Patients
This study was conducted at the Third Affiliated Hospital of Soochow University (Changzhou, China). It was reviewed and approved by the Ethics Committee of the Third Affiliated Hospital of Soochow University (Changzhou, China) and was conducted according to standard surgical procedures with informed consent. Fifteen tendon samples were collected from patients with shoulder cuff tears undergoing shoulder surgery. The mean age was 52 years (range, 35-67). Only patients with no clinical evidence of subscapularis tendinopathy on preoperative MRI scans or macroscopic subscapularis tendon injury on arthroscopy were included — they represent a true preclinical cohort according to these criteria. All patients in this cohort met the following criteria: 1) a history of shoulder pain and dysfunction, 2) no history of surgery in the affected shoulder; 3) no imaging signs of fracture in the shoulder, and 4) no history of rheumatoid or osteoarthritis. Another independent control package consisted of 10 subscapularis tendon samples from patients undergoing shoulder arthroscopy for stabilization of the shoulder, with arthroscopic confirmation of no tendon tear, no history of shoulder surgery, no previous history of rheumatoid or osteoarthritis, no radiographic evidence of shoulder fracture, and a control mean age of 32 years (25-42).
Cell preparation and flow cytometry quantification
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation (GE Healthcare, Tokyo, Japan) and immediately quantified by flow cytometry, and for flow cytometry quantification, the cells were stained with fluorescein-conjugated monoclonal antibodies against the following in strict accordance with the instructions. The following antibodies were used in this study: Anti-Lineage-FITC CD2, CD3, CD14, CD16, CD19, CD56, and CD235a (eBioscience (San Diego, CA, USA), Anti-IL-23R-PE (BioLegend), and Anti-IL-2R-APC (BD Biosciences). After incubation, the samples were washed twice with phosphate-buffered saline (PBS) and resuspended with 300 microliters of PBS. For the control group, the corresponding isotype-matched antibody was used for each staining. Labeled cells were quantified by flow cytometry (BD Biosciences). Flow cytometry data were analyzed with Flowjo10 software.
Tissue collection and preparation
Arthroscopic tendon repair was performed using a standard three-door technique as previously described. The subscapularis tendon was collected arthroscopically from the superior border of the tendon 1 cm lateral to the glenoid cavity, and the suprascapular tendon was removed 1.5 cm from the tear edge before surgical repair. The tissues were divided into two aliquots, and one of the tissue specimens was placed in 4% formalin and fixed for 4 to 6 hours, followed by paraffin embedding for HampE staining. One milliliter of TRIzol (Invitrogen, USA) was then added to another specimen, which was cryopreserved at − 80 ° C for extraction of total RNA.
Histological examination
The tendons were fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin. The tissue was cut into 4-μm-thick sections and stained with hematoxylin and eosin (H&E) for evaluation of inflammation. Inflammation in H&E-stained tendon sections was evaluated by five independent, blinded readers according to a semiquantitative scoring system. The results were calculated as the percentage of positive cells in each group. Data are presented as medians (range). All experiments were performed three times.
RNA extraction and fluorescence quantitative PCR
Total RNA from tendon tissue was extracted with TRIzol in strict accordance with the instructions, and the concentration and purity of RNA were measured by a NanoDrop 2000c and diluted to 500 ng/microliter per specimen with RNase-free water. Subsequently, equal amounts of RNA were analyzed by quantitative fluorescence PCR using the SYBR Green Premix EX Taq kit. Each RNA sample was tested for relative expression of CD45, RORC, IL-23, IL-22, and IL-17A mRNA and normalized to β-actin as a housekeeping gene, with comparative threshold cycles calculated with the Ct value method. All sequences of primers are shown in Table 1. Each sample was analyzed in duplicate with the CFXA96 Cycler (Thermal).
Statistical analysis
All data were statistically analyzed using Prism 5 (Graph Pad Software, La Jolla, CA, USA), and the data are expressed as the mean ± standard deviation. Student's unpaired t-test was used for difference analysis, Spearman's analysis was used for correlation analysis between two variables, and the difference was statistically significant when the p value was less than 0.05.