Rheological Characterization and 3D Fabrication of Artificial Bacterial Biofilms

Biofilms are significantly involved in the progression of many diseases, such as cancer and upper respiratory infections, due to their ability to adhere to soft tissues. Factors influencing biofilm development have been extensively studied on planar substrates; however, there is limited understanding regarding biofilm growth and interactions within 3D matrices. Developing biofilm models that closely mimic natural bacterial communities' chemical and mechanical properties in soft tissues is essential for developing next-generation antibacterial compounds and therapeutics, as 3D biofilms are more complex and less susceptible to treatment than their 2D counterparts. Here, to understand environmental viscoelastic effects on biofilms within 3D matrix environments, two types of alginate-based hydrogels are formulated and used to encapsulatevarying concentrations of Salmonella Typhimurium. We explore the effects of increasing S. Typhimurium concentrations on hydrogel rheological properties and assess the impact of printing parameters on bacterial viability. Results show that hydrogels exhibit shear thinning behavior and that increasing the bacterial concentration up to 1 × 10⁷ CFU mL^-1 has no significant effect on the hydrogel precursor moduli and low shear viscosity. However, increasing the bacterial concentration to 1 × 10¹⁰ CFU mL^-1 significantly decreases the hydrogel shear viscosity and modulus. Utilizing extrusion-based bioprinting, the optimal printing parameters (Pr > 0.8) have minimal effects on bacterial viability (>80%) over a 4 day incubation period. Additionally, we find that lower concentrations of bacteria form larger aggregates over time than hydrogels with higher cell concentrations. We show that biofilm growth in 3D depends on both initial bacterial density and matrix rigidity. Further development of physicochemically tuned bioprinted bacterial communities will aid our understanding of bacterial interactions within their 3D environments and enable the use of in vitro tissue models that incorporate biofilms for high-throughput therapeutic screening.