ECM protein quantification

The amount of the ECM proteins elastin, soluble/insoluble collagen and sulfated glycosaminoglycans (GAGs) was quantified with commercially available kits (n = 5 for all assays). Elastin was quantified with the Fastin Elastin Assay (Biocolor, UK) by extracting elastin from 20–30 mg wet ECM at 100°C in 750 μL 0.25M oxalic acid. This procedure was repeated twice, and extracts pooled. Then, 100 μ of extract were mixed with 100 μL precipitation reagent and supplied protocol followed, after which absorbance was measured at 513 nm. Soluble collagen was quantified with the Sircol Soluble Collagen Assay (Biocolor, UK), by extracting soluble collagen from 20–30 mg wet ECM at RT in 1 mL 0.1M HCl containing 0.1 mg/mL pepsin for 2 days. Then, 50 μL extract were used for the assay, following the suppliers’ protocol, after which absorbance was measured at 555 nm. Insoluble collagen was quantified with the Sircol Insoluble Collagen Assay (Biocolor, UK). Briefly, 15–20 mg wet ECM was incubated at 65°C for 3 h with supplied fragmentation reagent at a ratio of 50 μL reagent per mg ECM. Then, 50 μL extract were used for the assay, following the suppliers’ protocol, after which absorbance was read at 550 nm. Final results were obtained by multiplying with a correlation factor of 2.2, as denatured collagen obtained with this extraction method has 45% lower dye-binding affinity compared to native collagen. The amount of sulfated glycosaminoglycans (GAGs) in the ECM was quantified with the Blyscan Sulfated GAG Kit (Biocolor, UK), by extracting GAGs from 15–30 mg of wet tissue by incubation with 0.1 mg/mL papain (ThermoFisher, USA) in a sodium phosphate buffer at 65°C. When samples were completely digested, 50 μL and the samples were used for the assay, following the suppliers’ protocol, after which absorbance was measured at 656 nm. All assays were also performed on blanks and respective protein standard to allow quantification of protein content.