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.

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.

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.

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.

Effects of mixing techniques on vancomycin-impregnated polymethylmethacrylate.

The use of antibiotic-impregnated polymethylmethacrylate in joint arthroplasty is widespread. The Food and Drug Administration has approved commercially prepared antibiotic bone cement, but in a climate of increasingly drug-resistant bacteria, orthopedic surgeons often hand-mix their own. A recent study reported the effects on drug elution of different mixing methods designed to decrease antibiotic particle size and distribute those particles more uniformly. Theoretically, these mixing techniques could also improve antibiotic cement strength; however, the actual effects of these techniques on cement strength are undefined. In the present study, 3 different methods of mixing vancomycin with bone cement were compared. We conclude that the addition of vancomycin to polymethylmethacrylate at commonly accepted concentrations does substantially decrease cement strength and that more complex mixing techniques do not improve cement strength significantly.

Rotator cuff tear degeneration and the role of fibro-adipogenic progenitors.

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.

Comparison of Periarticular Infiltration and Combination Delivery of Local Anesthetics for Reducing Pain and Opioid Consumption after Total Knee Arthroplasty.

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.

Preconditioning improves muscle regeneration after ischemia-reperfusion injury.

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, ß3 adrenergic receptor (ß3AR) agonist amibegron and ß3AR 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 ß3AR signaling pathway-stimulated FAP-BAT differentiation.

Role of AP-1 and RE-1 binding sites in matrix metalloproteinase-2 transcriptional regulation in skeletal muscle atrophy.

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.