The Influence of Printing Parameters and Cell Density on Bioink Printing Outcomes

Abstract

Bioink printability persists as a limiting factor toward many bioprinting applications. Printing parameter selection is largely user-dependent, and the effect of cell density on printability has not been thoroughly investigated. Recently, methods have been developed to give greater insight into printing outcomes. This study aims to further advance those methods and apply them to study the effect of printing parameters (feedrate and flowrate) and cell density on printability. Two printed structures, a crosshatch and five-layer tube, were established as printing standards and utilized to determine the printing outcomes. Acellular bioinks were printed using a testing matrix of feedrates of 37.5, 75, 150, 300, and 600 mm/min and flowrates of 21, 42, 84, 168, and 336 mm³/min. Structures were also printed with cell densities of 5, 10, 20, and 40 × 10⁶ cell/mL at 150 mm/min and 84 mm³/min. Only speed ratios (defined as flowrate divided by feedrate) from 0.07 to 2.24 mm² were suitable for analysis. Increasing speed ratio dramatically increased the height, width, and wall thickness of tubular structures, but did not influence radial accuracy. For crosshatch structures, the area of pores and the frequency of broken filaments were decreased without impacting pore shape (Pr). Within speed ratios, feedrate and flowrate had negligible, inconsistent effects. Cell density did not affect any printing outcomes despite slight rheological changes. Printing outcomes were dominated by the speed ratio, with feedrate, flowrate, and cell density having little impact on printing outcomes when controlling for speed ratio within the ranges tested. The relevance of these results to other bioinks and printing conditions requires continued investigation by the bioprinting community, as well as highlight speed ratio as a key variable to report and suggest that rheology is a more sensitive measure than printing outcomes.

Keywords: bioink; bioprinting; cell density; federate; flowrate; hydrogel; printability.

FIG. 1.

Schematic diagram of speed ratio and cell density experiments. GelMA/GG composite bioinks were prepared for use in both experiments and tested both rheologically and by printing five-layer tube and crosshatch structures. The effect of printing conditions was examined by using a testing matrix of different feedrates and flowrates. For cell density experiments, MS1 cells were also included in the bioink at various cell densities. GelMA, gelatin methacrylate; GG, gellan gum.

FIG. 2.

(A) Representative images from different printing conditions. (B–G) Quantitative measures taken from five-layer tube and crosshatch structure, grouped by speed ratio, including (B) wall thickness, (C) radial accuracy, (D) tube width, (E) tube height, (F) Pr value, and (G) pore area. * Denotes statistical significance (p < 0.05). All data are represented as mean ± SD. SD, standard deviation.

FIG. 3.

Quantitative measures taken from five-layer tube and crosshatch structures at different feedrates and speed ratios, including (A) wall thickness, (B) radial accuracy, (C) tube width, (D) tube height, (E) Pr value, and (F) pore area. *Denotes statistical significance (p < 0.05). All data are represented as mean ± SD.

FIG. 4.

(A) Photographs grouped by bioink of the crosshatch structure (left), five-layer tube—top view (center), and five-layer tube—side view (right). Labels indicate the cell density of each bioink and scale bars represent 3 mm. Quantitative measures taken from five-layer tube and crosshatch structures at different cell densities, including (B) wall thickness, (C) radial accuracy, (D) tube width, (E) tube height, (F) Pr value, and (G) pore area. All data are represented as mean ± SD.

FIG. 5.

Rheological results from strain sweep and frequency sweep tests of bioinks with varying cell densities, including (A) storage modulus, (B) loss modulus, (C) yield stress, and (D) shear-thinning behavior. All data are represented as mean ± SD.