Chondroitin sulfate sodium salt

BC-GAG nanocomposite scaffolds were prepared by incubation of BC scaffolds (2×2 mm dimensions, ~50 mg) with chondroitin sulfate sodium salt (0.5 mL of 10 mg/mL stock, Sigma-Aldrich Co., St Louis, MO, USA) by shaking continuously for 24 hours. The scaffolds were then washed 2–3 times with ultrapure water to remove the unbound chondroitin sulfate. The chondroitin sulfate bound to BC was estimated using 1,9-dimethylmethylene blue (DMMB) dye.25 (link) Briefly, 6 mL of DMMB dye was added to each BC scaffold and vortexed vigorously for 30 minutes to promote complete complexation of bound chondroitin sulfate to the dye. The insoluble GAG–DMMB complex was separated by centrifugation (12,000× g, 10 minutes) and dissolved in 6 mL decomplexation solution (50 mM sodium acetate buffer, pH 6.8, containing 10% n-propanol and 4 M guanidine hydrochloride). Absorbance was measured at 656 nm. Assays were performed in triplicate.

GAG content in the culture medium and in tissues was quantified using 1,9-dimethyl-methylene blue (DMMB, Sigma-Aldrich) assay, as previously described.^{20 (link),21 (link),30 (link)} Microtissues, and amalgamated microtissues, were digested overnight at 60°C in 1 mL solution containing 1.6 U/mL papain and L-cysteine (both from Sigma-Aldrich). Twenty-five microliter of tissue digest or the medium collected during medium exchanges was combined with 200 µL of DMMB dye in 96 well plates (Nunc), and absorbance was measured at 590 nm in a plate reader (ThermoFisher’s Multiscan GO). A standard curve was generated using dilutions of chondroitin sulfate sodium salt extracted from shark cartilage (Sigma-Aldrich). DNA content in the tissues was estimated using the PicoGreen assay, as per the manufacturer’s instructions (ThermoFisher). PicoGreen solution was added to the tissue digests in half volume black 96 well plates (Costar) and samples read at 480 nm excitation and 520 nm emission on a plate reader (OMEGA FLUOstar multi-plate reader, BMG Labtech). A DNA standard curve was generated using λ-DNA (Quant-iT™ PicoGreen™ dsDNA Assay Kit, ThermoFisher).

Mineralized collagen scaffolds were fabricated using previously described procedures.^{10,11,21,27,40–45 (link)} In a cooled, jacketed vessel, 1.9 w/v% type I bovine collagen (Sigma-Aldrich, Missouri, USA), 0.84 w/v% chondroitin-6-sulfate (Sigma-Aldrich), and Ca(OH)₂, H₃PO₄, and Ca(NO₃)₂·4H₂O were thoroughly homogenized, making sure to prevent collagen clumping. The mineralized collagen suspension was transferred to 7.62 × 7.62 cm aluminum molds to create isotropic scaffolds. Alternatively, the suspension was pipetted into Teflon molds with a copper base to create anisotropic scaffolds.^{46 (link)} The suspensions were cooled at a constant rate of 1 °C min⁻¹ from 20 °C to −10 °C. Once frozen, the suspension was held at this temperature for 2 hours then lyophilized (0.2 torr pressure, 0 °C) in a VirTis Genesis 25XL Lyophilizer (SP Industries, Inc., Pennsylvania, USA).
Anisotropic mineralized collagen scaffolds with disparate GAG content were fabricated following the same method. However initial mineralized collagen suspensions were created using one of three potential glycosaminoglycans: chondroitin-6-sulfate (chondroitin sulfate sodium salt from shark cartilage, Sigma-Aldrich), chondroitin-4-sulfate (sodium chondroitin sulfate A, Toronto Research Chemicals Inc., Ontario, Canada), or heparin sulfate (Heparin sodium salt from porcine intestinal mucosa, Sigma-Aldrich).