2.17. Hydroxyproline Assay

A hydroxyproline assay was performed to confirm the total tendon collagen content. The sacrificed tissue was rapidly frozen in liquid nitrogen and stored at −80 °C. Tissue disruption and experimental methods were performed following the manufacturer’s instructions for a hydroxyproline colorimetric assay (Abcam; ab222941). After breaking the tissue, proteins were treated with 10 N NaOH and HCl and then centrifuged at 10,000× g for 5 min. After collecting the supernatant, an oxidation mixture was added and reacted at room temperature for 20 min. The developer and DMAB concentrate were added at 50 μL per vial and reacted at 65 °C for 45 min. Measurements were obtained at 560 nm using a Synergy Mix Multi-Mode Reader (BioTek).