Culture of 3D bioprinted bone constructs requires an increased fluid dynamic stimulation

In vitro flow-induced mechanical stimulation of developing bone tissue constructs has been shown to favor mineral deposition in scaffolds seeded with cells directly exposed to the fluid flow. However, the effect of fluid dynamic parameters, such as shear stress (SS), within 3D bioprinted constructs is still unclear. Thus, this study aimed at correlating the SS levels and the mineral deposition in 3D bioprinted constructs, evaluating the possible dampening effect of the hydrogel. Human mesenchymal stem cells (hMSCs) were embedded in 3D bioprinted porous structures made of alginate and gelatin. 3D bioprinted constructs were cultured in an osteogenic medium assessing the influence of different flow rates (0, 0.7 and 7 ml/min) on calcium and collagen deposition through histology, and bone volume (BV) through micro-computed tomography. Uniform distribution of calcium and collagen was observed in all groups. Nevertheless, BV significantly increased in perfused groups as compared to static control, ranging from 0.35±0.28 mm 3 , 11.90±8.74 mm 3 and 25.81±5.02 mm 3 at week 3 to 2.28±0.78 mm 3 , 22.55±2.45 mm 3 and 46.05±5.95 mm 3 at week 6 in static, 0.7 and 7 ml/min groups, respectively. SS values on construct fibers in the range 10-100 mPa in 7 ml/min samples were twice as high as those in 0.7 ml/min samples showing the same trend of BV. The obtained results suggest that it is necessary to enhance the flow-induced mechanical stimulation of cell-embedding hydrogels to increase the amount of mineral deposited by hMSCs, compared to what is generally reported for the development of in vitro bone constructs. In this study, we evaluated for the first time how the hydrogel structure dampens the effect of flow-induced mechanical stimulation during the culture of 3D bioprinted bone tissue constructs. By combining computational and experimental techniques we demonstrated that those shear stress thresholds generally considered for culturing cells seeded on scaffold surface, are no longer applicable when cells are embedded in 3D bioprinted constructs. Significantly, more bone volume was formed in constructs exposed to shear stress values generally considered as detrimental than in constructs exposed shear stress values generally considered as beneficial after 3 weeks and 6 weeks of dynamic culture using a perfusion bioreactor .