Design of experiments approach to developing a robust ink for bioprinting

Despite advancements in tissue engineering, the methods used to generate three-dimensional (3D)in vitromodels for rapid screening and characterization studies remain time and labor intensive. Bioprinting offers an opportunity to offset these limitations by providing a scalable, high-throughput method with precise control over biomaterial scaffold and cellular deposition. However, the process of formulating bioinks can be complex in terms of balancing the mechanical integrity of a bioscaffold and viability of cells. One key factor, especially in alginate-based bioinks, is the rate of bioscaffold dissolution. It must allow cells to replace the bioscaffold with extracellular matrix (ECM), yet remain durable during extended tissue culture. This study uses a Design of Experiments (DoE) approach to understand the dependencies of multiple variables involved in the formulation and processing of an alginate-based bioink. The focus of the DoE was to understand the effects of hydrogel composition on bioink durability while maintaining cell viability. Three ingredients were varied in all: alginate, nanocellulose, and fibrinogen. Their effects on the bioink were then measured with respect to extrudability, strength, and stiffness as determined by dynamic mechanical analysis (DMA). The DoE demonstrated that mechanical integrity increased with increasing alginate concentration. In contrast, fibrinogen and nanofibril concentration had no statistically significant effect. The optimized ink containing fibroblasts was printable using multiple nozzle sizes while also supporting fibroblast cell viability. DMA characterization further showed that the composition of the cell culture medium did not modulate the degradation rate of the hydrogel. Ultimately, the study outlines a methodology for formulating a bioink that will result in robust bioscaffolds forin vitromodel development.