3D Bioprinting of Dense Cellular Structures within Functional Hydrogels with User-Defined Heterogeneity

3D bioprinting has a strong potential to address tissue and organ shortage for transplantation. Successful fabrication of a functional tissue requires bioprinting strategies that can replicate the biological and structural features of the native tissue including formation of dense cellular structures. Embedded 3D printing approaches advanced extrusion-based bioprinting technology for fabrication of architecturally and biologically complex structures, yet this technology requires the use of support baths which generally hinders the relevant bioactivity and heterogeneity. In this study, we developed a new method to create dense cellular structures where cell-only bioinks could be deposited into photocurable support hydrogels with tunable stiffness, degradation and bioactivity. Our approach can utilize multiple cell types or functional hydrogels to create highly heterogeneous and complex structures that could potentially be used to fabricate functional tissue models, as well as tissue interfaces, with relevant biochemical and physical complexities.<br/><br/>In this study, we demonstrate bioprinting of dense cellular structures using fibroblasts (NIH 3T3), human umbilical vein endothelial cells (HUVECs), and human mesenchymal stem cells (hMSCs) within support or functional hydrogels. To demonstrate the potential of our approach and importance of matrix functionalization, we investigate the osteogenic differentiation of hMSCs bioprinted within MeHA hydrogels with controlled stiffness and bioactivity. To control stem cell aspect ratio initial concentration of the MeHA hydrogel is varied (5-15%). Bioactivity is varied by tethering RGD and BMP-2. Dense hMSCs structures show great overall cell viability (&gt;80%) and inclusion of RGD and then BMP-2 significantly enhanced osteogenic differentiation. This is confirmed with ALP assay, Alizarin Red staining and osteocalcin immunostaining. Finally, we fabricated dense cellular structures with spatially controlled cellular composition and matrix composition towards creating tissue interfaces. <br/><br/><b>Conflict of Interest: </b>New Jersey Institute of Technology has a financial interest in the bioprinting approach reported in this manuscript and has already filed a patent application for the technology described in this paper (U.S. Application No. 17/588,998). <br/><br/><b>Acknowledgements: </b>We acknowledge the funding from NSF/DMR - CAREER 2044479.