Volumetric bioprinting of the osteoid niche

Volumetric bioprinting has revolutionized the field of biofabrication by enabling the creation of cubic centimeter-scale living constructs at faster printing times (in the order of seconds). However, a key challenge remains: developing a wider variety of available osteogenic bioinks that allow osteogenic maturation of the encapsulated cells within the construct. Herein, the bioink exploiting a step-growth mechanism (norbornene-norbornene functionalized gelatin in combination with thiolated gelatin-GelNBNBSH) outperformed the bioink exploiting a chain-growth mechanism (gelatin methacryloyl-GelMA), as the necessary photo-initiator concentration was three times lower combined with a more than 50% reduction in required light exposure dose resulting in an improved positive and negative resolution. To mimic the substrate elasticity of the osteoid, two concentrations of the photo-initiator Li-TPO-L (1 and 10 mg ml^-1) were compared for post-curing whereby the lowest concentration was selected since it resulted in attaining the osteogenic substrate elasticity combined with excellent biocompatibility with HT1080 cells (>95%). Further physico-chemical testing revealed that the volumetric printing (VP) process affected the degradation time of the constructs with volumetric constructs degrading slower than the control sheets which could be due to the introduced fibrillar structure inherent to the VP process. Moreover, GelNBNBSH volumetric constructs significantly outperformed the GelMA volumetric constructs in terms of a 2-fold increase in photo-crosslinkable moiety conversion and a 3-fold increase in bulk stiffness of the construct. Finally, a 21-day osteogenic cell study was performed with highly viable dental pulp-derived stem cells (>95%) encapsulated within the volumetric printed constructs. Osteogenesis was greatly favored for the GelNBNBSH constructs through enhanced early (alkaline phosphatase activity) and late maturation (calcium production) osteogenic markers. After 21 d, a secretome analysis revealed a more mature osteogenic phenotype within GelNBNBSH constructs as compared to their chain-growth counterpart in terms of osteogenic, immunological and angiogenic signaling.