Multi-Material Volumetric Additive Manufacturing of Hydrogels Using Gelatin as a Sacrificial Network and 3D Suspension Bath

Volumetric additive manufacturing (VAM) is an emerging layerless method for the rapid processing of reactive resins into 3D structures, where printing is much faster (seconds) than other lithography and direct ink writing methods (minutes to hours). As a vial of resin rotates in the VAM process, patterned light exposure defines a 3D object and then resin that has not undergone gelation can be washed away. Despite the promise of VAM, there are challenges with the printing of soft hydrogel materials from non-viscous precursors, including into multi-material constructs. To address this, we used a sacrificial gelatin matrix to modulate resin viscosity to support the cytocompatible VAM printing of macromers based on poly(ethylene glycol), hyaluronic acid (HA), and polyacrylamide. After printing, gelatin and unreacted resin are removed by washing at an elevated temperature (37°C). These constructs are soft due to the low polymer concentration and would be difficult to print in other types of additive manufacturing. Cell viability of various cell types (bovine mesenchymal stromal cells (MSCs), chondrocytes, and meniscal fibrochondrocytes) was also confirmed over 7 days of culture with an average viability of 96.4%. To print multi-material constructs, the gelatin-containing resin is also used as a shear-yielding suspension bath (including high molecular weight HA to further modulate bath properties) where an ink (also containing gelatin and high molecular weight HA) with a different photoreactive monomer can be extruded into the bath to define a multi-material resin that can then be processed with VAM into a singular defined object. Multi-material constructs of methacrylated HA (MeHA) used as the bath and gelatin methacrylamide (GelMA) used as the ink are printed (as proof-of-concept) with encapsulated MSCs, where the local hydrogel properties define cell spreading behavior with culture. After 1 day of culture, the cells spread in the GelMA regions and stayed rounded in the MeHA regions, due to the adhesion ligands and degradability present in GelMA. In future studies, this process can be applied to tissue engineering to help recapitulate the heterogenous nature of complex tissues such as the meniscus.