Spatial confinement enables reproducible engineering of Staphylococcus aureus biofilms by 3D bioprinting

Biofilm research is limited by the lack of standardized experimental models capable of generating mature and reproducible bacterial biofilms, hindering quantitative comparison across laboratories and the development of reliable anti-biofilm strategies. In this study, a novel three-dimensional (3D)-Sandwich biofilm model, in which Staphylococcus aureus cells were spatially confined within the central layer of an alginate construct, was compared with a homogeneous 3D bioprinted model (3D-Mixed) and two conventional two-dimensional (2D) models (2D-Multiwell and 2D-PET models). The 3D-Sandwich model promoted earlier biofilm development, achieving after 48 h culturable bacterial counts comparable to those observed in the 3D-Mixed model after 96 h, together with a more extensive and continuous bacterial organization. Specifically, CLSM revealed differences in three-dimensional bacterial organization, while SEM observations and quantitative nearest-neighbor distance (NND) analysis documented progressive surface colonization and enhanced local bacterial packing. Raman spectroscopy further revealed temporal changes in the detectable biochemical fingerprint. Collectively, these findings demonstrate that the initial spatial bacterial confinement during bioprinting is a fundamental bioengineering design parameter influencing biofilm development, structural organization, and experimental reproducibility. Overall, this work establishes a framework for engineering standardized and reproducible biofilm models that reduce the gap between conventional in vitro systems and translational biofilm research, providing a candidate reference platform for quantitative biofilm research and inter-laboratory comparison.