Bones are complex and hierarchical tissues involved in the structural and mechanical support of human body.¹ Every year thousands of people are affected by skeletal debilitating conditions, the treatment of which often involves symptoms palliation instead of disease/disorder cure. Recently, new tissue engineering and regenerative medicine strategies have been explored. Among different types of biomaterials available for scaffold fabrication, silk fibroin has shown the capacity to induce both in vitro and in vivo osteogenic signalling,² thus making it a suitable candidate for bone-related applications, such as three-dimensional (3D) tissue substitute models.³ In this study, we aim to develop a new bioactive silk fibroin (SF) hydrogel for application in the development of 3D in vitro models. An enzymatic crosslinking approach in the presence of horseradish peroxidase and calcium peroxide as substrate has been used for SF hydrogels production. Previously characterized hydrogen peroxide-crosslinked hydrogels have been used as a control.⁴ The in vitro bioactivity has been assessed by means of soaking the SF hydrogels in a Simulated Body Fluid (SBF) solution for 7 and 14 days. Hydrogels have been analysed by means of the energy-dispersive X-ray (EDX) feature of the Scanning Electron Microscope (SEM) in order to investigate the apatite-like crystals formation and deposition. Calcium peroxide-crosslinked SF hydrogels have shown calcium incorporation and have exhibited faster apatite-like crystals formation as compared to the controls, suggesting higher mineralization ability. Our results indicate that SF hydrogels enzymatically-crosslinked using calcium peroxide as oxidizer can be used as matrices for bone tissue engineering application and possibly as mimetic 3D in vitro bone models.

Acknowledgements: This work has been funded under the project FROnTHERA-RL1 (Ref. NORTE-01-0145-FEDER-000023) and supported by EU Framework Programme for Research and Innovation H2020 on Forefront Research in 3D Disease Cancer Models as in vitro Screening Technologies (FoReCaST) under grant agreement no.668983.

References

1) Florencio-Silva, R., Sasso, G. R. da S., Sasso-Cerri, E., Simões, M. J. & Cerri, P. S. Biology of Bone Tissue: Structure, Function, and Factors That Influence Bone Cells. BioMed Research International 2015, 421746 (2015)

2) Midha, S., Murab, S. & Ghosh, S. Osteogenic signaling on silk-based matrices. Biomaterials 97, 133–153 (2016)

3) Melke, J., Midha, S., Ghosh, S., Ito, K. & Hofmann, S. Silk fibroin as biomaterial for bone tissue engineering. Acta Biomater. 31, 1–16 (2016)

4) Yan, L.-P. et al. Tumor Growth Suppression Induced by Biomimetic Silk Fibroin Hydrogels. Sci. Rep. 6, 31037 (2016)