Cell-Laden Biomimetially Mineralized Shark-Skin-Collagen-Based 3D Printed Hydrogels for the Engineering of Hard Tissues

Mineralization processes based on co-precipitation methods have been applied as a promising alternative to the most commonly used methods of polymerâ ceramic combination, direct mixing, and incubation in simulated body fluid (SBF) or modified SBF. In the present study, for the first time, the in situ mineralization (ideally hydroxyapatite formation) of blue shark (Prionace glauca (PG)) collagen to fabricate 3D printable cell-laden hydrogels is proposed. In the first part, several parameters for collagen mineralization were tested until optimization. The hydroxyapatite formation was confirmed by FTIR, XRD, and TEM techniques. In the second part, stable bioinks combining the biomimetically mineralized collagen with alginate (AG) (1:1, 1:2, 1:3, and AG) solution were used for 3D printing of hydrogels. The addition of Ca2+ ions into the system did present a synergistic effect: by one side, the in situ mineralization of the collagen occurred, and at same time, they were also useful to ionically crosslink the blends with alginate, avoiding the addition of any cytotoxic chemical crosslinking agent. Mouse fibroblast cell line survival during and after printing was favored by the presence of PG collagen as exhibited by the biological performance of the hydrogels. Inspired in a concept of marine byproduct valorization, 3D bioprinting of in situ mineralized blue shark collagen is thus proposed as a promising approach, envisioning the engineering of mineralized tissues.This study was funded by the Portuguese Foundation forScience and Technology (FCT) under the scope of the BiogenInk project (M-ERA-NET2/0022/2016), by the Euro-pean Union Seventh Framework Programme (FP7/2007-2013) through grant agreement n. ERC-2012-ADG 20120216-321266 (ERC Advanced Grant ComplexiTE) and by theINTERREG Atlantic Area Programme under project BlueHu-man (EAPA_151/2016). The Doctoral Programme NORTE-08-5369-FSE-000044 and Structured Projects NORTE-01-0145-FEDER-000021 and NORTE-01-0145-FEDER-000023,supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020Partnership Agreement, through the European Social Fund, are also greatly acknowledged. R.P. thanks FCT for the contractIF/00347/2015. The authors acknowledge also the Centro Tecnológico del Mar - Fundación CETMAR (Vigo, Spain) andCOPEMAR SA (fishing company, Spain) for the kind offer offish byproducts