Bioink formulations to ameliorate bioprinting-induced loss of cellular viability

Extrusion bioprinting, the most affordable and convenient bioprinting modality, is also associated with high process-induced cell deaths. Mechanical stresses on the cells during pneumatic or piston extrusion generate excessive reactive oxygen species and activate apoptosis, inflammatory pathways in the cells. In this study, a bioink formulation is augmented with an antioxidant, N-acetyl cysteine (NAC) as a possible solution to abrogate the effect of bioprinting-associated cell survival losses. The NAC addition to bioinks did not affect the bioprinting process, shape fidelity, or the mechanical properties of the constructs to any large extent. However, the bioprinting process conducted at 0.30 MPa pressure and 410 μm nozzle inner diameter with bioinks of 3% w/v alginate, 10⁵ cells/ml resulted in survival losses of up to 25% for MC3T3 cells. In contrast, NAC bioinks showed a significant (p < 0.01) improvement in day 1 cell survival (91%), while the enhancement in day 3 cell viability was still greater. It was further observed that the reactive oxygen species (ROS) load of bioprinted constructs was approximately 1.4 times higher compared to control, whereas NAC containing constructs reduced the ROS load at levels comparable to control samples. The effect on apoptosis and inflammation markers showed that NAC had a greater role in modulating apoptosis. It is concluded that the presented approach to preserve cell viability and functionality would be advantageous over other contemporary methods (like alterations in extrusion pressure, nozzle diameter, polymer concentration, etc.) as viability can be preserved without compromising the fabrication time or the resolution/mechanical properties of the constructs with this bioink formulation approach.