Overexpression of PRDM16 improves muscle function after rotator cuff tears.

BACKGROUND: Muscle atrophy, fibrosis, and fatty infiltration are commonly seen in rotator cuff tears (RCTs), which are critical factors that directly determine the clinical outcomes for patients with this injury. Therefore, improving muscle quality after RCT is crucial in improving the clinical outcome of tendon repair. In recent years, it has been discovered that adults have functional beige/brown adipose tissue (BAT) that can secrete batokines to promote muscle growth. PRDM16, a PR-domain-containing protein, was discovered with the ability to determine the brown fat cell fate and stimulate its development. Thus, the goal of this study was to discover the role of PRDM16 in improving muscle function after massive tendon tears using a transgenic mouse model with an elevated level of PRDM16 expression. METHODS: Transgenic aP2-driven PRDM16-overexpressing mice and C57BL/6J mice underwent unilateral supraspinatus (SS) tendon transection and suprascapular nerve transection (TTDN) as described previously (n = 8 in each group). DigiGait was performed to evaluate forelimb function at 6 weeks post the TTDN injury. Bilateral SS muscles, interscapular brown fat, epididymal white fat, and inguinal beige fat were harvested for analysis. The expression of PRDM16 in adipose tissue was detected by Western blot. Masson Trichrome staining was conducted to evaluate the muscle fibrosis, and Oil Red O staining was used to determine the fat infiltration. Muscle fiber type was determined by major histocompatibility complex (MHC) expression via immunostaining. All data were presented in the form of mean ± standard deviation. t test and 2-way analysis of variance was performed to determine a statistically significant difference between groups. Significance was considered when P < .05. RESULTS: Western blot data showed an increased expression of PRDM16 protein in both white and brown fat in PRDM16-overexpressing mice compared with wild-type (WT) mice. Even though PRDM16 overexpression had no effect on increasing muscle weight, it significantly improved the forelimbs function with longer brake, stance, and stride time and larger stride length and paw area in mice after RCT. Additionally, PRDM16-overexpressing mice showed no difference in the amount of fibrosis when compared to WT mice; however, they had a significantly reduced area of fatty infiltration. These mice also exhibited abundant MHC-IIx fiber percentage in the supraspinatus muscle after TTDN. CONCLUSION: Overexpression of PRDM16 significantly improved muscle function and reduced fatty infiltration after rotator cuff tears. Promoting BAT activity is beneficial in improving rotator cuff muscle quality and shoulder function after RCT.

The Role of Matrix Metalloproteinase-13 (MMP13) in TGFß/BMP Pathway Regulation of Fibro-Adipogenic Progenitor (FAP) Differentiation.

