(M-486) Rheological Characterization and Fabrication of S. Typhimurium laden Alginate-based Hydrogels

Introduction:: Bacteria are ubiquitous on Earth and can form complex communities called biofilms, which allow them to survive in extreme conditions. Biofilms consist of microbial cell aggregates surrounded by a self-produced extracellular matrix of proteins and polysaccharides, enabling bacterial adhesion to other cells and surrounding surfaces. Bacterial biofilms are known to colonize biomedical surfaces, leading to severe infections that can be fatal. Biofilms are also involved in the progression of many other diseases, such as cancer and upper respiratory infections, due to their ability to adhere to soft tissues. The heterogeneity of biofilms makes them more resistant to immune defenses and antimicrobial therapeutics. However, much is still unknown about how biofilms form into 3D structured microenvironments, in contrast to the extensive knowledge about mammalian cells encapsulated in 3D constructs. To develop next-generation antibacterial compounds and therapeutics, it is essential to fabricate biofilm models that closely mimic the chemical and mechanical properties of natural bacterial communities in soft tissues. In this study, we investigate the rheological properties of bacteria-laden alginate-gelatin hydrogels with varying concentrations of Salmonella Typhimurium and assess the impact of printing parameters on bacterial viability.


Materials and Methods:: Different concentrations of Salmonella Typhimurium (1x10⁵, 10⁶, 10⁷, and 10¹⁰ cells/mL) were encapsulated in alginate-gelatin hydrogels consisting of two different molecular weight alginate. The molecular weights of the two types of alginate used were 24kDa (Low) and 774kDa (Mid), respectively. Oscillatory shear measurements were conducted using an MCR 302 Anton Paar Rheometer to assess the rheological properties of the bioinks. Bioinks were then fabricated using an extrusion-based bioprinter (SunP Biomaker) with a print speed of 5 mm/s and an extrusion speed of 0.6 mm³/s. To characterize the hydrogel printability, hydrogel samples were extruded in lattice patterns and imaged to assess strand width and pore size. Bioprinted constructs were cross-linked with 5 w/v% calcium chloride and incubated in DMEM medium at 37 °C and 5% CO₂ for a 4-day culture period. Bacteria-laden constructs were assessed for cell viability using a Live/Dead (LD) cytotoxicity assay and aggregate formation during the culture period. Finally, scanning electron microscopy was used to visualize cell-matrix interaction and morphological changes.


Results, Conclusions, and Discussions:: The hydrogels formulated exhibited shear thinning behavior. Results indicate that increasing the bacterial concentration in the hydrogels up to 1x10⁷ cells/mL had no significant effect on the hydrogel precursors' storage and loss modulus or shear viscosity. However, increasing the bacterial concentration to 1x10¹⁰ cells/mL significantly decreased the hydrogel shear viscosity and modulus. This decrease can be attributed to a higher cell volume fraction modifying the microstructure of the hydrogels. We also observed that the selected printing parameters had minimal effects on bacterial viability, which was maintained at >80% during the 4-day incubation period. Additionally, we found that lower concentrations of bacteria formed larger aggregates over time compared to higher cell concentrations. SEM imaging showed that cells formed colonies within the pore network of the hydrogels over time.
This investigation demonstrates the importance of understanding the rheological properties of bacterial-laden hydrogels for the fabrication of biofilm models that closely mimic natural bacterial communities. Our findings reveal that incorporating higher concentrations of Salmonella Typhimurium in hydrogels can significantly modify the hydrogel microstructure and affect their rheological properties. Moreover, we show that bacterial viability is maintained during incubation, indicating the feasibility of using extrusion-based bioprinting for bacterial-laden hydrogel fabrication. Our work also provides insights into the formation of bacterial aggregates in hydrogel constructs over time, which is crucial for understanding biofilm formation and progression in soft tissue-related infections for developing next-generation antibacterial compounds and therapeutics.




Acknowledgements (Optional): : This work was funded by the National Science Foundation (HDR-2000202, CMMI-2000330) and support from the NSF FAMU CREST Center award (HDR-1735968) JA acknowledges support from the Florida A&M University Office of Graduate Studies and Research, Florida A&M University Division of Research, and the Florida-California Cancer Research, Education & Engagement (CaRE2) Health Equity program funded by the National Cancer Institute (5U54CA233396-02). All the work was performed at the National High Magnetic Field Laboratory, which is supported by National Science Foundation Cooperative Agreement No. DMR-2128556 and the State of Florida


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