Cryogenic Collagen-nanoHA Scaffold Synthesis

Cryogels were produced as previously described (Rodrigues et al., 2013 (link); Salgado et al., 2016 (link), 2019 (link)). Briefly, bovine collagen Type I (Sigma-Aldrich, Germany) was homogenized (Ultra Turrax T25, IKA) at 10000 rpm, in 5 mM HCl (36.5–38% grade, Sigma-Aldrich, Germany) at a concentration of 2% (w/v). Collagen-nanoHA biocomposites were prepared by mixing the collagen solution with 1% nanoHA (particle size 5.0 ± 1.0, nanoXIM.HAp202, FLUIDINOVA, S.A, Portugal), final composition Collagen-nanoHA 50:50 w/w%). O-phospho-L-serine (OPS, ≥98% grade, Sigma-Aldrich, Germany) was added to the nanoHA suspension (0.5% w/w%) with the final mass proportion of 1:1:0.5 for the Coll-nanoHA/OPS scaffold. For the preparation of cryogels, materials were crosslinked with 10 mM of N-hydroxysuccinimide (NHS, 98% grade, Sigma-Aldrich, Germany) and 20 mM of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, ≥98% grade, Sigma-Aldrich, Germany), at the final mass proportion of 1:0.012:0.031 (collagen/NHS/EDC), and were kept in a freezer at −18°C for 24 h to complete the crosslinking. Afterward, materials were thawed at room temperature and the scaffolds were washed with distilled water and finally dried in a freeze-dryer (Labconco, FreeZone 6) at −80°C for 24 h.