BACKGROUND/AIMS: Muscle fibrosis and fatty infiltration (FI) are common complications seen in various muscle disease states. Recent studies indicate that muscle residential fibro/adipogenic progenitors (FAPs) are the major cellular source for muscle fibrosis and FI. We previously showed that MMP13 knockout (KO) mice have significantly increased FI, suggesting an important role of MMP13 in muscle FI. However, how MMP13 affects the differentiation of FAPs remains unknown. METHODS: In order to assess the role of MMP-13 on FAP differentiation, we isolated FAPs from wildtype C57BL/6 and MMP13 knock out mice with FACS using CD31-, CD45-, Integrin α7- and Sca-1+ markers. FAPs were cultured in 24 well plate after FACS.in standard media till 80% confluent and then switched to adipogenic medium. In order to study the role of TGFß and BMP in their differentiation, FAPs from both wildtype and MMP13 KO mice were treated with TGFß1 (5 ng/ml). For MMP13 inhibitor treatment, FAPs from wildtype mice were incubated in adipogenic medium containing 10 µM MMP13 inhibitor (or vehicle) for 2 weeks. Immunofluorescence and gene expression analysis were used to assess FAP adipogenic and fibrogenic differentiation. FAPs were stained with Perilipin A (FITC, adipogenesis marker) and αSMA (Red, fibroblast marker), and DAPI. Real time PCR was performed for gene expression evaluation. A two-tailed Anova was used for statistical comparisons between groups, withp ≤ 0.05. Data are presented as mean ± standard deviation. RESULTS: In this study, we isolated FAPs from wildtype C57BL/6 and MMP13 KO mice and evaluated their adipogenic and fibrogenic differentiation in vitro. MMP13 KO FAPs demonstrated enhanced adipogenesis but reduced fibrogenesis compared to wildtype FAPs. Treating wildtype FAPs with an MMP13 inhibitor simulated phenotypes seen in MMP13 KO FAPs. In order to assess the role of MMP13 on TGFß/BMP signaling in regulating FAP differentiation, we treated wildtype and MMP13 KO FAPs with TGFß1, BMP7, TGFß inhibitor, and BMP inhibitor. TGFß1 treatment significantly enhanced fibrogenesis, but inhibited adipogenesis of wildtype FAPs. However, treatment with BMP7 showed the opposite effect. Interestingly, the effect of TGFß1/BMP7 was voided in MMP13 KO FAPs. Treating wildtype FAPs with MMP13 inhibitor also abolished the effect of TGFß1/BMP7 in FAP differentiation. CONCLUSION: Results from this study showed that TGFß1 inhibits FAP adipogenesis but stimulates FAP fibrogenesis. BMP7 was shown to promote FAP adipogenesis but reduce its fibrogenesis. The role of the TGFß/BMP signaling pathway regulating FAP differentiation was found to be MMP13 dependent. This study suggests that MMP13 is a critical downstream effector in TGFß/BMP pathway which may serve as a new therapeutic target for muscle fibrosis and FI.

Comparison of BMI on operative time and complications of robotic inguinal hernia repair at a VA medical center.

BACKGROUND: BMI is a risk factor for recurrence and post-operative complications in both open and laparoscopic totally extraperitoneal approach (TEP) repair. Robotic surgery using the transabdominal preperitoneal approach (TAPP) is a safe and viable option for inguinal hernia repair (IHR). The objective of this study is to determine how difference in BMI influences rate of operative time, complications, and rate of recurrence in a robotic TAPP IHR. METHODS: We performed a retrospective review of patients who underwent robotic inguinal hernia repair between 2012 and 2019 at a Veterans Health Administration facility (N = 304). The operating time, outcomes, and overall morbidity and mortality for robotic IHR were compared between three different BMI Groups. These groups were divided into: "Underweight/Normal Weight" (BMI < 25) n = 102, "Pre-Obese" (BMI 25-29.9) n = 120, and "Obese" (BMI 30 +) n = 82. RESULTS: The average operating time of a bilateral IHR by BMI group was 83.5, 98.4, and 97.8 min for BMIs < 25, 25-29.9, and 30 +, respectively. Operating time was lower in the Underweight/Normal BMI group compared to the Pre-Obese group (p = 0.006) as well as the Obese group (p = 0.001). For unilateral repair, the average operation length by group was 65.2, 70.9, and 85.6 min for BMIs < 25, 25-29.9, and 30 +, respectively, demonstrating an increased time for Obese compared to Underweight/Normal BMI (p = 0.001) and for Obese compared to Pre-Obese (p = 0.01). Demographic/comorbidity variables were not significantly different, except for a higher percentage of white patients in the Underweight/Normal BMI group compared to the Pre-Obese and Obese groups (p = 0.02 and p = 0.0003). There was no significant difference in complications or recurrence. CONCLUSION: BMI has a significant impact on the operating time of both unilateral and bilateral robotic hernia repair. Despite this increased operative time, BMI group did not differ significantly in postoperative outcomes or in recurrence rates.

ß3-Adrenergic receptor agonist treats rotator cuff fatty infiltration by activating beige fat in mice.

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 ß3-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 BAT activity, amibegron, a selective ß3-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.

