Sircol collagen assay s1000

Collagen content was quantified as described previously with a Sircol Collagen Assay (S1000; Biocolor, Carrickfergus, U.K., http://www.biocolor.co.uk). Briefly, parts of the 4‐ or 12‐weeks samples were cut, minced into 1‐mm³ pieces, and then digested with pepsin (1 mg/ml in 0.5 M acetic acid; MP, Biomedicals, Santa Ana, CA, http://www.mpbio.com/), the obtained supernatant was used for testing samples according to kit instructions. Collagen standard curve was calculated, from which total collagen per construct wet weight was obtained.
Sulfated glycosaminoglycans (sGAGs) quantification was processed by using a Blyscan sGAG Assay Kit (B1000; Biocolor) as described elsewhere 21. Briefly, sGAG was extracted from tiny pieces of specimens at 4 or 12 weeks after injection after being digested with papain extraction (Sigma‐Aldrich) reagent at 65°C for 18 hours. Measurement of sGAG concentration in the supernatant was then performed according to the kit instructions, followed by calculation of sGAG per wet weight construct from the sGAG standard curve.

Secreted collagen proteins I-V were detected in culture medium using the Sircol Collagen Assay (S1000; Biocolor, Carrickfergus, UK), according to the manufacturer’s instructions.