Optimizing gelatin methacryloyl-based bioinks with nanohydroxyapatite for 3D bioprinting in bone tissue engineering

In recent years, the incorporation of a ceramic phase such as nanohydroxyapatite (nHA) into gelatin methacryloyl (GelMA)-based bioinks has gained attention for extrusion-based bioprinting to replicate the inorganic mineral phase of bone tissue. However, the effects of different nHA concentrations in GelMA bioinks on printability, shape fidelity, long-term stability, cell viability, and osteogenic activity have not been fully investigated. Therefore, the current study investigates the incorporation of different concentrations of nHA (0, 1, 3 and 5% w/v) into GelMA/gelatin/nanocellulose bioink for printing human bone marrow-derived stromal cells for bone tissue engineering applications. As the concentration of nHA increased, the viscosity of the bioink also increased significantly. The bioink with the highest concentration of nHA failed to maintain the full height of the printed objects, while the bioinks containing only 1% nHA showed superior buildability and shape fidelity. Moreover, nHA particles hindered the photocrosslinking process in a concentration-dependent manner. Higher concentrations of nHA resulted in poor cross-linking, which negatively affected the stability of the printed structures under culture conditions. In combination, the increased viscosity and inadequate crosslinking at higher nHA concentrations had a detrimental effect on the cells: the 5% nHA bioink resulted in reduced cell viability and limited osteogenic differentiation. In contrast to the higher concentrations, the 1% nHA bioink supported high cell viability, maintained good metabolic activity and promoted osteogenic protein production (Runx2, Col-I, DMP1, OCN). This study highlights the importance of incorporating low concentrations of the nHA phase in multi-component bioinks for bone tissue engineering applications.