Increased mixing improves hydrogel homogeneity and quality of three-dimensional printed constructs

As the tissue engineering and drug delivery communities place greater emphasis on producing constructs of prescribed geometry and organization, three-dimensional printing is becoming as an increasingly important technique. While numerous tissue printing techniques have emerged, little has been done to characterize the properties of printing inks and the resultant effects on geometric fidelity, cell viability, and mechanical integrity. These questions have been neglected largely because of the lack of methods to characterize the real-time properties of printing inks. We present a novel technique for characterizing the homogeneity of hydrogel tissue printing inks that measures loads during ink deposition and its temporal variation, called, mechanical noise. We then used this technique to determine the effects of increased mixing on the homogeneity of alginate hydrogels and determined whether this results in improved geometric fidelity of printed constructs. We also studied potential adverse effects on cell viability and mechanical integrity of printed parts. Increased mixing between alginate and crosslinker to 128 cycles yielded an 82% reduction in mechanical noise. Geometric fidelity also improved with this increased mixing, in terms of a smoother surface texture, better matching of the target geometry, and fewer point defects. Viability was not adversely affected by increased mixing, and it actually improved by 34% with a 45 min curing time. As mixing before printing was increased from 8 to 200 cycles, the modulus also increased by 110% from 4.0  ±  0.1 to 8.4  ±  1.0 kPa. The results presented herein motivate a radical shift in alginate printing protocol, and also propose a useful methodology for characterizing three-dimensional printing materials.