Compositional analysis was performed as per previous reports [39 (link)]. Briefly, constructs were lyophilized at –55 °C and 0.2 mbar for 4–5 h and then 20 mg of each sample was digested (9 mM di-sodium EDTA, 20 mM sodium acetate, 20 mM l -cysteine (MP Biomedicals, Solon, OH, USA), 2 mg papain (MP Biomedicals) per gram lyophilized sample) at 60 °C for 10 h [40 (link), 41 (link)]. Digested samples were vigorously vortexed and then centrifuged (4000 × g, 10 min). Supernatants were stored at –80 °C until analysis. Double-stranded DNA was determined with a commercially available kit (Quant-iT™ PicoGreen® Kit, Invitrogen, Carlsbad, CA, USA) [41 (link)]. Hydroxyproline content as a measure of total collagen was quantified via Ehrlich’s colorimetric assay and absorbance at 550 nm based on trans-4-hydroxy-l -proline (ACROS Organics™, Morris Plains, NJ, USA) standards [41 (link), 42 (link)]. Sulfated glycosaminoglycan (sGAG) was quantified with a dimethylmethylene blue (DMMB) assay, with absorbance at 520 nm and a chondroitin sulfate standard curve [41 (link), 43 (link)]. Lowry’s total protein assay with Biuret’s and Folin-Ciocalteu’s reagents, absorbance at 650 nm, and bovine serum albumin standards, was used to quantify sample protein [44 (link)].
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