Bovine collagen type 1

C₁₆GSH stock solutions were prepared at 2 wt% in MilliQ water at pH 4 and sterilized by filtration through a 0.8/0.2 μm Supor^® Membrane filter (Pall Life Sciences, Port Washington, NY). PA solutions were heated to 50°C and sonicated for 5 min to ensure complete dissolution. PA solutions were then cooled to room temperature and further diluted with MilliQ water to specified concentrations before use. For in vivo studies, C₁₆GSH stock solutions were diluted to 2 and 0.2 wt% in water, raised to pH 6.5 using 1 M NaOH (Sigma Aldrich, Milwaukee, WI), and loaded into 1-mL sterile syringes. A buffer solution of 2×phosphate-buffered saline (PBS) was added and mixed thoroughly to achieve a concentration of 1 and 0.1 wt%, respectively, and a pH of 7.4.
Type I bovine collagen (Sigma Aldrich) was used as received (0.3 wt%). The manufacturer's instructions were followed for the gelling procedure. Briefly, the acidic collagen solution was mixed 8:1 with 10× PBS, and the pH was adjusted to 7.4 using 1 μL drops of 0.1 M NaOH. Collagen solutions were kept on ice before use to prevent gelation. Collagen solutions were gelled by incubation at 37°C for 1 h.

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).