Gelatin methacryloyl (GelMA) hydrogels is a widely used bioink because of its good biological properties and tunable physicochemical properties, which has been widely used in a variety of tissue engineering and tissue regeneration. However, pure GelMA is limited by the weak mechanical strength and the lack of continuous osteogenic induction environment, which is difficult to meet the needs of bone repair. Moreover, GelMA hydrogels are unable to respond to complex stimuli and therefore are unable to adapt to physiological and pathological microenvironments. This review focused on the functionalization strategies of GelMA hydrogel based bioinks for bone regeneration. The synthesis process of GelMA hydrogel was described in details, and various functional methods to meet the requirements of bone regeneration, including mechanical strength, porosity, vascularization, osteogenic differentiation, and immunoregulation for patient specific repair, etc. In addition, the response strategies of smart GelMA-based bioinks to external physical stimulation and internal pathological microenvironment stimulation, as well as the functionalization strategies of GelMA hydrogel to achieve both disease treatment and bone regeneration in the presence of various common diseases (such as inflammation, infection, tumor) are also briefly reviewed. Finally, we emphasized the current challenges and possible exploration directions of GelMA-based bioinks for bone regeneration.

We fabricated a pre-vascularized bioprinted hydrogel construct via coculture of human umbilical vein endothelial cells (HUVECs) and dental pulp stem cells (DPSCs) to investigate the role of ephrinB2 in 3D capillary-like cord self-assemble. After bioprinting gelatin methacrylate (GelMA) hydrogels, we performed a morphology observation of the inside capillary-like cords, quantitative analysis of branch points and cord length, and examination of pericyte markers in DPSCs. Next, we assessed the influence of DPSCs: HUVECs ratio and degree of functionalization (DoF) of GelMA on the cord formation. To investigate the role of ephrinB2, we used either EphB4/ephrinB2 inhibitor peptide, or knocked down or upregulated ephrinB2 in DPSCs, and evaluated their effects on these cords’ generation. Finally, bioprinted hydrogels were subcutaneously transplanted into mice to assess in vitro vascularization. Abundant capillary-like cords containing lumen were found in bioprinted hydrogels. DPSCs wrapped outside the cords and expressed pericyte markers. A 1:3 DPSCs: HUVECs ratio and a 30% DoF of GelMA led to significant increase in branch points and cord length by over 2-fold and 3-fold, respectively. Adding EphB4/ephrinB2 inhibitor peptide or knockdown of ephrinB2 in DPSCs impaired capillary-like cord generation, whereas overexpression of ephrinB2 in DPSCs promoted cord formation. In in vivo experiments, a significant over 3-fold increase in vessel number was observed in hydrogels when ephrinB2 was upregulated in DPSCs compared with wild-type DPSCs. Overexpression of ephrinB2 in DPSCs promoted in vitro capillary-like cord formation and in vivo vascularization in a 3D bioprinted hydrogel.