(D-123) 3D-bioprinting of Streptococcus mutans to create model oral biofilms

Biofilms are a high dynamic community of microorganisms imbibed in self-generated exopolysaccharide matrix(1-3). The predominance of certain microorganisms in biofilms leads to changes in exopolysaccharides matrix density, permeability, and viscoelasticity. These changes impact biofilm virulence(4-6). One of the most prevalent human disease biofilm-based in humans is dental caries, afflicting 3.5 billion people and costing approximately $387 billion globally(7).  Streptococcus mutans has the ability to survive in lower pH environment for having acid tolerance response mechanism and resisting environmental changes by controlling the cytoplasmatic pH(5, 8, 9), consequently, dominating the microorganism population within dental caries (9-11).

Screening new natural or synthetic products to mitigate biofilm formation without creating bacterial resistance is critical(12). The manual methodology currently used to screen potential drugs and compare the interactions against biofilm in vitro and in vivo is time and resource consuming(13). Therefore, the ability to automate biofilm formation and drug screening is a new approach to hasten testing and development(14), single and dual-layer strains(14, 15). This bio-ink approach is printed on agar surfaces as a primary source of sugar for growth(15). However, agar has limited relevance for reducing the research possibilities, it can miss important interactions between microorganism-substrate interface. Therefore, we have developed a strategy to use the bio-ink technique on any substrate, updating the culture medium solution to allow the hydrogel to be printed directly on hydroxyapatite surface, also creating a proper environment for biofilm development. The next challenging is adapting the 3D printing bio-ink technique on different substrates.

Hydroxyapatite discs (HA) were treated with 0.1 M calcium chloride solution for 10 min before introducing Streptococcus mutans UA159. Groups were divided into 1. Bio-ink; 2. Control sodium citrate; 3. Control calcium; and 4. Control. The culture medium was 2.5% tryptone, 1.5% yeast extract supplemented by 1% sucrose (all groups). The culture medium was supplemented by 0.1 M calcium chloride (groups 1, 2, and 3), and 1.5% alginate (group 1). Following incubation with 0.5 M sodium citrate for 2 h  to remove the alginate, analysis commenced. Culture medium was changed twice a day and pH was analyzed after media changes at 19, 27, and 43 h. The resulting biofilm characteristics were also analyzed including insoluble dry-weight, population, water- and alkali-soluble exopolysaccharide matrix, and morphology via confocal microscopy (19 h and 43 h). Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software, Inc., La Jolla, CA) after passing D'Agostino-Pearson and Shapiro-Wilk normality tests.  One-way ANOVA was performed employing a significance level fixed at 5% followed by post-hoc Tukey’s to multi comparison test.

Standard and bio-printed biofilms were analyzed at 43 h and 67 h in duplicate for at least three different experiments. Confocal microscopy was used to track growth at 19 h and 67 h. Bio-printed biofilms had a significantly longer lag phase prior to pH reduction versus standard biofilms but pH matched with standard biofilm after 27 h (pH ≤ 5.28), likely due to the difference microorganism density. Insoluble dry weight showed differences between alginate-removed (0.5 M sodium citrate) and alginate containing groups  at 43 h (p ≤ 0.03) . However, no statistical differences were identified in dry weight at 67 h. Furthermore, S. mutans density did not vary across the biofilm formation approaches. Water-soluble polysaccharides (WSP) showed a higher concentration within bio-inks compared to all groups in both time points. The alkali-soluble polysaccharides (ASP) showed a statistical difference between the bio-ink versus control sodium citrate at 43 h (p ≤ 0.04) and bio-ink versus control calcium at 67 h (p ≤ 0.016). The deposits of ASP from all groups were almost 2 times higher than the WSP deposits. Confocal microscopy showed a clear difference between standard deposition and bio-ink. The standard deposition shows S. mutans growing in a semi-circular pattern with the flat surface facing the HA surface, growing even in the edges of the disc with no restrictions. The bio-ink showed a radial expansion that was restricted from the HA surface by the hydrogel. Micro-colonies merged to form larger colonies, and, finally, biofilms.

            The updated bio-ink printing approach is amenable  biofilm deposition on hydroxyapatite surfaces (a non-sugar-dependent surface). Moreover, the approach offers versatility to incorporate and pattern various biofilm-related bacterial strains to investigate multi-species interactions. Several virulence factors were measured to describe the new environment created by the bio-ink, which showed similar growth to standard biofilms. The biofilm bioprinting approach enables automation of biofilm formation in vitro, which should enable robust drug screening and/or biofilm characterization.

The authors gratefully thank the University Research Award from the University of Rochester, the National Institutes of Health (R01 DE018023 to DB), the National Science Foundation (Convergence Accelerator Phase II 2230641 and Convergence Accelerator Phase I 2137561 to ASM), the Arnold and Mabel Beckman Foundation for an Instrumentation Grant to ASM, and Ram Gona for sharing his expertise during project development. We would also like to thank The Center for Translational Biomedical Research at the University of Rochester, PeaceHealth, and the University of Oregon’s Phil and Penny Knight Campus for Accelerating Scientific Impact to support GR on his Postdoctoral fellowship at University of Oregon.

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