Horseradish peroxidase-crosslinked calcium-containing silk fibroin hydrogels as artificial matrices for bone cancer research

"Online ahead of print" Hydrogels, being capable of mimicking the extracellular matrix composition of tissues, are greatly used as artificial matrices in tissue engineering applications. In this study, we report the generation of horseradish peroxidase (HRP)-crosslinked silk fibroin (SF) hydrogels, using calcium peroxide as oxidizer. The proposed fast forming calcium-containing SF hydrogels spontaneously undergo SF conformational changes from random coil to β-sheet during time, exhibiting ionic and pH stimuli responsiveness. In vitro response shows calcium-containing SF hydrogelsâ encapsulation properties and their ability to promote SaOs-2 tumor cells death after 10 days of culturing, upon complete β-sheet conformation transition. Calcium-containing SF hydrogelsâ angiogenic potential investigated in an in ovo chick chorioallantoic membrane (CAM) assay, show a high number of converging blood vessels as compared to the negative control, although no endothelial cells infiltration is observed. The in vivo response evaluated in subcutaneous implantation in CD1 and nude NCD1 mice shows that calcium-containing SF hydrogels are stable up to 6 weeks after implantation. However, an increased number of dead cells are also present in the surrounding tissue. The results suggest the potential of calcium-containing SF hydrogels to be used as novel in situ therapeutics for bone cancer treatment applications, particularly to osteosarcoma. L.P. thanks the International mobility program of the University of Trento, Italy. J.S.-C. and J.M.O. thank the Portuguese Foundation for Science and Technology (FCT) for the funds provided under the program Investigador FCT 2015 (IF/00115/2015 and IF/01285/2015, respectively). This research was funded by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) (NORTE-01-0145-FEDER-000023). The project BAMOS (H2020-MSCARISE-2016-734156) funded by the European Union under the Horizon 2020 program, and the EU Framework Programme for Research and Innovation H2020 on FoReCaST (Grant Agreement No.668983), are also greatly acknowledged. V.P.R. acknowledge for the Junior Researcher contract (POCI-01-0145-FEDER-031367) attributed by the Portuguese Foundation for Science and Technology to Fun4TE project (PTDC/EMD-EMD/31367/2017).