Bioprinting EphrinB2-Modified Dental Pulp Stem Cells with Enhanced Osteogenic Capacity for Alveolar Bone Engineering

Impact statementIn this work, we first optimized printing parameters of 5% Gelatin methacrylate (GelMA) and investigated stability of bioprinted constructs. Then, comparison of cell properties between bioprinted and casted hydrogel indicated that bioprinting had no obvious side effects on viability, proliferation, and osteogenic differentiation of dental pulp stem cells (DPSCs). Finally, bioprinted constructs loaded with EphrinB2-DPSCs exhibited enhanced osteogenic potential in vitro. In conclusion, our study reported optimized bioprinting parameters of low-concentrated GelMA, and provided a promising strategy for alveolar bone regeneration by combining bioprinting and EphrinB2 gene modification. Bioprinting, a technology that allows depositing living cells and biomaterials together into a complex tissue architecture with desired pattern, becomes a revolutionary technology for fabrication of engineered constructs. Previously, we have demonstrated that EphrinB2-modified dental pulp stem cells (DPSCs) are expected to be promising seed cells with enhanced osteogenic differentiation capability for alveolar bone regeneration. In this study, we aimed to bioprint EphrinB2-overexpressing DPSCs with low-concentrated Gelatin methacrylate (GelMA) hydrogels into three-dimensional (3D) constructs. The printability of GelMA (5% w/v) and the structural fidelity of bioprinted constructs were examined. Then, viability, proliferation, morphology, and osteogenic differentiation of DPSCs in bioprinted constructs were measured. Finally, the effect of EphrinB2 overexpression on osteogenic differentiation of DPSCs in bioprinted constructs was evaluated. Our results demonstrated that GelMA (5% w/v) in a physical gel form was successfully bioprinted into constructs with various shapes and patterns using optimized printing parameters. Embedded DPSCs showed round-like morphology, and had a high viability (91.93% +/- 8.38%) and obvious proliferation (similar to 1.9-fold increase) 1 day after printing. They also showed excellent osteogenic potential in bioprinted constructs. In bioprinted 3D constructs, EphrinB2-overexpressing DPSCs expressed upregulated osteogenic markers, including ALP, BMP2, RUNX2, and SP7, and generated more mineralized nodules, as compared with Vector-DPSCs. Taken together, this study indicated that fabrication of bioprinted EphrinB2-DPSCs-laden constructs with enhanced osteogenic potential was possible, and 3D bioprinting strategy combined with EphrinB2 gene modification was a promising way to create bioengineered constructs for alveolar bone regeneration.