an analogous promyogenic exosome-rich subpopulation. Study Design: Controlled laboratory study. Methods: FAPs from UCP1 reporter mice were isolated via fluorescence-activated cell sorting and sorted according to the differential intensity of the UCP1 signal observed: negative for UCP1 (UCP1–), intermediate intensity (UCP1+), and high intensity (UCP1++). Bulk RNA sequencing was performed on UCP1–, UCP1+, and UCP1++ FAPs to evaluate distinct characteristics of each population. Exosomes were harvested from UCP1++ FAP-BAT exosomes (Exo-FB) as well as UCP1– non–FAP-BAT exosomes (Exo-nFB) cells using cushioned-density gradient ultracentrifugation and used to treat C2C12 cells and mouse embryonic fibroblasts in vitro, and the myotube fusion index was assessed. Exo-FB and Exo-nFB were then used to treat wild type C57B/L6J mice that had undergone a massive rotator cuff tear. At 6 weeks mice were sacrificed, and supraspinatus muscles were harvested and analyzed for muscle atrophy, fibrosis, fatty infiltration, and UCP1 expression. Single-cell RNA sequencing was then performed on FAPs isolated from human muscle that were treated with the beta-agonist formoterol or standard media to assess for the presence of a parallel promyogenic subpopulation of FAP-BAT cells in humans. Results: Flow cytometry analysis of sorted UCP1 reporter mouse FAPs revealed a trimodal distribution of UCP1 signal intensity, which correlated with 3 distinct transcriptomic profiles characterized with bulk RNA sequencing. UCP1++ cells were marked by high mitochondrial gene expression, BAT markers, and exosome surface makers; UCP1– cells were marked by fibrogenic markers; and UCP1+ cells were characterized differential enrichment of white adipose tissue markers. Exo-FB treatment of C2C12 cells resulted in robust myotube fusion, while treatment of mouse embryonic fibroblasts resulted in differentiation into myotubes. Treatment of cells with Exo-nFB resulted in poor myotube formation. Mice that were treated with Exo-FB at the time of rotator cuff injury demonstrated markedly reduced muscle atrophy and fatty infiltration as compared with treatment with Exo-nFB or phosphate-buffered saline. Single-cell RNA sequencing of human FAPs from the rotator cuff revealed 6 distinct subpopulations of human FAPs, with one subpopulation demonstrating the presence of UCP1+ beige adipocytes with a distinct profile of BAT, mitochondrial, and extracellular vesicle–associated markers. Conclusion: FAP-BAT cells form a subpopulation of FAPs with upregulated beige gene expression and exosome production that mediate promyogenic effects in vitro and in vivo, and they are present as a transcriptomically similar subpopulation of FAPs in humans. Clinical Relevance: FAP-BAT cells and their exosomes represent a potential therapeutic avenue for treating rotator cuff muscle degeneration.

OBJECTIVE This study examines the role of electronic learning platforms for medical knowledge acquisition in orthopedic surgery residency training. This study hypothesizes that all methods of medical knowledge acquisition will achieve similar levels of improvement in medical knowledge as measured by change in orthopedic in-training examination (OITE) percentile scores. Our secondary hypothesis is that residents will equally value all study resources for usefulness in acquisition of medical knowledge, preparation for the OITE, and preparation for surgical practice. DESIGN 9 ACGME accredited orthopedic surgery programs participated with 95% survey completion rate. Survey ranked sources of medical knowledge acquisition and study habits for OITE preparation. Survey results were compared to OITE percentile rank scores. PARTICIPANTS 386 orthopedic surgery residents SETTING 9 ACGME accredited orthopaedic surgery residency programs RESULTS 82% of participants were utilizing online learning resources (Orthobullets, ResStudy, or JBJS Clinical Classroom) as primary sources of learning. All primary resources showed a primary positive change in OITE score from 2018 to 2019. No specific primary source improved performance more than any other sources. JBJS clinical classroom rated highest for improved medical knowledge and becoming a better surgeon while journal reading was rated highest for OITE preparation. Orthopedic surgery residents’ expectation for OITE performance on the 2019 examination was a statistically significant predictor of their change (decrease, stay the same, improve) in OITE percentile scores (p CONCLUSIONS Our results showed that no specific preferred study source outperformed other sources. Significantly 82% of residents listed an online learning platform as their primary source which is a significant shift over the last decade. Further investigation into effectiveness of methodologies for electronic learning platforms in medical knowledge acquisition and in improving surgical competency is warranted.

Background: Fatty infiltration of rotator cuff muscle is a limiting factor in the success of repairs. Fibroadipogenic progenitors (FAPs) are a population of stem cells within the rotator cuff that can differentiate into white adipocytes, fibroblasts, and beige adipocytes. The effects of patient age and rotator cuff tendon tear size on the number, differentiation patterns, and gene expression profiles of FAPs have not yet been analyzed. Purpose: To determine if patient age and rotator cuff tear size independently regulate FAP number, differentiation patterns, and gene expression profiles. Study Design: Controlled laboratory study. Methods: Supraspinatus muscle samples were collected from 26 patients between the ages of 42 and 76 years with partial- or full-thickness rotator cuff tears. FAPs were quantified using fluorescence-activated cell sorting. Gene expression analysis was performed across a custom 96-gene panel using NanoString. In vitro differentiation assays of FAPs were conducted using adipogenic, fibrogenic, and beige-inducing (amibegron-treated) media, and quantitative polymerase chain reaction was used to assess gene expression differences between adipogenic and amibegron media conditions. Multivariable linear regressions were performed using Stata to independently analyze the effects of age and rotator cuff tear size on FAP number, differentiation, and gene expression. Results: Increasing age and tear size were independently correlated with increased FAP number (βage = 0.21, P = .03; βtear size = 3.86, P = .05). There was no clear association between age and gene expression of freshly sorted FAPs. Under adipogenic and fibrogenic media conditions, increasing age and tear size were independently associated with increased adipogenic and fibrogenic differentiation of FAPs. Under amibegron treatment conditions, age positively correlated with increased beige differentiation (β = 1.03; P < .0001), while increasing tear size showed a trend toward decreased beige differentiation (β = −4.87; P = .1). When gene expression patterns between adipogenic and amibegron media conditions were compared, larger tear size strongly inhibited beige gene expression, while advanced age did not. Conclusion: Patient age and rotator cuff tear size independently regulated FAP number, differentiation, and gene expression. Age and tear size were positively correlated with increased FAP number and fibrogenic/adipogenic differentiation. Advancing patient age did not limit FAP beige differentiation and gene expression, while increasing rotator cuff tear size strongly inhibited these processes.

Ischemia-reperfusion injury (IRI) is a critical condition associated with serious clinical manifestations. Extensive research has focused on the strategies increasing organ tolerance to IRI. Preconditioning (PC) has been shown to provide protection to various organs toward IRI. However, the underlying mechanisms remain unknown. This study aimed to evaluate the role of PC on muscle regeneration after IRI and the potential underlying mechanisms. Three-month-old male UCP-1 reporter mice underwent unilateral hindlimb IRI with or without PC, the tissue viability and injury index were measured at 24 h after IRI. Hindlimb gait, muscle contractility, muscle histology were analyzed at 2 weeks after IRI. In another group of animals, β3 adrenergic receptor (β3AR) agonist amibegron and β3AR antagonist SR-59230A were administrated before PC/IRI, the hindlimb function and muscle regeneration were evaluated at 2 weeks after IRI. Our results showed that PC has little effect on improving the tissue viability at the acute phase of IRI, but it showed a long-term beneficial role of improving hindlimb function and muscle regeneration as evidenced by increased central nuclei regenerating myofibers. The effects of PC are related to inducing muscle fibro-adipogenic progenitor (FAP) brown/beige-like adipocyte (BAT) differentiation. Amibegron treatment displayed a similar role of PC while SR-59230A abolished the effect of PC. This study suggests PC has a beneficial role in promoting muscle regeneration after IRI through β3AR signaling pathway-stimulated FAP-BAT differentiation.

