Silk Fibroin Tissue Engineering-based Approaches for The Treatment of Degenerated Intervertebral Disc

Lower back pain and intervertebral disc degeneration represent a global socio-economical problem affecting 266 million people annually, always increasing due to aging of the population. No restorative treatments are available. In case of chronic degeneration, surgical operation with spinal fusion or total disc replacement represents the best alternative. This leads to pain relief but reduces the patient’s mobility. Moreover, follow-ups and re-intervention due to weak osteointegration are common consequences of currently used metal prostheses. For this reason, there is an urgent need to develop customized regenerative approaches aimed at the restoration of IVD function, as well as the optimization of osteointegration in actual vertebral prostheses by creating hybrid metal implants with infill materials to better induce bone ingrowth. In this work, tissue engineering-based approaches have been exploited by tuning the remarkable properties of silk fibroin for two purposes, disc restoration via in situ 3D printing technique, and improvement of osteointegration of vertebral prostheses. In situ 3D printing is the most promising strategy for the development of a personalized medicine approach aimed at the restoration of IVD. However, silk fibroin application as pristine ink in 3D printing technique is hindered by its low viscosity. For this reason, the aim of the first part of the work has been the design and development of silk fibroin-based inks in situ applications, overcoming its intrinsic limitations. Specifically, a covalent crosslinking process consisting of a pre-photo-crosslinking prior to printing and in situ enzymatic crosslinking was designed. Two different silk fibroin molecular weights were characterized. We proved that despite the use of low concentration silk solutions, the synergistic effect of the covalent bonds with the shear forces applied in the nozzle enhanced silk secondary structure shift toward β-sheets conformation. The resultant hydrogels exhibited good mechanical properties, stability over time, and resistance to enzymatic degradation over 14 days, with no significant changes over time in their secondary structure and swelling behavior. The designed process was tunable and versatile, leading to good shape fidelity and printing resolutions, making real the application of silk fibroin-based inks for in situ applications. The results obtained represent an important step for further studies on the mimicry of the whole IVD structure. 2 In the second part of the work, silk fibroin has been evaluated as candidate infill material for metal prostheses to improve bone ingrowth and osteointegration. In two independent works, silk fibroin-based foams and methacrylate silk fibroin sponges were biologically characterized and the differentiation of bone marrow-derived human mesenchymal stem cells (hBM-MSCs) toward osteogenic phenotype was studied. Silk fibroin foams have been demonstrated to induce and support cells adhesion, migration, and differentiation, and to induce early mineralization phase since day 7 during the differentiative culture. Methacrylate silk fibroin foams have been fabricated with different photo-initiator concentrations and in presence/absence of a porogen. The impact of the composition on the pore size, mechanical properties, and stem cells differentiation was deeply investigated. We demonstrated that despite all the conditions well-supported cells differentiation, the lowest photo-initiator concentration in combination with the porogen used enhanced osteogenic differentiation as confirmed by gene expression tests.