Volumetric Tomographic 3D Bioprinting of Heterocellular Bone-like Tissues in Seconds

3D bioprinting has emerged as a powerful tool for custom fabrication of biomimetic hydrogel constructs that support the differentiation of stem cells into functional bone tissues. Existing stem cell-derived in vitro bone models, however, often lack terminally differentiated bone cells named osteocytes which are crucial for bone homeostasis. Here, we report ultrafast volumetric tomographic photofabrication of centimeter-scale heterocellular bone models that enabled successful 3D osteocytic differentiation of human mesenchymal stem cells (hMSCs) within hydrogels after 42 days co-culture with human umbilical vein endothelial cell (HUVECs). It is hypothesized that after 3D bioprinting the paracrine signaling between hMSCs and HUVECs will promote their differentiation into osteocytes while recreating the complex heterocellular bone microenvironment. To this, we formulated a series of bioinks with varying concentrations of gelatin methacryloyl (GelMA) and lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP). A bioink comprising 5% GelMA and 0.05% LAP was identified as an optimal material with high cell viability (>90%) and excellent structural fidelity. Increasing LAP concentration led to much lower degree of cell spreading, presumably due to phototoxicity effects. Biochemical assays evidenced significantly increased expression of both osteoblastic markers (collagen-I, ALP, osteocalcin) and osteocytic markers (Podoplanin, PDPN; dentin matrix acidic phosphoprotein 1, Dmp1) after 3D co-cultures for 42 days. Additionally, we demonstrate volumetric 3D bioprinting of perfusable, pre-vascularized bone models where HUVECs self-organized into an endothelium-lined channel within 2 days. Altogether, this work leverages the benefits of volumetric tomographic bioprinting and 3D co-culture, offering a promising platform for scaled biofabrication of 3D bone-like tissues with unprecedented long-term functionality.