Development of bio-inks for bioprinting applications

Abstract

Bioprinting allows cells and materials to be deposited to create tissue pre-cursors. However, the quality of the bioprinted constructs depends on bio-inks, which need optimisation to enable repeatable processes, achieve physiological cell densities and tailored structures. The primary aim of this research was to develop reliable bio-inks for printing single cells and cells in gels. A novel bio-ink formulation has been developed to allow reliable inkjet bioprinting of small cell numbers, allowing single cell deposition. Poly-L-lysine (PLL) was investigated as temporary speckled coating, which would allow to avoid nozzle blockage during the printing process. Different PLL concentrations were evaluated, with 10 µg/mL exhibiting the highest viability and metabolic activity similar to control samples on three different cell lineages. At the optimised concentration, fast internalisation and metabolisation of PLL was observed with no subsequent effect on cell behaviour. Repeatable inkjet cell printing was achieved, whilst maintaining cell function. High cell density cell-laden hydrogels were produced using a bioprinting technique called Reactive Jet Impingement (ReJI), developed at Newcastle University as part of a related project. A protocol for using ReJI to produce a collagen-alginate-fibrin (CAF) gel was established and used to create cell-laden gels. The fabricated hydrogels exhibited rapid solution uptake and porous structures, both essential for cell migration. When culturing mesenchymal stem cells in the CAF gel a cell density of 40 million cells/ml supported faster osteogenic differentiation than a cell density of 4 million cells/ml. Additionally, increased cell density produced more organised tissue structures and increased expression of osteogenic biomarkers, indicative of accelerated bone tissue formation. This research has developed and characterised the performance of two bio-inks for two bioprinting techniques. It is concluded that careful consideration of the processing techniques and bio-ink formulation can allow for the creation of effective cell printing techniques for both single cells and cell-laden hydrogels.