Bovine achilles tendon

Collagen solution was prepared by dissolving type I bovine collagen (Bovine Achilles tendon, Sigma-Aldrich, St. Louis, Missouri, USA) in 0.5 mol/L acetic acid to the final concentration of 0.5% (w/v %) and stirring with high speed Turrax (T25D, IKA^®, Janke and Kunkel IKA-Labortechnik, Staufen, Germany) at 10,000 rpm and 4 °C for 2–3 h.
B. mori silk fibroin was prepared by initially degumming by boiling silkworm cocoons in Na₂CO₃ solution at 1 g/L for 30 min at 85 °C. This procedure was repeated two times and, finally, the cocoons were boiled in distilled water for 30 min in order to separate the glue-like sericin from fibroin. After adequate washing with distilled water, the obtained silk fibroin fibers were dried at room temperature. Finally, silk fibroin fibers were milled to facilitate their dissolution process. Silk fibroin was dissolved in a solution of ternary solvent containing CaCl₂:CH₃CH₂OH:H₂O (1:2:8 molar ratio), at 85 °C in order to obtain the final concentration of 0.5% (w/v %). The fibroin solution was dialyzed against distilled water for 72 h.

0.2 µM fluorescently labeled ColG-variants were incubated at 25 °C for 30 min with prewetted insoluble type I collagen from bovine Achilles tendon (Sigma, Germany) (0, 2, 4, 6, and 8 mg or 6 mg) in 50 mM HEPES pH 7.5, 100 mM NaCl, 10 mM CaCl₂, 0.1% Tween-20, 1% fraction V of bovine serum albumin, 3 mM NaN₃, ±10 mM ß-mercaptoethanol. The reactions were centrifuged at 13,000×g for 5 min at RT and the collagen-binding ability was determined by monitoring the free fluorescence intensity in the supernatant. More details can be found in SI Appendix.

Hybrid electrospun scaffold sheets were prepared using high-molecular-weight poly(L-lactide) (PL18, Purac, The Netherlands) solutions containing HA nanoparticles (Sigma-Aldrich, USA) and/or bovine collagen (Type I) (Sigma-Aldrich, USA), with an average fiber diameter of 200–950 nm. The electrospun scaffolds were fabricated using the following solution compositions: For PLLA/Col/HA (100∶10∶15) scaffold, 2.4 g of PLLA+240 mg of collagen (Bovine Achilles Tendon) in hexafluoroisopropanol (HFIP) (Sigma-Aldrich, USA) (total weight: 30 g) and 360 mg of HA (200-nm particles) were used; for PLLA/Col (100∶10) scaffold, 2.4 g of PLLA+240 mg of collagen (Bovine Achilles Tendon) in HFIP (total weight: 30 g) were used; and for PLLA/HA (100∶15) scaffold, 2.4 g of PLLA in HFIP (total weight: 30 g) and 336 mg of HA (200-nm particles) were used. In each case, 5 ml of the solution was electrospun onto an aluminum-foil-covered, earthed rotating mandrel collector (rotation speed = 60 rpm) in an environmentally controlled cabinet (temperature = 25°C and relative humidity = 25%).