Microbiome-friendly PS/PVP electrospun fibrous membrane with antibiofilm properties for dental engineering

Abstract

Dental caries is one of the most prevalent and biofilm-associated oral diseases in humans. Streptococcus mutans, with a high ability to form biofilms by adhering to hard surfaces, has been established as an important etiological agent for dental caries. Therefore, it is crucial to find a way to prevent the formation of cariogenic biofilm. Here, we report an electrospun fibrous membrane that could inhibit the adhesion and biofilm formation of S. mutans. Also, the polystyrene (PS)/polyvinyl pyrrolidone (PVP) electrospun fibrous membrane altered the 3D biofilm architecture and decreased water-insoluble extracellular polysaccharide production. Notably, the anti-adhesion mechanism which laid in Coulomb repulsion between the negatively charged PS/PVP electrospun fibrous membrane and S. mutans was detected by zeta potential. Furthermore, metagenomics sequencing analysis and CCK-8 assay indicated that PS/PVP electrospun fibrous membrane was microbiome-friendly and displayed no influence on the cell viability of human gingival epithelial cells and human oral keratinocytes. Moreover, an in vitro simulation experiment demonstrated that PS/PVP electrospun fibrous membrane could decrease colony-forming unit counts of S. mutans effectively, and PS/PVP electrospun fibrous membrane carrying calcium fluoride displayed better anti-adhesion ability than that of PS/PVP electrospun fibrous membrane alone. Collectively, this research showed that the PS/PVP electrospun fibrous membrane has potential applications in controlling and preventing dental caries.

Keywords: Coulomb repulsion; PS/PVP electrospun fibrous membrane; Streptococcus mutans; anti-adhesion; biofilm(s).

Graphical abstract

Figure 1.

Electrospun fibrous membrane inhibits biofilm formation and adhesion of S.mutans UA159. (A) Growth curves of S.mutans grown in BHI medium containing electrospun fibrous membranes, glass coverslips and hydroxyapatite disc for 12 h. (B) Quantitative results were collected to evaluate biofilm formation on different surfaces by crystal violet staining. Data were obtained in triplicate and are presented as mean ± SD (*P ˂ 0.05). (C) Water-insoluble EPS content of biofilms quantified using anthrone–sulfuric method. Data were obtained in triplicate and are presented as mean ± SD (*P ˂ 0.05). (D) Double-labeled imaging of biofilms showing EPS (red, Alexa Fluor 647) and bacteria (green, SYTO 9). Images were taken using a ×60 oil immersion objective lens. Representative images from at least five randomly selected positions of each sample are shown.

Figure 2.

CFU counts of S.mutans biofilms on HA and PS/PVP electrospun fibrous membrane grown in different culture medium. CFU counts of S.mutans biofilms cultured at (A) different concentration of sucrose of medium and (B) different pH of medium. Data were obtained in triplicate and are presented as mean ± SD (*P ˂ 0.05).

Figure 3.

The influence of surface potential of electrospun fibrous membrane on the adhesion of S.mutans. (A) Biofilms incubated on PS/PVP and PS/PVP+TBAB, and images taken at ×5000 magnification. The red arrows indicated the bacterial cells. (B) CFU counts of S.mutans biofilms. Data were obtained in triplicate and are presented as mean ± SD (*P ˂ .05). (C) Zeta potential of different electrospinning solution. (D) Absolute zeta potential values.

Figure 4.

The impact of PS/PVP electrospun fibrous membrane on oral microbiota composition. (A) Relative abundance of oral bacteria at phylum level. (B) Distance matrix and principal coordinates analysis (PCoA). (C) Hierarchical clustering analysis. (D) The heatmap consisted of relative abundance of the 20 most predominant oral bacteria at genus level. ESF, electrospinning fiber; HA, hydroxyapatite disc; Sali, saliva.

Figure 5.

The effect of PS/PVP electrospun fibrous membrane on HGEs and HOKs. Results of CCK-8 assays performed on (A) HGEs and (B) HOKs. Data were obtained in triplicate and are presented as mean ± SD.

Figure 6.

The PS/PVP electrospun fibrous membrane carrying calcium fluoride nanoparticles suppress biofilm formation of S.mutans UA159. (A) Electrospun fibrous membrane carrying calcium fluoride nanoparticles and images taken at ×10 000 magnification. (B) Fluoride release from modified electrospun fibrous membrane. (C) Data were obtained in triplicate and are presented as mean ± SD (*P ˂ 0.05). Quantitative results were collected to evaluate biofilm formation by crystal violet staining. (D) CFU counts of biofilms. Data were obtained in triplicate and are presented as mean ± SD (*P ˂ 0.05).

Figure 7.

The evaluation on the antibiofilm effect of different retainers. (A) Schematic illustration displaying the experimental process. (B) CFU counts and (C) water-insoluble EPS content of biofilms in different groups. Data were obtained in triplicate and are presented as mean ± SD (*P ˂ 0.05).