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ABSTRACT Novel approaches are needed to study relationships between oral biofilm strains, enable three-dimensional oral biofilm deposition, and hasten the rigor and pace of basic and translational biofilm studies. Previously, 3D-bioprinters were leveraged to deposit spatially patterned biofilms onto sugar-rich agar surfaces to study how the underlying spatial organization of various microbes impacts biofilm persistence and virulence. Herein, we have developed a new method to adapt this process from limited, soft agar surfaces to biomimetic solid substrates submerged in aqueous solutions for studying oral biofilmsin vitro. Streptococcus mutansUA159 was used to compare standardin vitrobiofilm development with our new 3D-printed bio-ink hydrogels on hydroxyapatite disks, which mimic tooth surfaces. Biofilms formed using the bio-ink methodology showed minimal quantitative differences in virulence factors, including environmental pH, biomass, and cell density, compared to biofilms formed using the standardin vitromethodology. The bio-ink technique resulted in higher exopolysaccharide deposition, a key virulence factor for biofilm cohesion and protection, as well as more homogeneous spatial distribution of bacterial microcolonies. Our newly developed technique produces 3D-printable model biofilms that match the virulence benchmarks of the standard method, opening possibilities to print biofilms onto any substrate and a new way to study multidimensional biofilm dynamics.IMPORTANCEDental caries is the most common oral disease caused by biofilms in humans with cost limitations. Changes in the human diet have increased the exposure to sugar-rich processed food, increasing the incidence and severity of dental caries and creating greater rationale for understanding biofilm deposition, microbial interactions, and maintenance of quiescence of the oral microbiota. Recent 3D-printing techniques have been leveraged to develop the first model biofilms, providing spatial control over microbe deposition and enabling unprecedented investigation of the impact of cell-cell interactions and spatial organizationupon biofilm persistence, sensitivity to drugs, and virulence. Here, we have developed new methods to extend bioprinting to oral biofilms using cariogenicStreptococcus mutans. Our technique is an attempt to establish an alternative method for oral biofilm formationin vitrothat uses 3D-printing tools, preserving the virulence of standardin vitrobiofilms while amplifying the availability and versatility of methods for understanding the microbiome.