Beige fibro-adipogenic progenitor transplantation reduces muscle degeneration and improves function in a mouse model of delayed repair of rotator cuff tears.

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 <.05 was used to determine statistical significance. RESULTS: Cell transplantation diminished fibrosis, FI, and atrophy and enhanced vascularization in both delayed repair models. Cell transplantation resulted in improved shoulder function as assessed with gait analysis in both the delayed repair models. CONCLUSIONS: BAT-FAPs significantly reduced muscle degeneration and improved shoulder function after RC repair. BAT-FAPs hold significant promise as a therapeutic adjunct to repair for patients with advanced RC pathology.

Reversal of Fatty Infiltration After Suprascapular Nerve Compression Release Is Dependent on UCP1 Expression in Mice.

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.

Complications of Morbid Obesity in Total Joint Arthroplasty: Risk Stratification Based on BMI.

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 (P<0.05) in combined complications, acute kidney injury (AKI), cardiac arrest (CA), reintubation, reoperation, and superficial infection (SI). Univariate analysis for BMI>40 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 BMI>40 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.

Upregulation of transforming growth factor-ß signaling in a rat model of rotator cuff tears.

BACKGROUND: Muscle atrophy, fatty infiltration, and fibrosis of the muscle have been described as important factors governing outcome after rotator cuff injury and repair. Muscle fibrosis is also thought to have a role in determining muscle compliance at the time of surgery. The transforming growth factor-ß (TGF-ß) pathways are highly conserved pathways that exert a potent level of control over muscle gene expression and are critical regulators of fibrosis in multiple organ systems. It has been shown that TGF-ß can regulate important pathways of muscle atrophy, including the Akt/mammalian target of rapamycin pathway. The purpose of this study was to evaluate the expression of TGF-ß and its downstream effectors of fibrosis after a massive rotator cuff tear (RCT) in a previously established rat model. METHODS: To simulate a massive RCT, infraspinatus and supraspinatus tenotomy and suprascapular nerve transection were performed on Sprague-Dawley rats with use of a validated model. Two and 6 weeks after surgery, supraspinatus muscles were harvested to study alterations in TGF-ß signaling by Western blotting, quantitative polymerase chain reaction, and histologic analysis. RESULTS: There was a significant increase in fibrosis in the rotator cuff muscle after RCT in our animal model. There was a concomitant increase in TGF-ß gene and protein expression at both 2 and 6 weeks after RCT. Evaluation of the TGF-ß signaling pathway revealed an increase in SMAD2 activation but not in SMAD3. There was an increase in profibrotic markers collagen I, collagen III, and α-smooth muscle actin. CONCLUSIONS: TGF-ß signaling is significantly upregulated in rat supraspinatus muscles after RCTs.

Defining Endogenous Mitochondrial Transfer in Muscle After Rotator Cuff Injury.

