Biochemistry samples were weighed wet, then frozen and lyophilized to acquire dry weights. Collagen content was measured with the use of a Sircol standard (Biocolor) and a modified chloramine-T colorimetric hydroxyproline assay (Cissell et al., 2017 (link)). GAG content was quantified using the Blyscan Glycosaminoglycan assay kit (Biocolor). All quantification measurements for collagen and GAG content were performed with a GENios spectrophotometer/spectrofluorometer (TECAN).
Quantification of pyridinoline crosslink content was performed via a liquid chromatography mass spectrometry (LC-MS) assay (Naffa et al., 2019 (link)). Lyophilized samples were hydrolyzed in 6N HCl at 105°C for 18 h. After evaporation, dried hydrolysates were resuspended in 25% (v/v) acetonitrile and 0.1% (v/v) formic acid in water, centrifuged at 15,000 g for 5 min, and the supernatant was transferred to a LCMS autosampler vial. Liquid chromatography was carried out on a Cogent Diamond Hydride HPLC Column (2.1 mm × 150 mm, particle size 2.2 μm, pore size 120 Å, MicroSolv) and a pyridinoline standard (BOC Sciences) as previously described (Gonzalez-Leon et al., 2020 (link)).
Quantification of pyridinoline crosslink content was performed via a liquid chromatography mass spectrometry (LC-MS) assay (Naffa et al., 2019 (link)). Lyophilized samples were hydrolyzed in 6N HCl at 105°C for 18 h. After evaporation, dried hydrolysates were resuspended in 25% (v/v) acetonitrile and 0.1% (v/v) formic acid in water, centrifuged at 15,000 g for 5 min, and the supernatant was transferred to a LCMS autosampler vial. Liquid chromatography was carried out on a Cogent Diamond Hydride HPLC Column (2.1 mm × 150 mm, particle size 2.2 μm, pore size 120 Å, MicroSolv) and a pyridinoline standard (BOC Sciences) as previously described (Gonzalez-Leon et al., 2020 (link)).
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