Comparative Analysis of Tendon Cell Responses

Tendon-derived stromal cells from healthy hamstring (n = 5) and diseased supraspinatus tendons (n = 5) were seeded at a density of 15,000 cells per well in a 24 well plate. Tendon cells were allowed to reach 80% confluence prior to stimulation with IL-1β (Merck, 10ngml^—1) in DMEM F12 phenol red free medium (Gibco) containing 1% heat inactivated human serum (Sigma) and 1% penicillin-streptomycin. Non-treated cells (vehicle only, containing 0.1% endotoxin free BSA, Sigma) served as controls for each experiment. After cytokine treatment, cells were then incubated at 37 °C and 5% CO₂ until harvest of the cell lysate for mRNA after 24 h. For tissues, samples of healthy subscapularis (n = 4) and diseased supraspinatus tendons (n = 14) were snap frozen and stored at −80 °C. RNA isolation, cDNA synthesis and quantitative PCR were performed using previously published protocols^{9 (link)}. Pre-validated Qiagen primer assays (15-PGDH, STAT-1, IL6, PDPN, β-actin and GAPDH) were used for qPCR. Results were calculated using the ddCt method using reference genes for human β-actin and GAPDH. Results were consistent using these reference genes and data are shown normalized to β-actin.