Rotator cuff (RC) muscle fatty infiltration (FI) is an important factor that determines the clinical outcome of patients with RC repair. There is no effective treatment for RC muscle FI at this time. The goal of this study is to define the role Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor in regulating muscle fibro/adipogenic progenitors (FAPs) adipogenesis and treating muscle fatty degeneration after massive RC tears in a mouse model. We hypothesize that TSA reduces muscle FI after massive RC tears. HDAC activity was measured in FAPs in RC muscle after tendon/nerve transection or sham surgery. FAPs were treated with TSA for 2 weeks and FAP adipogenesis was evaluated with perilipin and Oil Red O staining, as well as reverse transcript-polymerase chain reaction for adipogenesis-related genes. About 0.5 mg/kg TSA or dimethyl sulfoxide was administered to C57B/L6 mice with massive rotator cuff tears through daily intraperitoneal injection for 6 weeks. Supraspinatus muscles were harvested for biochemical and histology analysis. We found that FAPs showed significantly higher HDAC activity after RC tendon/nerve transection. TSA treatment significantly reduced HDAC activity and inhibited adipogenesis of FAPs. TSA also abolished the role of bone morphogenetic protein-7 in inducing FAP adipogenesis and promoted FAP brown/beige adipose tissue (BAT) differentiation. TSA injection significantly increased histone H3 acetylation and reduced FI of rotator cuff muscles after massive tendon tears. Results from this study showed that TSA can regulate FAP adipogenesis and promote FAP BAT differentiation epigenetically. HDAC inhibition may be a new treatment strategy to reduce muscle FI after RC tears and repair.

BACKGROUND: Muscle atrophy and fatty infiltration (FI) are common occurrences following rotator cuff (RC) tears. Tears of all sizes are subject to muscle degeneration. The degree of muscle degeneration following RC tears is highly correlated with repair success and functional outcomes. We have recently discovered that muscle fibro-adipogenic progenitors (FAPs) can differentiate into uncoupling protein 1 (UCP-1)–expressing beige adipocytes and induce muscle regeneration. This study evaluated the potential of local cell transplantation of beige adipose FAPs (BAT-FAPs) to treat RC muscle degeneration in a murine model of RC repair. METHODS: BAT-FAPs were isolated from muscle in UCP-1 reporter mice by flow cytometry as UCP-1(+)/Sca1(+)/PDGFR(+)/CD31(−)/CD45(−)/integrin α7(−). C57/BL6J mice underwent supraspinatus tendon tear with suprascapular nerve transection followed by repair 2 or 6 weeks after the initial injury. At the time of repair, mice received either no additional treatment, phosphate-buffered saline injection, or BAT-FAP injection. Functional outcomes were assessed by gait analysis. Mice were humanely killed at 6 weeks after cell transplantation. Supraspinatus muscle FI, fibrosis, muscle fiber size, and vascularity were analyzed and quantified via ImageJ. Analysis of variance with post hoc Tukey test and P

Surgical-site delivery of local anesthetics decreases pain and opioid consumption after total knee arthroplasty (TKA). The optimal route of administration is unknown. We compared local anesthetic delivery using periarticular soft-tissue infiltration to delivery using a combination of preimplantation immersion and intra-articular injection (combination treatment). The records of patients who underwent unilateral, cemented, primary TKA with spinal anesthesia and adductor canal blocks at a single Veterans Affairs Medical Center were retrospectively reviewed. Three subgroups were compared, including controls who did not receive additional local anesthetics, patients who received periarticular infiltration, and patients who received combination treatment. Mean daily pain scores and mean 24-hour opioid consumption on postoperative days (PODs) 0 and 1 were calculated, and analysis of variance was used to assess for significant differences. Factors that were associated with lower pain scores and opioid consumption were then identified using multivariate stepwise regression. There were 26 controls, 25 periarticular infiltration patients, and 39 combination patients. The periarticular infiltration cohort had significantly lower mean pain scores and opioid consumption than controls on POD 0, but not on POD 1. The combination cohort had significantly lower mean pain scores and opioid consumption than controls on PODs 0 and 1. There were no significant differences between the infiltration and combination groups on either day. Multivariate regression analysis showed that infiltration was associated with significantly decreased opioid consumption on both days and decreased pain on POD 0. Combination treatment was associated with significantly decreased pain and opioid consumption on both days. Both local anesthetic periarticular infiltration and combination treatment are associated with decreased pain and opioid consumption after TKA. The stronger effects of the combination treatment compared with periarticular infiltration on POD 1 suggests that combination delivery may have a longer duration of action.

Microenergy acoustic pulses (MAP) is a modified low-intensity extracorporeal shock wave therapy that currently used for treating musculoskeletal disorders. However, its function on muscle regeneration after ischemia-reperfusion injury (IRI) remains unknown. This study aimed to explore the effect of MAP on muscle injury after IRI and its underlying mechanisms. Ten-week-old C57BL/6J mice underwent unilateral hindlimb IRI followed with or without MAP treatment. Wet weight of tibialis anterior muscles at both injury and contralateral sides were measured followed with histology analysis at 3 weeks after IRI. In in vitro study, the myoblasts, endothelial cells and fibro-adipogenic progenitors (FAP) were treated with MAP. Cell proliferation and differentiation were assessed, and related gene expressions were measured by real-time PCR. Our results showed that MAP significantly increased the muscle weight and centrally nucleated regenerating muscle fiber size along with a trend in activating satellite cells. In vitro data indicated that MAP promoted myoblast proliferation and differentiation and endothelial cells migration. MAP also induced FAP brown/beige adipogenesis, a promyogenic phenotype of FAPs. Our findings demonstrate the beneficial function of MAP in promoting muscle regeneration after IR injury by inducing muscle stem cells proliferation and differentiation.

Rotator cuff (RC) tears are a common cause of upper extremity disability. Any tear size can result in subsequent muscle atrophy and fatty infiltration (FI). Preoperative muscle degeneration can predict repair and postoperative functional outcomes. Muscle residential fibro-adipogenic progenitors (FAPs) are found to be capable of differentiating into beige adipocytes that release factors to promote muscle growth. This study evaluated the regenerative potential of local cell transplantation of beige FAPs to mitigate muscle degeneration in a murine massive RC tear model. Beige FAPs were isolated from muscle in UCP-1 reporter mice by flow cytometry as UCP-1+ /Sca1+ /PDGFR+ /CD31- /CD45- /integrin α7- . C57/BL6J mice undergoing supraspinatus tendon tear with suprascapular nerve transection (TT + DN) received either no additional treatment, phosphate-buffered saline injection, or beige FAP injection 2 weeks after the initial injury. Forelimb gait analysis was used to assess shoulder function with DigiGait. Mice were sacrificed 6 weeks after cell transplantation. FI, fibrosis, fiber size, vascularity were analyzed and quantified via ImageJ. Our results showed that beige FAP transplantation significantly decreased fibrosis, FI, and atrophy, enhanced vascularization compared with saline injection and non-treatment groups. Beige FAP transplantation also significantly improved shoulder function as measured by gait analysis. This study suggests that beige-differentiated FAPs may serve as a treatment option for RC muscle atrophy and FI, thus improving shoulder function in patients with massive RC tendon tears. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1159-1166, 2020.

Background Volumetric muscle loss (VML) often leads to chronic muscle weakness, impaired limb function, and even permanent disability. Recent studies suggest muscle residential fibro/adipogenic progenitors (FAPs) can adopt novel beige fat differentiation promoting muscle regeneration. The goal of this study is to define the role of FAP beige adipogenesis in muscle regeneration after VML in a mouse model.Methods Three months old male C57BL/6J mice, PDGFRα-GFP reporter mice, UCP-1 reporter mice, PDGFRα-CREERT/DTA inducible FAP depletion mice and NSG immune-deficient mice were used in this study. Volumetric muscle loss (VML) was created on tibialis anterior (TA) muscle with a punch. To induce FAP beige adipogenesis, Amibegron, a beta 3 adrenergic receptor (B3AR) agonist was administered to mice with I.P. injection. In a separate group, murine and human beige adipogenic FAPs was transplanted to mouse muscle after VML. Limb function was measured with gait analysis at 2 and 6 weeks after VML. Muscle histology and FAP gene expression analysis was also conducted at 2 and 6 weeks after VML.Results After VML, we observed robust proliferation of FAPs in PDGFRα-GFP reporter mice. PDGFRα-CREERT/DTA mice inducible FAP depletion mice showed reduced muscle regeneration after FAPs are depleted, suggesting a positive role of FAP in muscle regeneration after VML. Both Amibegron treatment and beige FAP transplantation significantly improved muscle regeneration and limb function after VML.Conclusion Stimulating FAP beige adipogenesis with B3AR agonists or transplantation of beige adipogenic FAPs could serve as new strategies in treating VML.

