The influence of viscoelastic properties of bioinks on 3D bioprinted tissue models - A study of cell behaviour and printability

Abstract

The recent advancement within the field of 3D bioprinting has enabled its appli cation in areas such as the pharmaceutical industry, tissue engineering and many types of cell-based research. The principle is to utilize 3D printing technology to print a variety of biomaterials together with viable cells, to produce accurate tissue models by mimicking the in vivo cell environment. Bioinks used for 3D bioprint ing are commonly composed of hydrogels based on naturally derived polymers like gelatin, collagen, alginate and nanofibrillated cellulose (NFC). Bioinks are viscoelas tic materials which can be crosslinked after being printed to keep their structure and shape. The crosslinking method and conditions determine the stiffness of the result ing tissue construct, which can in turn also affect the behaviour of incorporated cells. This study aims to investigate the influence of the bioink’s viscoelastic properties on both cell behaviour and printability of the bionink. The studied bioinks include CELLINK Bioink, GelMA, GelMA C, GelXA and Photogel95, which are crosslinked either ionically using a CaCl2 solution or using photo-crosslinking, or a combination of the two. The bioinks’ viscoelastic properties, as well as stiffness after crosslinking at two different conditions for each bioink were investigated using rheological measure ments, showing that different stiffnesses could be achieved. 3D bioprinting of the bioinks with mesenchymal stem cells (MSC) was used to produce samples which were crosslinked at the same two conditions, cultured over 14 days and analyzed at several time points. The cell viability was evaluated by fluorescent staining using Calcein-AM and propidium iodide (PI), and the cell morphology by using Actin Green and DAPI, followed by fluorescent microscopy imaging. The stiffness of the cell samples over time was also evaluated by measurements at the same time points. The stiffness of the cell samples over time showed some unexpected results and high variation between samples, which can to some extent be explained by the method not being fully suitable and well-adapted for these samples. The cell viability was relatively high at day 1 for all bioinks and crosslinking conditions, above 90 % for most samples but around 80 % for a few. A decrease in cell viability was then observed for all samples at day 7 and day 14. The cell morphology analysis showed cells spreading in all gelMA-based bioinks at day 7 and day 14, except for Photogel95. However, no distinct correlations between the stiffnesses achieved at the different crosslinking conditions and the cell behaviour could be determined.