Collagen from bovine achilles tendon

Collagen has both a reducing and a stabilizing role in Au NP formation. A stock solution of gold salt was prepared by dissolving 1 g hydrogen tetrachloroaurate (III) hydrate (99.9% metal basis, Alfa Aesar, Karlsruhe, Germany) in 50 mL ultrapure water. A solution of collagen was prepared by mixing 10 mL ultrapure water with 0.02 g of collagen from bovine Achilles tendon (Sigma-Aldrich, Darmstadt, Germany) in the presence of 500 µL hydrochloric acid 37% (Sigma-Aldrich). One-point-five milliliters of collagen solution (0.02 g collagen in 10 mL water) was mixed with 0.5 mL ethanol. Subsequently, 90 mL of ultrapure water were added to the collagen-ethanol solution, which was then mixed with 1 mL of gold salt solution. The mixture was heated and stirred until boiling. When it started to boil, the solution was neutralized by quickly adding 2 mL of 1% Sodium hydroxide, and the heating was turned off. Instantly, the solution turned into a wine-red color, and its pH was 7. Sodium hydroxide (Sigma-Aldrich), ethanol and sodium chloride (Merck, Darmstadt, Germany) were of analytical grade. All solutions were prepared in ultrapure water with a resistance higher than 18 MΩ (Direct-Q 3 UV, Merck Millipore, Darmstadt, Germany). The protocol is given in full detail in the Supporting Information. The Raman spectrum of collagen-coated Au NPs is presented in Figure S1.

Anisotropic collagen scaffolds were synthesized using our freeze drying method^{21 (link)}. Briefly, collagen from bovine Achilles tendon (Sigma Aldrich, UK) was added to 0.05 M acetic acid overnight, homogenised and aspirated into engineered molds containing a copper pin. Using a freeze drier ice was then sublimated and the scaffolds cross-linked with 70% ethanol +33 mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride +6 mM N-hydroxysuccinimide and sterilized in 70% ethanol overnight. Scaffolds were washed in phosphate buffered saline (PBS) and incubated in maintenance medium (MM) (DMEM (Gibco, 41965), 10% heat inactivated foetal bovine serum (FBS) (Gibco, 10500064) and 1x penicillin/streptomycin (Gibco, 15140–122)) overnight to assure sterility.