The high prevalence of rotator cuff tears poses challenges to individual patients and the healthcare system at large. This orthopedic injury is complicated further by high rates of retear after surgical repair. Outcomes following repair are highly dependent upon the quality of the injured rotator cuff muscles, and it is, therefore, crucial that the pathophysiology of rotator cuff degeneration continues to be explored. Fibro-adipogenic progenitors, a major population of resident muscle stem cells, have emerged as the main source of intramuscular fibrosis and fatty infiltration, both of which are key features of rotator cuff muscle degeneration. Improvements to rotator cuff repair outcomes will likely require addressing the muscle pathology produced by these cells. The aim of this review is to summarize the current rotator cuff degeneration assessment tools, the effects of poor muscle quality on patient outcomes, the role of fibro-adipogenic progenitors in mediating muscle pathology, and how these cells could be leveraged for potential therapeutics to augment current rotator cuff surgical and rehabilitative strategies.

Spinal cord injury (SCI) is accompanied by rapid loss of bone and increased risk of low impact fractures. Current pharmacological treatment approaches have proven to be relatively ineffective in preventing or treating bone loss after SCI. Dietary supplementation with dried plum (DP) has been shown to have dramatic effects on bone in various other disease models. In this study, we tested the efficacy of DP in preventing bone loss after SCI and restoring bone that has already been lost in response to SCI. Male C57BL/6J mice (3‐month‐old) underwent SCI and were fed a diet containing 25% DP by weight or a control diet for up to 4 weeks to assess whether DP can prevent bone loss. To determine whether DP could restore bone already lost due to SCI, mice were put on a control diet for 2 weeks (to allow bone loss) and then shifted to a DP supplemented diet for an additional 2 weeks. The skeletal responses to SCI and dietary supplementation with DP were assessed using microCT analysis, bone histomorphometry and strength testing. Dietary supplementation with DP completely prevented the loss of bone and bone strength induced by SCI in acutely injured mice. DP also could restore a fraction of the bone lost and attenuate the loss of bone strength after SCI. These results suggest that dietary supplementation with DP or factors derived from DP may prove to be an effective treatment for the loss of bone in patients with SCI., Spinal cord injury (SCI) is accompanied by rapid loss of bone and increased risk of low impact fractures. In this study, we showed that diet supplement of dried plum (DP) completely prevented bone loss and restored a fraction of the bone lost after SCI in a mouse model. These results suggest that dietary supplementation with DP or factors derived from DP may be an effective treatment for the loss of bone after SCI.

Author(s): Freshman, Ryan D; Cortez, Xavier C; Kim, Hubert T; Feeley, Brian T; Zhang, Alan L; Lansdown, Drew A | Abstract: PurposeTo evaluate research listed as "Submitted" on orthopaedic surgery residency applications for eventual publication rates and quality.SignificanceAs the orthopaedic surgery residency selection process becomes increasingly competitive, the number of research publications listed on applications continually increases. However, the utility of using publications listed as "Submitted" in the applicant evaluation process remains unknown.MethodsDemographic and publication data were retrospectively collected from 1303 applications to an orthopaedic surgery residency program. The PubMed database was used to verify "Submitted" publications for (1) publication fruition or (2) publication mismatch, defined as discordance between the listed journal of submission and the eventual journal of publication.ResultsA total of 594 applications (45.6%) listed ≥1 publication as "Submitted." Out of 1636 "Submitted" publications, 565 were unverifiable (32.5%). Of the 1071 verified publications, 362 (33.8%) experienced publication mismatch. Within this subgroup, a significant difference existed between the mean impact factors of the listed journal of submission and the eventual journal of publication (1.5 ± 2.7 versus 3.0 ± 2.5, P l 0.01). Demographic data were not predictive of having an unverified publication.ConclusionPublications listed as "Submitted" in orthopaedic surgery residency applications frequently remain unpublished or are published in less impactful journals than originally intended.

BACKGROUND: Rotator cuff (RC) muscle atrophy and fatty infiltration (FI) are independent factors correlated with failure of attempted tendon repair in larger RC tears. However, there is no effective treatment for RC muscle atrophy and FI at this time. The recent discovery of beige adipose tissue (BAT) in adults shed light on a new avenue in treating obesity and excessive fat deposition by promoting BAT activity. The goal of this study was to define the role of intramuscular BAT in RC muscle FI and the effect of β(3)-adrenergic receptor agonists in treating RC muscle FI by promoting BAT activity. MATERIALS AND METHODS: Three-month-old wild-type C57BL/6J, platelet derived growth factor receptor-alpha (PDGFRα) green fluorescent protein (GFP) reporter and uncoupling protein 1 (UCP-1) knockout mice underwent a unilateral RC injury procedure, which included supraspinatus (SS) and infraspinatus tendon resection and suprascapular nerve transection. To stimulate BATactivity, amibegron, a selective β(3)-adrenergic receptor agonist, was administered to C57BL/6J mice either on the same day as surgery or 6 weeks after surgery through daily intraperitoneal injections. Gait analysis was conducted to measure forelimb function at 6 weeks or 12 weeks (in groups receiving delayed amibegron treatment) after surgery. Animals were killed humanely at 6 weeks (or 12 weeks for delayed amibegron groups) after surgery. SS muscles were harvested and analyzed histologically and biochemically. RESULTS: Histologic analysis of SS muscles from PDGFRα-GFP reporter mice showed that PDGFRα-positive fibroadipogenic progenitors in RC muscle expressed UCP-1, a hallmark of BAT during the development of FI after RC tears. Impairing BAT activity by knocking out UCP-1 resulted in more severe muscle atrophy and FI with inferior forelimb function in UCP-1 knockout mice compared with wild-type mice. Promoting BAT activity with amibegron significantly reduced muscle atrophy and FI after RC tears and improved forelimb function. Delayed treatment with amibegron reversed muscle atrophy and FI in muscle. CONCLUSIONS: Fat accumulated in muscle after RC tears possesses BAT characteristics. Impairing BAT activity results in worse RC muscle atrophy and FI. Amibegron reduces and reverses RC atrophy and FI by promoting BAT activity. LEVEL OF EVIDENCE: Basic Science Study; Histology; In Vivo Animal Model

Absract Background Paraspinal muscles are crucial for vertebral stabilization and movement. These muscles are prone to develop fatty infiltration (FI), fibrosis, and atrophy in many spine conditions. Fibro‐adipogenic progenitors (FAPs), a resident muscle stem cell population, are the main contributors of muscle fibrosis and FI. FAPs are involved in a complex interplay with satellite cells (SCs), the primary myogenic progenitor cells within muscle. Little is known about the stem cell composition of the multifidus. The aim of this study is to examine FAPs and SCs in the multifidus in disc herniation patients. Multifidus muscle samples were collected from 10 patients undergoing decompressive spine surgery for lumbar disc herniation. Hamstring muscle was collected from four patients undergoing hamstring autograft ACL reconstruction as an appendicular control. Multifidus tissue was analyzed for FI and fibrosis using Oil‐Red‐O and Masson's trichrome staining. FAPs and SCs were visualized using immunostaining and quantified with fluorescence‐activated cell sorting (FACS) sorting. Gene expression of these cells from the multifidus were analyzed with reverse transcription‐polymerase chain reaction and compared to those from hamstring muscle. FI and fibrosis accounted for 14.2%± 7.4% and 14.8%±4.2% of multifidus muscle, respectively. The multifidus contained more FAPs (11.7%±1.9% vs 1.4%±0.2%; P, Paraspinal muscles are known to develop muscle fatty infiltration (FI), fibrosis, and atrophy in a number of spine conditions, including intervertebral disc herniation. In this study, we histologically examined multifidus muscle specimens harvested from patients undergoing spine surgery for the degree of degenerative muscle pathology and characterized fibro‐adipogenic progenitors (FAPs) and Satellite cells (SCs) within the muscle in terms of quantity and gene expression profiles. We discovered a high degree of FI and fibrosis in the multifidus as well as large quantities of FAPs and SCs that displayed different gene expression profiles than those taken from healthy hamstring muscle controls.