BACKGROUND: Rotator cuff muscle degeneration leads to poor clinical outcomes for patients with rotator cuff tears. Fibroadipogenic progenitors (FAPs) are resident muscle stem cells with the ability to differentiate into fibroblasts as well as white and beige adipose tissue. Induction of the beige adipose phenotype in FAPs has been shown to improve muscle quality after rotator cuff tears, but the mechanisms of how FAPs exert their beneficial effects have not been fully elucidated. PURPOSE: To study the horizontal transfer of mitochondria from FAPs to myogenic cells and examine the effects of ß-agonism on this novel process. STUDY DESIGN: Controlled laboratory study. METHODS: In mice that had undergone a massive rotator cuff tear, single-cell RNA sequencing was performed on isolated FAPs for genes associated with mitochondrial biogenesis and transfer. Murine FAPs were isolated by fluorescence-activated cell sorting and treated with a ß-agonist versus control. FAPs were stained with mitochondrial dyes and cocultured with recipient C2C12 myoblasts, and the rate of transfer was measured after 24 hours by flow cytometry. PdgfraCreERT/MitoTag mice were generated to study the effects of a rotator cuff injury on mitochondrial transfer. PdgfraCreERT/tdTomato mice were likewise generated to perform lineage tracing of PDGFRA+ cells in this injury model. Both populations of transgenic mice underwent tendon transection and denervation surgery, and MitoTag-labeled mitochondria from Pdgfra+ FAPs were visualized by fluorescent microscopy, spinning disk confocal microscopy, and 2-photon microscopy; overall mitochondrial quantity was compared between mice treated with ß-agonists and dimethyl sulfoxide. RESULTS: Single-cell RNA sequencing in mice that underwent rotator cuff tear demonstrated an association between transcriptional markers of adipogenic differentiation and genes associated with mitochondrial biogenesis. In vitro cocultures of murine FAPs with C2C12 cells revealed that treatment of cells with a ß-agonist increased mitochondrial transfer compared to control conditions (17.8% ± 9.9% to 99.6% ± 0.13% P < .0001). Rotator cuff injury in PdgfraCreERT/MitoTag mice resulted in a robust increase in MitoTag signal in adjacent myofibers compared with uninjured mice. No accumulation of tdTomato signal from PDGFRA+ cells was seen in injured fibers at 6 weeks after injury, suggesting that FAPs do not fuse with injured muscle fibers but rather contribute their mitochondria. CONCLUSION: The authors have described a novel process of endogenous mitochondrial transfer that can occur within the injured rotator cuff between FAPs and myogenic cells. This process may be leveraged therapeutically with ß-agonist treatment and represents an exciting target for improving translational therapies available for rotator cuff muscle degeneration. CLINICAL RELEVANCE: Promoting endogenous mitochondrial transfer may represent a novel translational strategy to address muscle degeneration after rotator cuff tears.

Evaluation of cartilage degeneration in a rat model of rotator cuff tear arthropathy.

BACKGROUND AND HYPOTHESIS: Rotator cuff tears are the most common injury seen by shoulder surgeons. Glenohumeral osteoarthritis develops in many late-stage rotator cuff tear patients as a result of torn cuff tendons, termed "cuff tear arthropathy." However, the mechanisms of cuff tear arthropathy have not been fully established. It has been hypothesized that a combination of synovial and mechanical factors contribute equally to the development of cuff tear arthropathy. The goal of this study was to assess the utility of this model in investigating cuff tear arthropathy. MATERIALS AND METHODS: We used a rat model that accurately reflects rotator cuff muscle degradation after massive rotator cuff tears through either infraspinatus and supraspinatus tenotomy or suprascapular nerve transection. Using a modified Mankin scoring system, we found significant glenohumeral cartilage damage after both rotator cuff tenotomy and suprascapular nerve transection after only 12 weeks. RESULTS: Cartilage degeneration was similar between groups and was present on both the humeral head and the glenoid. Denervation of the supraspinatus and infraspinatus muscles without opening the joint capsule caused cartilage degeneration similar to that found in the tendon transection group. CONCLUSIONS: Our results suggest that altered mechanical loading after rotator cuff tears is the primary factor in cartilage degeneration after rotator cuff tears. Clinically, understanding the process of cartilage degeneration after rotator cuff injury will help guide treatment decisions in the setting of rotator cuff tears. LEVEL OF EVIDENCE: Basic science study, animal model.

New-onset synovial chondromatosis after total knee arthroplasty.

Total joint arthroplasty is commonly recommended as a definitive treatment for synovial chondromatosis refractory to other treatment. We describe a unique case of synovial chondromatosis developing after total joint arthroplasty in a patient presenting 5 years after total knee arthroplasty for osteoarthritis. This case illustrates that the diagnosis of synovial chondromatosis cannot be excluded in a patient with chronic, painful swelling of a joint, even after total joint arthroplasty.

Polarized macrophages regulate fibro/adipogenic progenitor (FAP) adipogenesis through exosomes.

