The effect of culture conditions on the bone regeneration potential of osteoblast-laden 3D bioprinted constructs

Three Dimensional (3D) bioprinting is one of the most recent additive manufacturing technologies and enables the direct incorporation of cells within a highly porous 3D-bioprinted construct. While the field has mainly focused on developing methods for enhancing printing resolution and shape fidelity, little is understood about the biological impact of bioprinting on cells. To address this shortcoming, this study investigated the in vitro and in vivo response of human osteoblasts subsequent to bioprinting using gelatin methacryloyl (GelMA) as the hydrogel precursor. First, bioprinted and two-dimensional (2D) cultured osteoblasts were compared, demonstrating that the 3D microenvironment from bioprinting enhanced bone-related gene expression. Second, differentiation regimens of 2-week osteogenic pre-induction in 2D before bioprinting and/or 3-week post-printing osteogenic differentiation were assessed for their capacity to increase the bioprinted construct's biofunctionality towards bone regeneration. The combination of pre-and post-induction regimens showed superior osteogenic gene expression and mineralisation in vitro. Moreover, a rat calvarial model using microtomography and histology demonstrated bone regeneration potential for the pre-and post-differentiation procedure. This study shows the positive impact of bioprinting on cells for osteogenic differentiation and the increased in vivo osteogenic potential of bioprinted constructs via a pre-induction method. 3D bioprinting, one of the most recent technologies for tissue engineering has mostly focussed on developing methods for enhancing printing properties, little is understood on the biological impact of bioprinting and /or subsequent in vitro maturation methods on cells. Therefore, we addressed these fundamental questions by investigating osteoblast gene expression in bioprinted construct and assessed the efficacy of several induction regimen towards osteogenic differentiation in vitro and in vivo. Osteogenic induction of cells prior to seeding in scaffolds used in conventional tissue engineering applications has been demonstrated to increase the osteogenic potential of the resulting construct. However, to the best of our knowledge, pre-induction methods have not been investigated in 3D bioprinting.