Background: Successful repair of large and massive rotator cuff (RC) tears remains a challenge at least partially because of secondary muscle atrophy and fatty infiltration. β3 Adrenergic agonists are a group of drugs that promote fat resorption through “white fat browning” of intramuscular stem cells. Purpose: To test the role of a β3 adrenergic receptor agonist, amibegron, in improving muscle quality and forelimb function in a delayed RC repair model via promoting brown/beige adipose tissue activation. Study Design: Controlled laboratory study. Methods: Three-month-old PDGFRα-GFP reporter mice, wild type C57BL/6J mice, and uncoupling protein 1 (UCP-1) knockout mice underwent unilateral supraspinatus tendon transection with a 6-week delayed tendon repair. Animals with sham surgery served as controls. Amibegron was given either immediately after tendon transection or after repair. Gait analysis was conducted to measure forelimb function at 6 weeks after tendon repair. Animals were sacrificed at 6 weeks after repair. Supraspinatus muscles were harvested and analyzed histologically. Reverse transcription polymerase chain reaction was performed to quantify gene expression related to atrophy, fibrosis, and fatty infiltration. Results: Histology of PDGFRα reporter mice showed significantly increased UCP-1 expression, suggesting white fat browning in muscle after RC repair. As administered either immediately after tendon transection or after tendon repair, amibegron significantly reduced muscle atrophy and fatty infiltration and resumed normal upper extremity gait in wild type mice. However, the effect of amibegron was not present in UCP-1 knockout mice, suggesting that the effect of amibegron in treating RC muscle atrophy and fatty infiltration is through a UCP 1–dependent mechanism. Conclusion: Amibegron reduced muscle atrophy and fatty infiltration and improved forelimb function after delayed RC repair through a UCP 1–dependent mechanism. This may be an effective clinical treatment strategy for patients to improve muscle quality after RC repair. Clinical Relevance: β3 Adrenergic agonists may serve as a new pharmacologic modality to treat RC muscle atrophy and fatty infiltration to improve clinical outcome of RC repair.

Fatty infiltration (FI) of rotator cuff (RC) muscles is common in patients with RC tears. Studies have demonstrated that fibro-adipogenic progenitors (FAPs), a population of resident muscle stem cells, are the main contributors of FI, which adversely affects muscle quality and RC repair success. Although FI is common in RC injuries, it is not frequently reported after other musculotendinous injuries. Additionally, studies have shown the development of different pathology patterns across muscle groups suggestive of intrinsic differences in cellular composition and behavior. This study evaluates FAP distribution and differentiation properties across anatomic locations in mice. Muscles from seven different anatomic locations were harvested from PDGFRα-eGFP FAP reporter mice. FAPs were quantified using histology and FACS sorting with BD Aria II with CD31- /CD45- /Integrinα7- /Sca-1+ and PDGFRα reporter signal (n = 3 per muscle). The cells were analyzed for adipogenesis using immunocytochemistry and for proliferation properties with Brdu-Ki67 staining. In a separate group of mice, RC and tibialis anterior muscles received glycerol injection and were harvested after 2 weeks for FI quantification (n = 4). One-way analysis of variance was used for statistical comparisons among groups, with significance at p < 0.05. FAPs from the RC, masseter, and paraspinal muscles were more numerous and demonstrated greater proliferative capacity and adipogenic potency than those from the tibialis anterior and gastrocnemius. The RC demonstrated significantly greater levels of FI than the tibialis anterior after glycerol-injection injury. Clinical Significance: This study suggests differences in FAP distribution and differentiation characteristics may account for the propensity to develop FI in RC tears as compared with other musculotendinous injuries. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1113-1121, 2020.

Previous studies have suggested that macrophage-mediated chronic inflammation is involved in the development of rotator cuff muscle atrophy and degeneration following massive tendon tears. Increased RhoA signaling has been reported in chronic muscle degeneration, such as muscular dystrophy. However, the role of RhoA signaling in macrophage infiltration and rotator muscle degeneration remains unknown. Using a previously established rat model of massive rotator cuff tears, we found RhoA signaling is upregulated in rotator cuff muscle following a massive tendon-nerve injury. This increase in RhoA expression is greatly potentiated by the administration of a potent RhoA activator, lysophosphatidic acid (LPA), and is accompanied by increased TNFα and TGF-β1 expression in rotator cuff muscle. Boosting RhoA signaling with LPA significantly worsened rotator cuff muscle atrophy, fibrosis, and fatty infiltration, accompanied with massive monocytic infiltration of rotator cuff muscles. Co-staining of RhoA and the tissue macrophage marker CD68 showed that CD68+ tissue macrophages are the dominant cell source of increased RhoA signaling in rotator cuff muscles after tendon tears. Taken together, our findings suggest that LPA-mediated RhoA signaling in injured muscle worsens the outcomes of atrophy, fibrosis, and fatty infiltration by increasing macrophage infiltraion in rotator cuff muscle. Clinically, inhibiting RhoA signaling may represent a future direction for developing new treatments to improve muscle quality following massive rotator cuff tears. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1539-1547, 2017.

Background: Rotator cuff (RC) tears are common tendon injuries seen in orthopaedic patients. Successful repair of large and massive RC tears remains a challenge due to our limited understanding of the pathophysiological features of this injury. Clinically relevant small animal models that can be used to study the pathophysiological response to repair are limited by the lack of chronic repair models. Purpose: To develop a highly clinically relevant mouse model of delayed RC repair. Study Design: Controlled laboratory study. Methods: Three-month-old C57BL/6J mice underwent unilateral supraspinatus (SS) and infraspinatus (IS) tendon tear with immediate, 2-week delayed, or 6-week delayed tendon repair. Animals with no repair or sham surgery served as controls. Gait analysis was conducted to measure shoulder function at 2 weeks and 6 weeks after surgery. Animals were sacrificed 6 weeks after the last surgery. Shoulder joint, SS, and IS muscles were harvested and analyzed histologically. Ex vivo mechanical testing of intact and repaired SS and IS tendons was conducted. Reverse-transcriptase polymerase chain reaction was performed on SS and IS muscles to quantify atrophy, fibrosis, and fatty infiltration–related gene expression. Results: Histological and tendon mechanical testing showed that torn tendons could be successfully repaired as late as 6 weeks after transection. However, significant atrophy and fatty infiltration of muscle, with impaired shoulder function, were persistent in the 6-week delayed repair group. Shoulder function correlated with the severity of RC muscle weight loss and fatty infiltration. Conclusion: We successfully developed a clinically relevant mouse model of delayed RC repair. Six-week delayed RC repair resulted in persistent muscle atrophy and fatty infiltration with inferior shoulder function compared with acute repair. Clinical Relevance: Our novel mouse model could serve as a powerful tool to understand the pathophysiological and cellular/molecular mechanisms of RC muscle and tendon degeneration, eventually improving our strategies for treating and repairing RC tears.

