Targeting the oral plaque microbiome with immobilized anti-biofilm peptides at tooth-restoration interfaces

Abstract

Recurrent caries, the development of carious lesions at the interface between the restorative material and the tooth structure, is highly prevalent and represents the primary cause for failure of dental restorations. Correspondingly, we exploited the self-assembly and strong antibiofilm activity of amphipathic antimicrobial peptides (AAMPs) to form novel coatings on dentin that aimed to prevent recurrent caries at susceptible cavosurface margins. AAMPs are alternative to traditional antimicrobial agents and antibiotics with the ability to target the complex and heterogeneous organization of microbial communities. Unlike approaches that have focused on using these AAMPs in aqueous solutions for a transient activity, here we assess the effects on microcosm biofilms of a long-acting AAMPs-based antibiofilm coating to protect the tooth-composite interface. Genomewise, we studied the impact of AAMPs coatings on the dental plaque microbial community. We found that non-native all D-amino acids AAMPs coatings induced a marked shift in the plaque community and selectively targeted three primary acidogenic colonizers, including the most common taxa around Class II composite restorations. Accordingly, we investigated the translational potential of our antibiofilm dentin using multiphoton pulsed near infra-red laser for deep bioimaging to assess the impact of AAMPs-coated dentin on plaque biofilms along dentin-composite interfaces. Multiphoton enabled us to record the antibiofilm potency of AAMPs-coated dentin on plaque biofilms throughout exaggeratedly failed interfaces. In conclusion, AAMPs-coatings on dentin showed selective and long-acting antibiofilm activity against three dominant acidogenic colonizers and potential to resist recurrent caries to promote and sustain the interfacial integrity of adhesive-based interfaces.

Fig 1. Schematics for the preparation of an ex vivo simulated dentin-composite failed interface used to assess the impact of AAMPs-treated interfaces on dental plaque biofilms.

A reproducible artificial gap was introduced along the dentin-composite interface of the restored discs using a method adapted from [25]. The radicular dentin tubes were total-etched and filled with dental resin composite (Filtek™ Z250 Universal Restorative 3M, St. Paul, MN, USA) without adhesive. Clear matrix strips (4" x 3/8" x 0.002") (Patterson^® Mylar^® Matrix Strips, Patterson Dental, Dental Supply, St. Paul, MN, USA) were introduced in the dentin cylinders, against the dentin wall, before composite application. After curing of the composite, the matrix strips were pulled out leaving a gap of ~ 400 μm in thickness along the interface. Then, the restored dentin cylinders were transversally sliced using a pre-calibrated template into 2-mm thick dentin-composite discs. This model was used to assess the biofilm succession along the interface with and without D-GL13K treatment using multiphoton deep bioimaging as detailed in Fig 5.

Fig 2. Antimicrobial effect of peptide candidates on dental plaque communities.

A) The minimum inhibitory concentrations (MICs) of D-GL13K against 10 different supragingival plaque samples collected from caries active individuals. Bars show average ± standard deviation (n = 3). B) 6-day-old highly adherent plaque biofilm of #311 sample (top panels). Bottom panels show #311 sample treated with 25μg/ml D-GL13K [22]. C) MICs of antimicrobial and non-antimicrobial control peptides against #311 plaque sample [23]. Bars show average ± standard deviation (n = 3). D) X-ray photoelectron spectroscopy (XPS) spectra for peptide coatings on HA discs. Top panel shows the surface atomic composition of acid-etched (32% H₃PO₄) control HA discs. Middle and bottom panels show the surface atomic composition of etched HA discs after coating with D-GL13K (an all D-amino acids peptide) and 1018 (an all L-amino acids peptide), respectively. The red dashed line shows the position of the nitrogen-N1s peak. The appearance of the nitrogen peak in peptide-coated HA discs is indicative of the presence of the peptides on these discs. HA: Hydroxyapatite.

Fig 3. Antibiofilm effect of D-GL13K, DJK2, and 1018 coated HA discs on dental plaque biofilms.

A) Cell death quantification by counting colony forming units (CFU) for detached bacteria biofilms from HA discs coated with all AAMPs. Control group is 32% phosphoric acid-etched non-coated HA discs. N = 3. B) Merged live (green) and dead (red) bacteria viability assay images of 48-hour plaque biofilms grown on HA discs with (bottom image) and without (top image) D-GL13K coatings. C) Live and dead viability assay images of 48-hour (left), 5-day (middle), and 15-day-old (right) plaque biofilms grown on HA discs with (bottom images) and without (top images) D-GL13K coating. Middle panel shows regrown biofilms for 5 days after 45 min of ultrasonication (mechanically challenged D-GL13K coating). Right panel shows regrown biofilms for 15 days after 45 min immersion in 30% acetic acid (chemically challenged D-GL13K coating). Merged images are on the left side of each group and separated live (top)/dead (bottom) bacteria images are on the right side of each group.

Fig 4. Effect of D-GL13K, DJK2, and 1018 coated HA discs on the dental plaque biofilms microbial community composition using 16s ribosomal (rRNA) gene next generation sequencing.

A) Principal coordinates plots shows the distance and relatedness between taxonomic data of tested populations. B) Alpha diversity plots show the genus-level change in bacterial richness (number of bacterial taxa) and bacterial evenness (relative abundance of bacterial taxa). Shannon index (top panel) is sensitive to bacterial richness, Simpson index (middle panel) is sensitive to bacterial evenness, InvSimpson (bottom panel) is the inverse of the Simpson index diversity estimator. C) Alpha diversity plots show the change in bacterial richness and evenness of highly abundant Streptococcus and Veillonella combined. D) Analysis of composition of microbiomes (ANCOM) statistical framework shows the bacterial selectivity of tested AAMPs for Streptococcus, Veilonella, Actinomyces and Gemella.

Fig 5. Multiphoton fluorescence 3D rendering of plaque biofilms along dentin-composite interfacial artificial gaps up to 1 mm in depth created as shown in Fig 1.

A) 9-day-old plaque biofilm and B) 18-day-old plaque biofilm along D-GL13K treated (left) and control non-peptide treated (right) interfaces. Quantified dead cells in A) are average percentage values ± standard deviations. C) Zoomed-out images of the 18-day-old plaque biofilm grown at the interfacial gap between dentin and composite restorations. Top panel shows the plaque biofilm response to D-GL13K coated dentin and composite restoration; that is the straight surface and the curved surface of the artificial gap, respectively. Bottom panel shows a control non-coated sample. In A) and C), left channels (red+green = merged live and dead bacteria), central channels (Syto-9, green = live bacteria), and right channels (PI, red = dead bacteria).