Background

The muscle quality of the rotator cuff (RC), measured by atrophy and fatty infiltration (FI), is a key determinant of outcomes in RC injury and repair. The ability to regenerate muscle after repair has been shown to be limited.

Purpose

To determine if there is a source of resident endogenous stem cells, fibroadipogenic progenitor cells (FAPs), within RC injury patients, and if these cells are capable of adipogenic, fibrogenic, and pro-myogenic differentiation.

Study design

Controlled laboratory study.

Methods

A total of 20 patients between the ages of 40 and 75 years with partial- or full-thickness RC tears of the supraspinatus and evidence of atrophy and FI Goutallier grade 1, 2, or 3 were selected from 2 surgeons at an orthopaedic center. During the surgical repair procedure, supraspinatus muscle biopsy specimens were obtained for analysis as were deltoid muscle biopsy specimens to serve as the control. FAPs and satellite cells were quantified using fluorescence-activated cell sorting. Muscle FI and fibrosis was quantified using Oil Red O and Masson trichrome staining. FAP differentiation and gene expression profiles were compared across tear sizes after culture in adipogenic, fibrogenic, and beta-3 agonist (amibegron) conditions. Analysis of variance was used for statistical comparisons between groups, with P < .05 as statistically significant.

Results

Histologic analysis confirmed the presence of fat in biopsy specimens from patients with full-thickness tears. There were more FAPs in the full-thickness tear group compared with the partial-thickness tear group (9.43% ± 4.25% vs 3.84% ± 2.54%; P < .01). Full-thickness tears were divided by tear size, with patients with larger tears having significantly more FAPs than those with smaller tears. FAPs from muscles with full-thickness tendon tears had more adipogenic and fibrogenic potential than those with partial tears. Induction of a beige adipose tissue (BAT) phenotype in FAPs was possible, as demonstrated by increased expression of BAT markers and pro-myogenic genes including insulin-like growth factor 1 and follistatin.

Conclusion

Endogenous FAPs are present within the RC and likely are the source of FI. These FAPs were increased in muscles with in larger tears but are capable of adopting a pro-myogenic BAT phenotype that could be utilized to improve muscle quality and patient function after RC repair.

Introduction

Rotator cuff (RC) tears are common tendon injuries. Clinically, both muscle atrophy and fatty infiltration have generally been attributed to poor functional outcomes. Matrix metalloproteinase-13 plays a crucial role in extracellular matrix remodeling in many physiological and pathological processes. Nevertheless, its role in rotator cuff muscle atrophy and fatty infiltration remains unknown. The purpose of this study is to define the functional role of MMP-13 in rotator cuff muscle atrophy and fatty infiltration using a mouse RC tears model.

Materials and methods

Unilateral complete supraspinatus and infraspinatus tendon transection and suprascapular nerve transection was performed on nine of MMP-13 (-/-) knockout and nine of MMP-13 (+/+) wildtype mice at 3 months old. Mice were sacrificed 6 weeks after surgery. Supraspinatus (SS) and infraspinatus (IS) muscles were harvested for histology and gene expression analysis with RT-PCR.

Results

Six weeks after RC surgery, no significant difference in muscle atrophy and fibrosis between MMP-13 knockout and wild type mice was observed. However, there was a significant increase in the amount of fatty infiltration in MMP-13 knockout mice compared to the wild types. Muscles from MMP-13 knockout mice have significantly higher expression of fatty infiltration related genes.

Discussion

Results from this study suggest that MMP-13 plays a crucial role in rotator cuff muscle fatty degeneration. This novel finding suggests a new molecular mechanism that governs RC muscle FI and MMP-13 may serve as a target for therapeutics to treat muscle FI after RC 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.

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

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 β₃-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 β₃-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.

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.

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.

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.

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.

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.