BACKGROUND: In large rotator cuff tears, retraction of the supraspinatus muscle creates suprascapular nerve traction and compression. However, suprascapular nerve transection, when used in previous models, is different from chronic compression of the suprascapular nerve in patients. To define the role of suprascapular nerve chronic injury in rotator cuff muscle atrophy and fatty infiltration, we developed a novel reversible suprascapular nerve compression mouse model. QUESTIONS/PURPOSES: We asked: (1) Can suprascapular nerve injury be induced by compression but reversed after compression release? (2) Can muscle fatty infiltration be induced by suprascapular nerve compression and reversed after compression release? (3) Is white fat browning involved in fatty infiltration resorption? METHODS: Mice in a common strain of C57BL/6J were randomly assigned to suprascapular nerve transection (n = 10), nerve compression (n = 10), nerve compression and release (n = 10), or sham control (n = 10) groups. To study the role or white fat browning on muscle fatty infiltration, additional UCP1 reporter mice (n = 4 for nerve compression and n = 4 for nerve compression release) and knockout mice (n = 4 for nerve compression and n = 4 for nerve compression release) were used. Nerve injury was testified using osmium tetroxide staining and neural muscular junction staining and then semiquantified by counting the degenerating axons and disrupted junctions. Muscle fatty infiltration was evaluated using Oil Red O staining and then semiquantified by measuring the area fraction of fat. Immunofluorescent and Oil Red O staining on UCP1 transgenic mice was conducted to testify whether white fat browning was involved in fatty infiltration resorption. Ratios of UCP1 positively stained area and fat area to muscle cross-section area were measured to semiquantify UCP1 expression and fatty infiltration in muscle by blinded reviewers. Analysis of variance with Tukey post hoc comparisons was used for statistical analysis between groups. RESULTS: Suprascapular nerve injury was induced by compression but reversed after release. The ratios of degenerating axons were: sham control: 6% ± 3% (95% confidence interval [CI], 3%-10%); nerve compression: 58% ± 10% (95% CI, 45%-70% versus sham, p < 0.001); and nerve compression and release: 15% ± 9% (95% CI, 5%-26% versus sham, p = 0.050). The supraspinatus muscle percentage area of fatty infiltration increased after 6 weeks of nerve compression (19% ± 1%; 95% CI, 18%-20%; p < 0.001) but showed no difference after compression release for 6 weeks (5% ± 3%; 95% CI, 1%-10%; p = 0.054) compared with sham (2% ± 1%; 95% CI, 1%-3%). However, the fat area fraction in UCP1 knockout mice did not change after nerve compression release (6% ± 1%; 95% CI, 4%-8% at 2 weeks after compression and 5% ± 0.32%; 95% CI, 4%-6% after 2 weeks of release; p = 0.1095). CONCLUSIONS: We developed a clinically relevant, reversible suprascapular nerve compression mouse model. Fatty infiltration resorption after compression release was mediated through white fat browning. CLINICAL RELEVANCE: If the mechanism of browning of white fat in rotator cuff muscle fatty infiltration can be confirmed in humans, a UCP1 agonist may be an effective treatment for patients with suprascapular nerve injury.

This study stratifies complication risk in primary total joint arthroplasty (TJA) based on body mass index (BMI). Demographics, co-morbidities, perioperative variables, and complications were reviewed for 22,808 patients. Chi-squared, one-way ANOVA, univariate and multivariable regression analysis were performed. Increasing BMI led to an increase (P0.05) in combined complications, acute kidney injury (AKI), cardiac arrest (CA), reintubation, reoperation, and superficial infection (SI). Univariate analysis for BMI40 revealed an increase in combined complications (15.21-vs-17.40%), AKI (1.93-vs-3.87%), CA (0.22-vs-0.57%), reintubation (0.47-vs-0.95%), reoperation (2.36-vs-3.37%), and SI (0.82-vs-1.65%). Multivariable regression showed BMI40 as an independent predictor for combined complications (OR=1.18), AKI (OR=1.79), CA (OR=3.94), reintubation (OR=2.56), reoperation (OR=1.44), and SI (OR=2.11). Morbid obesity confers increased risk for complications in TJA.

Ischemia-reperfusion injury (IRI) occurs when blood returns to tissues following a period of ischemia. Reintroduction of blood flow results in the production of free radicals and reactive oxygen species that damage cells. Skeletal muscle IRI is commonly seen in orthopedic trauma patients. Experimental studies in other organ systems have elucidated the importance of extracellular and intracellular matrix metalloproteinase-2 (MMP-2) isoforms in regulating tissue damage in the setting of oxidant stress resulting from IRI. Although the extracellular full-length isoform of MMP-2 (FL-MMP-2) has been previously studied in the setting of skeletal muscle IRI, studies investigating the role of the N-terminal truncated isoform (NTT-MMP-2) in this setting are lacking. In this study, we first demonstrated significant increases in FL- and NTT-MMP-2 gene expression in C2C12 myoblast cells responding to re-oxygenation following hypoxia in vitro. We then evaluated the expression of FL- and NTT-MMP-2 in modulating skeletal muscle IRI using a previously validated murine model. NTT-MMP-2, but not FL-MMP-2 expression was significantly increased in skeletal muscle following IRI. Moreover, the expression of toll-like receptors (TLRs) -2 and -4, IL-6, OAS-1A, and CXCL1 was also significantly up-regulated following IRI. Treatment with the potent anti-oxidant pyrrolidine dithiocarbamate (PDTC) significantly suppressed NTT-MMP-2, but not FL-MMP-2 expression and improved muscle viability following IRI. This data suggests that NTT-MMP-2, but not FL-MMP-2, is the major isoform of MMP-2 involved in skeletal muscle IRI. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Available evidence indicates that some Tie2-expressing (Tie2(+) ) cells serve as multipotent progenitors that have robust BMP-dependent osteogenic activity and mediate heterotopic ossification (HO). Since signaling through the G protein Gi is required for cell motility, we hypothesized that blockade of endogenous Gi signaling in Tie2(+) cell populations would prevent HO formation. Blockade of Gi signaling in Tie2(+) cells was accomplished in transgenic mice with expression of pertussis toxin (PTX) under the control of the Tie2 promoter (Tie2(+) /PTX(+) ). Bone formation within HOs was evaluated 2 weeks after BMP injection. Expression of PTX in Tie2(+) cells significantly reduced the bone volume (BV) of HOs in male and female mice. Orthotopic bones were assessed at the distal femur and expression of PTX significantly increased trabecular bone fractional volume and bone formation rate in females only. In adult Tie2(+) /GFP(+) mice, GFP(+) cells appeared both inside and at the surfaces of bone tissue within HOs and in orthotopic bones. In summary, blockade of Gi signaling in Tie2(+) cells reduced the accrual of HOs and stimulated osteogenesis in orthotopic bones. Targeting of Gi protein coupled receptors in Tie2(+) cells may be a novel therapeutic strategy in states of abnormal bone formation such as osteoporosis and HO.

Rotator cuff tears (RCTs) are among the most common musculoskeletal injuries seen by orthopaedic surgeons. Clinically, massive cuff tears lead to unique pathophysiological changes in rotator cuff muscle, including atrophy, and massive fatty infiltration, which are rarely seen in other skeletal muscles. Studies in a rodent model for RCT have demonstrated that these histologic findings are accompanied by activation of the Akt/mammalian target of rapamycin (mTOR) and transforming growth factor-β (TGF-β) pathways following combined tendon-nerve injury. The purpose of this study was to compare the histologic and molecular features of rotator cuff muscle and gastrocnemius muscle--a major hindlimb muscle, following combined tendon-nerve injury. Six weeks after injury, the rat gastrocnemius did not exhibit notable fatty infiltration compared to the rotator cuff. Likewise, the adipogenic markers SREBP-1 and PPARγ as well as the TGF-β canonical pathway were upregulated in the rotator cuff, but not the gastrocnemius. Our study suggests that the rat rotator cuff and hindlimb muscles differ significantly in their response to a combined tendon-nerve injury. Clinically, these findings highlight the unique response of the rotator cuff to injury, and may begin to explain the poor outcomes of massive RCTs compared to other muscle-tendon injuries.

Heterotopic ossification (HO) develops in about 20% to 30% of patients with spinal cord injury (SCI) and significantly impairs their rehabilitation. There is no effective prevention or treatment for this condition at this time. Our current understanding of its etiology and pathophysiology is limited partially due to the lack of clinically relevant animal models. In this study, we report a novel mouse model of SCI-induced HO by administering a subthreshold dose of bone morphogenetic protein (BMP)-2 to muscles in mice after SCI. Micro-computed tomography scanning showed that an intramuscular injection of 0.25 micrograms of BMP-2 causes significant HO in mice with SCI but not in control (sham surgery) mice. Our analysis of gene expression showed significantly increased BMP signaling in quadriceps following SCI, suggesting that BMP signaling may play a role in SCI-induced HO. Administering 0.25 micrograms of BMP-2 to the front arms of the mice with SCI also results in the development of significant HO but not in control mice. This suggests that SCI causes a systematic osteogenic effect, which is not limited to paralyzed limbs. This novel mouse model will serve as a powerful tool in exploring the molecular mechanisms of SCI-induced HO, which may lead to novel treatment for this disease.