BACKGROUND: Macrophage polarization has been observed in the process of muscle injuries including rotator cuff (RC) muscle atrophy and fatty infiltration after large tendon tears. In our previous study, we showed that fibrogenesis and white adipogenesis of muscle residential fibro/adipogenic progenitors (FAPs) cause fibrosis and fatty infiltration and that brown/beige adipogenesis of FAPs promotes rotator cuff muscle regeneration. However, how polarized macrophages and their exosomes regulate FAP differentiation remains unknown. METHODS: We cultured FAPs with M0, M1, and M2 macrophages or 2 × 109 exosomes derived from M0, M1 and M2 with and without GW4869, an exosome inhibitor. In vivo, M0, M1, and M2 macrophages were transplanted or purified macrophage exosomes (M0, M1, M2) were injected into supraspinatus muscle (SS) after massive tendon tears in mice (n = 6). SS were harvested at six weeks after surgery to evaluate the level of muscle atrophy and fatty infiltration. RESULTS: Our results showed that M2 rather than M0 or M1 macrophages stimulates brown/beige fat differentiation of FAPs. However, the effect of GW4869, the exosome inhibitor, diminished this effect. M2 exosomes also promoted FAP Beige differentiation in vitro. The transplantation of M2 macrophages reduced supraspinatus muscle atrophy and fatty infiltration. In vivo injections of M2 exosomes significantly reduced muscle atrophy and fatty infiltration in supraspinatus muscle. CONCLUSION: Results from our study demonstrated that polarized macrophages directly regulated FAP differentiation through their exosomes and M2 macrophage-derived exosomes may serve as a novel treatment option for RC muscle atrophy and fatty infiltration.

Fibroblast growth factor 2 enhances the kinetics of mesenchymal stem cell chondrogenesis.

Treatment of mesenchymal stem cells (MSCs) with fibroblast growth factor 2 (FGF-2) during monolayer expansion leads to increased expression of cartilage-related molecules during subsequent pellet chondrogenesis. This may be due to faster differentiation and/or a durable change in phenotype. In order to evaluate changes over time, we assessed chondrogenesis of human MSCs at early and late time points during pellet culture using real-time PCR, measurement of glycosaminoglycan accumulation, and histology. Marked enhancement of chondrogenesis was seen early compared to controls. However, the differences from controls in gene expression dramatically diminished over time. Depending on conditions, increases in glycosaminoglycan accumulation were maintained. These results suggest that FGF-2 can enhance the kinetics of MSC chondrogenesis, leading to early differentiation, possibly by a priming mechanism.

Thermal stress potentiates bupivacaine chondrotoxicity.

PURPOSE: The primary objective of this study was to determine whether thermal stress potentiates the chondrotoxic effect of bupivacaine, resulting in decreased articular chondrocyte viability compared with exposure to bupivacaine alone. METHODS: Bovine articular cartilage explants and cultured chondrocytes were treated with a range of thermal exposures (10 to 20 minutes at 37°C to 65°C) to create time/temperature viability curves and to determine threshold conditions for cell death. A second set of experiments was performed to determine whether subthreshold thermal stress potentiates bupivacaine toxicity. Explants were exposed to 37°C or 55°C for 10 or 20 minutes, and cultured chondrocytes were exposed to 37°C or 45°C for 10 or 20 minutes. Thirty minutes later, the explants and chondrocytes were treated with either 0.9% normal saline solution or 0.5% bupivacaine for 30 minutes. Chondrocyte viability was quantified 24 hours after treatment. RESULTS: There was a temperature- and time-dependent decrease in chondrocyte viability above the thermo-toxicity threshold in both intact cartilage explants and cultured chondrocytes (55°C and 45°C, respectively; P < .05). Chondrocyte viability in cartilage explants was significantly lower after treatment with thermal stress for 10 or 20 minutes followed by bupivacaine for 30 minutes compared with treatment with bupivacaine at 37°C (bupivacaine and 55°C for 10 minutes, 0.09% ± 0%; bupivacaine and 55°C for 20 minutes, 0.08% ± 0%; bupivacaine and 37°C for 10 minutes, 37.4% ± 1.2% [P < .001]; and bupivacaine and 37°C for 20 minutes, 47.1% ± 0.8% [P < .001]). A similar trend was seen in cultured chondrocytes, although it was not statistically significant (P > .05). CONCLUSIONS: Thermal stress potentiates the chondrotoxic effects of bupivacaine in intact cartilage, leading to decreased chondrocyte viability compared with exposure to bupivacaine alone. CLINICAL RELEVANCE: Intra-articular injection of bupivacaine after arthroscopic procedures during which cartilage is exposed to elevated temperatures, such as with prolonged use of radiofrequency probes, may increase the risk of chondrocyte toxicity.

