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Bacterial biofilms associated with implants remain a significant source of infections in dental, implant, and other healthcare industries due to challenges in biofilm removal. Biofilms consist of bacterial cells surrounded by a matrix of extracellular polymeric substance (EPS), which protects the colony from many countermeasures, including antibiotic treatments. Biofilm EPS composition is also affected by environmental factors. In the oral cavity, the presence of sucrose affects the growth of Streptococcus mutans that produce acids, eroding enamel and forming dental caries. Biofilm formation on dental implants commonly leads to severe infections and failure of the implant. This work determines the effect of sucrose concentration on biofilm EPS formation and adhesion of Streptococcus mutans, a common oral colonizer. Bacterial biofilms are grown with varying concentrations of sucrose on titanium substrates simulating dental implant material. Strategies for measuring adhesion for films such as peel tests are inadequate for biofilms, which have low cohesive strength and will fall apart when tensile loading is applied directly. The laser spallation technique is used to apply a stress wave loading to the biofilm, causing the biofilm to delaminate at a critical tensile stress threshold. Biofilm formation and EPS structures are visualized at high magnification with scanning electron microscopy (SEM). Sucrose enhanced the EPS production of S. mutans biofilms and increased the adhesion strength to titanium, the most prevalent dental implant material. However, there exists a wide range of sucrose concentrations that are conducive for robust formation and adhesion of S. mutans biofilms on implant surfaces.
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Reproducible 3D bioprinting of Streptococcus mutans to create model oral biofilms
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.
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- Award ID(s):
- 2230641
- PAR ID:
- 10660628
- Editor(s):
- Kolodkin-Gal, Ilana
- Publisher / Repository:
- American Society for Microbiology Press
- Date Published:
- Journal Name:
- Microbiology Spectrum
- Volume:
- 13
- Issue:
- 11
- ISSN:
- 2165-0497
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1128/spectrum.00935-25