Joint instability and cartilage trauma have been previously studied and identified as key mediators in the development of posttraumatic osteoarthritis (PTOA). The purpose of this study was to use an in vivo model to compare the effect of joint instability, caused by the rupture of the anterior cruciate ligament (ACL), versus cartilage compression. In this study, mice were subjected to cyclical axial loads of twelve Newtons (N) for 240 cycles or until the ACL ruptured. One and eight weeks after this procedure, knees were sectioned coronally and evaluated for osteoarthritis by histology. Using a scoring scale established by [Pritzker K, Gay S, Jimenez S, et al. (2006): Osteoarthritis Cartilage 14:13-29], the articular cartilage across each surface was scored and combined to produce a total degeneration score. The ACL-ruptured group had a significantly greater total degeneration score than either control or compression treated joints at 1 and 8 weeks. Additionally, only sections from ACL-ruptured knees consistently showed synovitis after 1 week and osteophyte formation after 8 weeks. Thus, it appears using that ACL rupture consistently creates a severe osteoarthritis phenotype, while axial cartilage compression alone does not appear to be an appropriate method of inducing PTOA in vivo.

Severe soft tissue trauma is associated with heterotopic ossification (HO), the abnormal deposition of bone at extra-skeletal sites. The pathophysiology of the development of trauma-induced HO remains largely unknown due in part to the lack of appropriate animal models. In this study, we sought to develop a new trauma-induced HO mouse model using muscle impact injury combined with low dose BMP-2. BMP-2 at doses ranging from 0 to 2 µg was injected into quadriceps muscles of adult male C57/BL6 mice. Animals then received a one-time quadriceps impaction injury to mimic the trauma associated with severe injuries. HO was monitored using in vivo microCT scanning at 1, 2, 4, and 8 weeks after treatment. After trauma, the expression of BMP-2, -4, BMP receptor 1, SOX9 and RUNX2 were increased in muscle. Although little or no HO was observed in mice receiving 1 µg BMP-2, combining this dose with muscle trauma produced an abundance of HO. At higher doses of BMP-2, trauma did not augment mineral deposition. These results suggest that BMP-2 signaling can sensitize muscle to trauma-induced HO. They also provide the basis for a new model to study the pathogenesis of trauma-induced HO. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:183–188, 2014.

Previous studies have evaluated role of Akt/mTOR signaling in rotator cuff muscle atrophy and determined that there was differential in signaling following tendon transection (TT) and suprascapular nerve (SSN) denervation (DN), suggesting that atrophy following TT and DN was modulated by different protein degradation pathways. In this study, two muscle proteolytic systems that have been shown to be potent regulators of muscle atrophy in other injury models, the ubiquitin-proteasome pathway and autophagy, were evaluated following TT and DN. In addition to examining protein degradation, this study assessed protein synthesis rate following these two surgical models to understand how the balance between protein degradation and synthesis results in atrophy following rotator cuff injury. In contrast to the traditional theory that protein synthesis is decreased during muscle atrophy, this study suggests that protein synthesis is up-regulated in rotator cuff muscle atrophy following both surgical models. While the ubiquitin-proteasome pathway was a major contributor to the atrophy seen following DN, autophagy was a major contributor following TT. The findings of this study suggest that protein degradation is the primary factor contributing to atrophy following rotator cuff injury. However, different proteolytic pathways are activated if SSN injury is involved.

Background: Local anesthetics are frequently delivered intra-articularly to provide perioperative pain control. Previous studies have shown that the commonly used drugs lidocaine, ropivacaine, and bupivacaine can be toxic to human chondrocytes. The present study was conducted to determine whether the toxic effects of local anesthetics on human chondrocytes also extend to human mesenchymal stem cells. Methods: Human mesenchymal stem cells from three healthy donors were grown in tissue culture and exposed to the following anesthetic treatments for sixty minutes: (1) 1% lidocaine, (2) 2% lidocaine, (3) 0.25% bupivacaine, (4) 0.5% bupivacaine, (5) 0.2% ropivacaine, and (6) 0.5% ropivacaine. The cells were then allowed to recover for twenty-four hours in regular growth media, and viability was measured with use of fluorescent staining for live cells or a luminescence assay for ATP content. Results: The live cell counts and ATP content were correlated (r2 = 0.79), and 2% lidocaine was found to be significantly more toxic than all doses of bupivacaine and ropivacaine. Treatment with 1% lidocaine resulted in significantly fewer live cells (49%) compared with the control, and the live cell count was also significantly less than that for the other anesthetics. However, the ATP level in the 1% lidocaine group was not significantly lower than those in the other groups. Bupivacaine and ropivacaine did not exhibit significant differences in toxicity compared with the control or with each other. Conclusions: Ropivacaine and bupivacaine had limited toxicity in human mesenchymal stem cells. However, lidocaine could significantly decrease mesenchymal stem cell viability. Since other studies have shown ropivacaine to be less toxic to chondrocytes than bupivacaine, ropivacaine may be a safer intra-articular anesthetic. Clinical Relevance: Mesenchymal stem cells likely play a key role in healing following surgical procedures such as microfracture and ligament reconstruction. If local anesthetics are used following joint surgery, selection of an agent with low toxicity toward mesenchymal stem cells, such as ropivacaine, may maximize tissue healing potential.

Backgroundrotator cuff muscle atrophy, fibrosis and fatty infiltration are common complications after large and massive rotator cuff tears. Currently, there are no effective treatments for these muscle pathologies after injury. Furthermore, the cellular source for fibrotic and adipose tissues in rotator cuff muscle after injury remains unknown. In this study, we proposed that two groups of muscle resident progenitors, Tie2+ muscle mesenchymal progenitors and PDGFRα(+) fibro/adipogenic progenitor cells (FAPs), contribute significantly to rotator cuff muscle fibrosis and fatty infiltration.Methodswe tested our hypothesis using reporter mice. Rotator cuff muscles from Tie2-GFP and PDGFRα-GFP reporter mice were harvested at 2 and 6 weeks after unilateral massive rotator cuff tear surgeries. Immunofluorescent staining for fibroblast and adipocyte markers was conducted.Resultsour results showed significant co-localization of Tie2+ cells with fibrotic markers vimentin and αSMA. In the PDGFRα-GFP reporter mice, GFP signal was seen in only a small fraction of cells staining positive for vimentin and αSMA. However, PDGFRα showed significant co-localization with adipocyte markers, including PPAR-γ, adiponectin, and perilipin A. Oil red O staining confirmed that the mature adipocytes appearing in rotator cuff muscles after injury are also PDGFRα(+).Conclusionthese data demonstrated that the Tie2(+) muscle mesenchymal progenitors are the major source of fibroblasts while PDGFRα(+) FAPs are the major source of adipocytes in rotator cuff muscle fatty infiltration. Basic Science Study.

Previous studies have suggested that macrophage-mediated chronic inflammation is involved in the development of rotator cuff muscle atrophy and degeneration following massive tendon tears. Increased RhoA signaling has been reported in chronic muscle degeneration, such as muscular dystrophy. However, the role of RhoA signaling in macrophage infiltration and rotator muscle degeneration remains unknown. Using a previously established rat model of massive rotator cuff tears, we found RhoA signaling is upregulated in rotator cuff muscle following a massive tendon-nerve injury. This increase in RhoA expression is greatly potentiated by the administration of a potent RhoA activator, lysophosphatidic acid (LPA), and is accompanied by increased TNFα and TGF-β1 expression in rotator cuff muscle. Boosting RhoA signaling with LPA significantly worsened rotator cuff muscle atrophy, fibrosis, and fatty infiltration, accompanied with massive monocytic infiltration of rotator cuff muscles. Co-staining of RhoA and the tissue macrophage marker CD68 showed that CD68+ tissue macrophages are the dominant cell source of increased RhoA signaling in rotator cuff muscles after tendon tears. Taken together, our findings suggest that LPA-mediated RhoA signaling in injured muscle worsens the outcomes of atrophy, fibrosis, and fatty infiltration by increasing macrophage infiltraion in rotator cuff muscle. Clinically, inhibiting RhoA signaling may represent a future direction for developing new treatments to improve muscle quality following massive rotator cuff tears. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1539-1547, 2017.

