Print parameter optimisation for three dimensional bioprinting of hydrogel scaffolds

Abstract

In drug discovery and delivery research, there is a continuing need for valid and reliable alternatives for in vitro drug efficacy and toxicity studies, and consequently tissue engineering has caught the attention of researchers within this field. The mechanical properties, bioactivity and printability of these tissue constructs/scaffolds, however, still remain a challenge. Various studies have focused on investigating alternative printers, biopolymers and cross-linking methods of the hydrogels/bioinks and recent papers mainly focus on the final tissue constructs; nonetheless, few have focused on the performance of the printing process.The aim of this study was to systematically optimise the printing parameters required to successfully 3D bioprint a preselected computer-aided design (CAD) model into a sustainable scaffold with a preformulated hybrid hydrogel. The optimised parameters include nozzle size, printing speed, pneumatic extrusion pressure as well as temperature. The formulated hybrid hydrogel, which consisted of alginate, a naturally derived biomaterial and Pluronic F-127 (PF127), a synthetic poloxamer, had to undergo characterisation studies which included determining the optimal concentration of alginate, the ionic cross-linking solution used, as well as the cross-linking method. An alginate concentration between 4 and 6% (w/v) yielded successful cross-linking with a 2% (w/v) CaCl2 cross-linking solution. PF127 was consequently added in various concentrations, classifying it as a hybrid hydrogel, also had to undergo characterisation studies to determine the optimal PF127 concentration as well as determining the properties of the hybrid hydrogel. Porosity, degradation rate, viscosity and cross-linking capabilities were measured. A concentration of 6% (w/v) alginate (A) mixed with a 46% (w/v) PF127 in a 2:1 ratio, displayed the most positive results, with an average porosity of 50.50±3.1%, a low degradation rate that can be attributed to the PF127 addition, and a rheological characterisation of a non-Newtonian fluid. Cross-linking the 6%A:46%PF127 hybrid hydrogel had to be done with a 4% (w/v) CaCl2 cross linking solution as the 2% (w/v) CaCl2 caused insufficient cross-linking. This was ascribed to PF127's chemical inertness, as it does not undergo ionic cross-linking, but entangle within the polymer chains formed from the alginate. This hybrid hydrogel ( 6%A:46%PF127) was consequently used to systematically optimise the printing parameters. The parameter optimisation index (POI), described by Webb and Doyle (2017), was implemented in combination with a newly formulated scoring method to help determine the optimal printing parameters. v It was found that in combination with these grading methods, the visual appearance plays a similarly important role in selecting optimal printing parameters. The CAD model was printed to 8 to 12% completion, photographed and enlarged to ease line width and corner drag measurements as they determine the POI as well as the score allocation that form part of the newly formulated grading method. Optimal printing parameters to yield a successful scaffold print were found to be a nozzle size of 27G, extrusion pressure of 70kPa and a printing speed of 30mm/s at 37ºC. The printed scaffold was submerged into a 4% (w/v) CaCl2 cross-linking solution for 24h. The swelling ratio of the printed scaffold was also determined to be the better in 2% (w/v) CaCl2 at 37ºC. Although the printing parameters were successfully optimised with the formulated hybrid hydrogel, high concentrations of PF127 could possibly have a negative effect on cell proliferation in the event that they are included in the bioink, therefore a clear need for the design and development for alternative polymer support materials is needed.