Developing double-crosslinking 3D printed hydrogels for bone tissue engineering

Bone defects are one of the main causes of disability worldwide. Due to the disadvantages associated with autografts, the latest advances have been focused on tissue regeneration approaches that use injectable hydrogels or 3D printed hydrogel-based structures that could refill appropriately the bone gap area without the need for external fixatives, leading to bone formation in the long term. Injectable hydrogels could be applied in extrusion-based 3D printing as inks; in this sense, double-crosslinking hydrogels appear as ideal candidates. In this work, injectable and printable double crosslinkable hydrogels based on oxidized xanthan gum (XGox) and methacrylate polyaspartylhydrazide (PAHy-MA) were produced. The formation of dynamic hydrazone bonds, occurring between aldehyde groups on the polysaccharide backbone and hydrazine moieties of PAHy-MA, induced an instant gelation, conferring, also, injectability and self-healing properties to the hydrogels. The presence of methacrylic moieties on the synthetic polymer allowed further crosslinking upon UV irradiation that stabilized the hydrogel shape and mitigated its susceptibility to hydrolytic degradation. Obtained hydrogels showed pseudoplastic behaviour and good recovery of viscoelastic properties over time. The physicochemical and rheological characterization highlighted increased stability and higher viscoelastic moduli after photo-crosslinking. The hydrogels also showed good printability, cytocompatibility and the early formation of a bone-like matrix when osteosarcoma-derived cells (MG-63) were cultured in the scaffolds for 21 days, with an increased collagen I deposition, mineralization and the expression of characteristic osteogenic markers.