IntroductionCurrent tissue engineering strategies to heal critical-size bone defects through direct bone formation are limited by incomplete integration of grafts with host bone and incomplete graft vascularization. An alternative strategy for bone regeneration is the use of cartilage grafts that form bone through endochondral ossification. Endochondral cartilages stimulate angiogenesis and are remodeled into bone, but are found in very small quantities in growth plates and healing fractures. We sought to develop engineered endochondral cartilage grafts using osteoarthritic (OA) articular chondrocytes as a cell source. Such chondrocytes often undergo hypertrophy, which is a characteristic of endochondral cartilages.Materials and methodsWe compared the ability of unmodified human OA (hOA) cartilage and cartilage grafts formed in vitro from hOA chondrocytes to undergo endochondral ossification in mice. Scaffold-free engineered chondrocyte grafts were generated by pelleting chondrocytes, followed by culture with transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein 4. Samples derived from either primary or passaged chondrocytes were implanted subcutaneously into immunocompromised mice. Grafts derived from passaged chondrocytes from three patients were implanted into critical-size tibial defects in mice. Bone formation was assessed with histology after 4 weeks of implantation. The composition of tibial repair tissue was quantified with histomorphometry.ResultsEngineered cartilage grafts generated from passaged OA chondrocytes underwent endochondral ossification after implantation either subcutaneously or in bone. Cartilage grafts integrated with host bone at 15 out of 16 junctions. Grafts variably remodeled into woven bone, with the proportion of bony repair tissue in tibial defects ranging from 22% to 85% (average 48%). Bony repair tissue bridged the tibial defects in half of the animals. In contrast, unmodified OA cartilage and engineered grafts formed from primary chondrocytes did not undergo endochondral ossification in vivo.ConclusionshOA chondrocytes can adopt an endochondral phenotype after passaging and TGF-β superfamily treatment. Engineered endochondral cartilage grafts can integrate with host bone, undergo ossification, and heal critical-size long-bone defects in a mouse model. However, additional methods to further enhance ossification of these grafts are required before the clinical translation of this approach.

Rotator cuff tears (RCTs) are among the most common injuries seen in orthopedic patients. Chronic tears can result in the development of muscular atrophy and fatty infiltration. Despite the prevalence of RCTs, little is known about the underlying molecular pathways that produce these changes. Recently, we have shown that mammalian target of rapamycin (mTOR) signaling plays an important role in muscle atrophy that results from massive RCTs in a rat model. The purpose of this study was therefore to extend our understanding of mTOR signaling and evaluate its role in fatty infiltration after a combined tendon transection and suprascapular nerve denervation surgery. Akt/mTOR signaling was significantly increased and resulted in the up-regulation of two transcription factors: SREBP-1 and PPARγ. We also saw an increase in expression of adipogenic markers: C/EBP-α and FASN. Upon treatment with rapamycin, an inhibitor of mTOR, we observed a decrease in mTOR signaling, activity of transcription factors, and reduction in fatty infiltration. Therefore, our study suggests that mTOR signaling mediates rotator cuff fatty infiltration via SREBP-1 and PPARγ. Clinically, our finding may alter current treatment methods to address rotator cuff fatty infiltration.

Aim: Current synthetic tubular conduits are inferior to nerve autograft for the repair of segmental peripheral nerve injuries. We examined motor outcomes with the use of longitudinally aligned poly (L-lactide-co-caprolactone) nanofiber conduits for repair of nerve gap injury in a rat model. Methods: Ten-millimeter segments of sciatic nerve were resected in 44 Lewis rats. The gaps were either left unrepaired (n = 6), repaired with nerve autograft (n = 19), or repaired with conduit (n = 19). After 12 weeks, nerve conduction latency, compound muscle action potential amplitude, muscle force and muscle mass were measured. The numbers of axons and axon diameters both within the grafts and distally were determined. Results: After 12 weeks, gastrocnemius isometric tetanic force and muscle mass for the conduit group reached 85 and 82% of autograft values, respectively. Nerve conduction and compound muscle action potential were not significantly different between these two groups, although the latter approached significance. There was no recovery in the unrepaired group. Conclusion: Muscle recovery for the animals treated with this aligned nanofiber conduit approached that of autograft, suggesting the importance of internal conduit structure for nerve repair.

Human mesenchymal stem cells (hMSCs) are attractive candidates for tissue engineering and cell-based therapy because of their multipotentiality and availability in adult donors. However, in vitro expansion and differentiation of these cells is limited by replicative senescence. The proliferative capacity of hMSCs can be enhanced by ectopic expression of telomerase, allowing for long-term culture. However, hMSCs with constitutive telomerase expression demonstrate unregulated growth and even tumor formation. To address this problem, we used an inducible Tet-On gene expression system to create hMSCs in which ectopic telomerase expression can be induced selectively by the addition of doxycycline (i-hTERT hMSCs). i-hTERT hMSCs have inducible hTERT expression and telomerase activity, and are able to proliferate significantly longer than wild type hMSCs when hTERT expression is induced. They stop proliferating when hTERT expression is turned off and can be rescued when expression is re-induced. They retain multipotentiality in vitro even at an advanced age. We also used a selective inhibitor of telomere elongation to show that the mechanism driving immortalization of hMSCs by hTERT is dependent upon maintenance of telomere length. Thanks to their extended lifespan, preserved multipotentiality and controlled growth, i-hTERT hMSCs may prove to be a useful tool for the development and testing of novel stem cell therapies. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1879–1885, 2012

Peri-tendinous injection of local anaesthetic, both alone and in combination with corticosteroids, is commonly performed in the treatment of tendinopathies. Previous studies have shown that local anaesthetics and corticosteroids are chondrotoxic, but their effect on tenocytes remains unknown. We compared the effects of lidocaine and ropivacaine, alone or combined with dexamethasone, on the viability of cultured bovine tenocytes. Tenocytes were exposed to ten different conditions: 1) normal saline; 2) 1% lidocaine; 3) 2% lidocaine; 4) 0.2% ropivacaine; 5) 0.5% ropivacaine; 6) dexamethasone (dex); 7) 1% lidocaine+dex; 8) 2% lidocaine+dex; 9) 0.2% ropivacaine+dex; and 10) 0.5% ropivacaine+dex, for 30 minutes. After a 24-hour recovery period, the viability of the tenocytes was quantified using the CellTiter-Glo viability assay and fluorescence-activated cell sorting (FACS) for live/dead cell counts. A 30-minute exposure to lidocaine alone was significantly toxic to the tenocytes in a dose-dependent manner, but a 30-minute exposure to ropivacaine or dexamethasone alone was not significantly toxic.Dexamethasone potentiated ropivacaine tenocyte toxicity at higher doses of ropivacaine, but did not potentiate lidocaine tenocyte toxicity. As seen in other cell types, lidocaine has a dose-dependent toxicity to tenocytes but ropivacaine is not significantly toxic. Although dexamethasone alone is not toxic, its combination with 0.5% ropivacaine significantly increased its toxicity to tenocytes. These findings might be relevant to clinical practice and warrant further investigation.

Atrophy of the rotator cuff muscles is a factor that complicates the treatment of a massive rotator cuff tear (RCT). However, the molecular mechanisms that govern the development of muscle atrophy after RCTs have not been well defined. The Akt/mammalian target of rapamycin (mTOR) signaling pathway plays a central role in maintaining muscle mass in response to mechanical loading. The role of this pathway in the development of muscle atrophy after a massive RCT remains unknown. The purpose of this study was to investigate the regulation of the Akt/mTOR pathway in the development of muscle atrophy after a RCT and suprascapular nerve (SSN) injury. We evaluated the activity of the Akt/mTOR signaling pathway and how this pathway interacts with two atrophy-related genes, MuRF-1 and MAFbx, in supraspinatus muscles of rats that underwent unilateral complete rotator cuff tendon transection or SSN transection. Akt/mTOR activity was significantly reduced after tendon rupture, but increased after nerve injury. MuRF-1 and MAFbx were only up-regulated following denervation. These results suggest that tendon transection leads to a decrease in protein synthesis with down-regulation of the Akt/mTOR signaling pathway, whereas denervation leads to an increase in protein degradation via up-regulation of expression of MuRF-1 and MAFbx. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 30:1440–1446, 2012

Background: Recent basic science studies have demonstrated local anesthetic chondrotoxicity in vivo and in vitro in both human and animal cartilage. Clinically, chondrolysis associated with the use of intra-articular local anesthetic pain pumps has been described by several groups. This has raised concern regarding the clinical use of intra-articular local anesthetics. Methods: The authors undertook a review of the current orthopaedic literature on local anesthetic chondrotoxicity and its potential relationship to clinical chondrolysis. Results: Local anesthetics such as bupivacaine, lidocaine, and ropivacaine are chondotroxic to human articular cartilage in vitro, although ropivacaine is less so. The evidence suggests that there is a greater risk for chondrolysis with a longer exposure to a higher concentration of local anesthetic, such as with a pain pump, than with a single injection. However, late cellular and metabolic changes are seen after even a single injection of bupivacaine in animal models, and the loss of an intact cartilage matrix also leads to more extensive chondrocyte death. Some studies suggest that additives and the pH of the local anesthetic solution may also play a role in chondrotoxicity. Conclusion: Intra-articular local anesthetics should be used with caution, especially continuous infusions of bupivacaine and lidocaine at high concentrations in joints with compromised cartilage. The consequences of a single intra-articular injection of local anesthetic remains unclear and requires further investigation. Clinical Relevance: Intra-articular use of local anesthetics may have lasting detrimental effects on human articular cartilage and chondrocytes, although the clinical relationship between local anesthetic exposure and chondrolysis requires further study.

