The Serum from Patients with Secondary Frozen Shoulder Following Rotator Cuff Repair Induces Shoulder Capsule Fibrosis and Promotes Macrophage Polarization and Fibroblast Activation

Abstract

Disorders with systematic inflammation were prognostic for secondary frozen shoulder (sFS) following rotator cuff repair (RCR); however, how systematic inflammation affects sFS remains unclear. The aim of this study was to observe the effect of pre-operative serum from patients with sFS and the serum from those without on shoulder capsule in mice, and on macrophages and fibroblasts in vitro. Methods: Serum samples of a consecutive cohort of patients for RCR were collected preoperatively. Three months after RCR, patients who developed sFS (Group S) were identified. Serum samples from gender-and age-matched controls without sFS (group NS) were also picked out. Firstly, the effect of serum on shoulder capsule fibrosis was observed histologically and biomechanically in a mouse model of RCR. Secondly, the roles of the serum on macrophage polarization and fibroblast activation were investigated, and the potentially involved signaling pathways were identified. Finally, inflammation and fibrosis-related cytokines in serum were quantified. Results: In our cohort, all patients had free pre-operative shoulder range of motion. Seven patients developed sFS at 3 months after surgery. Seven matched patients without sFS were selected as control. The inter-group difference of basic characteristics was not significant. Compared to the serum of group NS, the serum of group S significantly induced hypercellularity, capsular thickening, and range of motion deficiency in mice shoulders after RCR. Compared to the serum of group NS, samples of group S significantly promoted M2 polarization of THP-1 human macrophages and the activation of human capsule-derived fibroblasts. Meanwhile, Smad3 and p-Smad3 in macrophages and fibroblasts were significantly up-regulated. On the other hand, levels of inflammation and fibrosis-related cytokines were not significantly different between serum in group S and group NS. Conclusion: Although all patients in this cohort had free range of motion pre-operatively, the pre-operative serum from patients with sFS at 3 months after RCR could act as a trigger of shoulder capsule fibrosis post-operatively. This effect may be related to its promotion on macrophage polarization to M2 phenotype and fibroblast activation.

Human bone marrow mesenchymal stem cell-derived extracellular vesicles inhibit shoulder stiffness via let-7a/Tgfbr1 axis

“…CDFs have been a commonly applied model in vitro for investigating fibrosis-related mechanisms, especially FS, which are abundant in capsule tissue and retain the original features when passaged [ 41 , 59 ]. As for fibroblasts with pro-fibrotic phenotype, cell viability, proliferation rate, and migration ability are always up-regulated [ 60 ].…”

“…Besides, α-SMA, Col 1, and VIM have been widely accepted hallmarks of fibrosis, and are the direct fibrogenesis evidence [ 61 ]. In addition, the contraction ability was assessed by contraction test due to typically, the contraction ability of fibrosis is enhanced in activated fibroblasts [ 41 ]. A comprehensive evaluation of the status of fibroblasts in our study could be reached based on these biomarkers.…”

“…To obtain the human capsule-derived fibroblasts (CDFs), fresh capsular samples were harvested in the RCT group, and 13 patients with the SS group. Then, the CDFs were collected following the published protocol [ 41 ]. Afterward, CDFs were cultured in high-glucose DMEM medium (Thermo Fisher Scientific) + 10% FBS (Gibco) + 1% penicillin/streptomycin solution.…”

“…A collagen contraction experiment was performed as described previously [ 41 ]. In general, CDFs (2.0 × 10 5 cells/ml) were suspended in 24-well plates with a 1 ml growth medium, which contained 300ul of 2 mg/ml concretionary collagen (Solarbio, Beijing, China) with or without other substances, such as TGF-β and EVs in each well.…”

Advances in traditional Chinese medicine as adjuvant therapy for diabetic foot

“…The main goal of therapy for type 2 diabetes is to prevent or delay complications and maintain quality of life[ 5 ]. There are many complications of DM, such as car-diovascular disorders[ 6 ], end-stage renal disease[ 7 ], retinopathy[ 8 ], neuropathy[ 9 ], mental illness[ 10 ], muscle atrophy[ 11 ], adhesive capsulitis[ 12 ] and even joint stiffness following surgery[ 13 , 14 ]. Diabetic foot (DF) is a frequent complication of DM due to vascular and neuropathological damage and is the main reason for amputation and death[ 15 ].…”