Muscle degeneration in rotator cuff tears.

Rotator cuff tears are among the most common injuries seen by orthopedic surgeons. Although small- and medium-sized tears do well after arthroscopic and open repair, large and massive tears have been shown to develop marked muscle atrophy and fatty infiltration within the rotator cuff muscles. These pathologic changes have been found to be independent predictors of failed surgical repair with poor functional outcomes. To understand the pathophysiology of rotator cuff disease, we must first develop an understanding of the changes that occur within the cuff muscles themselves. The purpose of this review is to summarize the molecular pathways behind muscular degeneration and emphasize new findings related to the clinical relevance of muscle atrophy and fatty infiltration seen with rotator cuff tears. Understanding these molecular pathways will help guide further research and treatment options that can aim to alter expression of these pathways and improve outcomes after surgical repair of massive rotator cuff tears.

Muscle-Derived Beige Adipose Precursors Secrete Promyogenic Exosomes That Treat Rotator Cuff Muscle Degeneration in Mice and Are Identified in Humans by Single-Cell RNA Sequencing.

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.

Microenergy acoustic pulses promotes muscle regeneration through in situ activation of muscle stem cells.

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 Tear Size Regulates Fibroadipogenic Progenitor Number and Gene Expression Profile in the Supraspinatus Independent of Patient Age.

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 (ßage = 0.21, P = .03; ßtear 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 (ß = 1.03; P < .0001), while increasing tear size showed a trend toward decreased beige differentiation (ß = -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.

Does the Preferred Study Source Impact Orthopedic In-Training Examination Performance?

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<0.001). 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.

Designing successful virtual residency interviews: a survey-based needs assessment of applicants across medical specialties.

Background: Residency selection in the United States relied on in-person interviews for many decades. The COVID-19 pandemic and recommendations from the Coalition for Physician Accountability (COPA) required programs to implement virtual interviews for the 2020-2021 residency selection cycle. Although virtual interviews may become the norm in the future, there is scant data at the institutional level to inform how to best approach this process. Objective: To describe the perceptions of applicants to several residency programs at one institution on the importance of virtual recruitment features and assess the impact on their overall ranking decisions. Methods: Applicants who interviewed for 12 medical and surgical residency programs during the 2020-2021 cycle at the University of California San Francisco were invited to participate in an anonymous survey in March 2021, after all interviews were completed. A survey consisting of 26 questions was administered to applicants on features that are important during interviews and the impact on their ranking decisions scored on a 5-point Likert scale. Results: Of the 1422 participating applicants, 303 (21%) completed the survey. The most important feature for applicants during the interview day was getting a feel of the program (92%). Conversations with residents (91%) and faculty (79%) were also highly rated. Respondents reported morale and happiness of residents (71%) as an extremely important factor in their overall ranking decision. Conclusion: Programs should consider prioritizing features that aid in alignment with getting to know residents and faculty and provide a sense of morale over emphasis on the institutional and location features. Supplementary Information: The online version contains supplementary material available at 10.1007/s44186-022-00004-5.