Certain clinical environments, including military field hospitals or rural medical centers, lack readily available surgical subspecialists. We hypothesized that telementoring by a surgical subspecialist using a robotic platform is feasible and can convey subspecialty knowledge and skill to a remotely located general surgeon.Eight general surgery residents evaluated the effect of remote surgical telementoring by performing 3 operative procedures, first unproctored and then again when teleproctored by a surgical subspecialist. The clinical scenarios consisted of a penetrating right ventricular injury requiring suture repair, an open tibial fracture requiring external fixation, and a traumatic subdural hematoma requiring craniectomy. A robotic platform consisting of a pan-and-tilt camera with laser pointer attached to an overhead surgical light with integrated audio allowed surgical subspecialists the ability to remotely teleproctor residents. Performance was evaluated using an Operative Performance Scale. Satisfaction surveys were given after performing the scenario unproctored and again after proctoring.Overall mean performance scores were superior in all scenarios when residents were proctored than when they were not (4.30 +/- 0.25 versus 2.43 +/- 0.20; p0.001). Mean performance scores for individual metrics, including tissue handling, instrument handling, speed of completion, and knowledge of anatomy, were all superior when residents were proctored (p0.001). Satisfaction surveys showed greater satisfaction and comfort among residents when proctored. Proctored residents believed the robotic platform facilitated learning and would be feasible if used clinically.This study supports the use of surgical teleproctoring in guiding remote general surgeons by a surgical subspecialist in the care of a wounded patient in need of an emergency subspecialty operation.

Previous work has suggested that an extracellular matrix degrading enzyme-matrix metalloproteinase-2 (MMP-2) plays an important role in the development of muscle atrophy. However, the transcriptional regulation mechanism of MMP-2 in skeletal muscle atrophy remains largely unknown. Using transgenic MMP-2 promoter reporter mice, we have demonstrated that AP-1 and RE-1 binding sites in the MMP-2 promoter region, coupled with increased binding of Fra-1, Fra-2 and AP-2, play a critical role in MMP-2 transcriptional regulation in muscle atrophy. Novel information gained from this study has improved our understanding of in vivo transcriptional regulation of MMP-2 in skeletal muscle atrophy.

Gelatinases are a subgroup of the family of matrix metalloproteinases, which contains two members—gelatinase A and B. These enzymes play an important role in basement membrane homeostasis. Previous studies have associated basement membrane degradation with skeletal muscle atrophy. However, the specific contribution of gelatinases to the pathobiology of muscle atrophy remains unknown. In this study we examined the specific roles of gelatinase A and B in disuse-induced skeletal muscle atrophy using knockout mice. Although both gelatinase A and B are highly upregulated in disused muscle, only gelatinase A null mice had significantly reduced muscle atrophy as compared to wildtype littermates. Type IV collagen and laminin, two major components of basement membrane, were relatively well-preserved in disused muscle in gelatinase A null mice, but not in gelatinase B null mice. These findings suggest that gelatinase A, and not gelatinase B, plays a critical role in disuse-induced skeletal muscle atrophy. Muscle Nerve, 2010

Minimally-invasive monitoring of regeneration in diseased tissue is an important aspect of stem cell therapy. Magnetic resonance imaging (MRI) based tracking of cells labelled with ferumoxides has the potential for non-invasive in vivo detection and longitudinal assessment of implanted cells. Cells labelled with ferumoxides appear as hypointense regions on MR images and thus can be distinguished from the surroundings. Application of this methodology to intervertebral disc degeneration (IVD), and detection of labelled cells implanted into the disc for tissue regeneration was examined. Mesenchymal stem cells labelled with a ferumoxide contrast agent were imaged in vitro to quantitatively characterize the signal intensity loss using MRI relaxation parameters (T1, T2, and T2*). To determine whether labelled cells could be detected within scaffolds suitable for implantation, labelled cells were seeded within both natural and synthetic polymers and imaged using MRI. Labelled cells were loaded within poly(ethylene glycol) hydrogels and imaged in vitro using both MRI and confocal microscopy. Labelled cells were also loaded into fibrin gels, and detected ex vivo within rat IVDs using MRI. Lastly, the effect of ferumoxide labelling on cell viability was investigated. Quantitatively, labelled cells demonstrate the greatest signal intensity loss and contrast on T2*-weighted images. Labelled cells can be detected in both synthetic and natural polymers, and can be distinguished from the native tissue environment of the rat IVD. Finally, labelling does not significantly impair cell viability. Consequently, this technique shows promise as a potential method for in vivo longitudinal tracking of stem cell based regeneration of the IVD.

Background: It has been shown that bupivacaine, the most commonly used local anesthetic for postoperative intra-articular use, is cytotoxic to bovine articular chondrocytes in vitro. Ropivacaine is as effective as bupivacaine for intra-articular analgesia and has less systemic toxicity. We compared the in vitro viability of human articular chondrocytes after exposure to bupivacaine, ropivacaine, and saline solution control. Methods: Macroscopically normal human articular cartilage was harvested from the femoral head or tibial plateau of five patients. Full-thickness cartilage explants and cultured chondrocytes isolated from these patients were treated with 0.9% normal saline solution, 0.5% ropivacaine, or 0.5% bupivacaine for thirty minutes. Twenty-four hours after treatment, chondrocyte viability was measured with use of the LIVE/DEAD Viability/Cytotoxicity Kit for cartilage explants and with use of the CellTiter-Glo Luminescent Cell Viability Assay for cultured chondrocytes. Results: Chondrocyte viability in cartilage explants was significantly greater after treatment with ropivacaine as compared with bupivacaine (94.4% ± 9.0% compared with 78% ± 12.6%; p = 0.0004). There was no difference in viability after treatment with ropivacaine as compared with saline solution (94.4% ± 9.0% compared with 95.8% ± 5.7%; p = 0.6). The viability of cultured chondrocytes was significantly greater after treatment with ropivacaine as compared with bupivacaine (63.9% ± 19% as compared with 37.4% ± 12% of the value in the saline solution group; p < 0.0001). Conclusions: In vitro, 0.5% ropivacaine is significantly less toxic than 0.5% bupivacaine in both intact human articular cartilage and chondrocyte culture. Clinical Relevance: Although bupivacaine is the most commonly used local anesthetic for intra-articular analgesia, the demonstrated toxicity to human articular chondrocytes is cause for concern. The present study demonstrated that ropivacaine is less chondrotoxic than bupivacaine and, therefore, may be safer for intra-articular analgesia.

A retrospective outcomes study was performed on 25 consecutive acetabular reconstructions of major segmental defects by using an oblong-shaped cementless implant. All patients had combined acetabular defects (type III) as defined by the American Academy of Orthopaedic Surgeons classification of acetabular bone deficiency. Long-term follow-up was performed at an average of 11 years postoperatively. Clinical and radiographic outcomes were measured. Failures were defined by component revision or clear radiographic evidence of loosening. Six patients died before final evaluation, and 4 patients did not have complete radiographic data, leaving 14 patients (15 hips) for final analysis. At final follow-up, only 3 of the implants had failed and were revised. There was 1 case of a well-functioning implant with circumferential radiolucency; otherwise, there was no evidence of loosening among the remaining implants.