Functional signatures of ex-vivo dental caries onset.

Background: The etiology of dental caries remains poorly understood. With the advent of next-generation sequencing, a number of studies have focused on the microbial ecology of the disease. However, taxonomic associations with caries have not been consistent. Researchers have also pursued function-centric studies of the caries microbial communities aiming to identify consistently conserved functional pathways. A major question is whether changes in microbiome are a cause or a consequence of the disease. Thus, there is a critical need to define conserved functional signatures at the onset of dental caries. Methods: Since it is unethical to induce carious lesions clinically, we developed an innovative longitudinal ex-vivo model integrated with the advanced non-invasive multiphoton second harmonic generation bioimaging to spot the very early signs of dental caries, combined with 16S rRNA short amplicon sequencing and liquid chromatography-mass spectrometry-based targeted metabolomics. Findings: For the first time, we induced longitudinally monitored caries lesions validated with the scanning electron microscope. Consequently, we spotted the caries onset and, associated with it, distinguished five differentiating metabolites - Lactate, Pyruvate, Dihydroxyacetone phosphate, Glyceraldehyde 3-phosphate (upregulated) and Fumarate (downregulated). Those metabolites co-occurred with certain bacterial taxa; Streptococcus, Veillonella, Actinomyces, Porphyromonas, Fusobacterium, and Granulicatella, regardless of the abundance of other taxa. Interpretation: These findings are crucial for understanding the etiology and dynamics of dental caries, and devising targeted interventions to prevent disease progression.

Current State and Challenges of the Global Outcomes of Dental Caries Research in the Meta-Omics Era.

Despite significant healthcare advances in the 21st century, the exact etiology of dental caries remains unsolved. The past two decades have witnessed a tremendous growth in our understanding of dental caries amid the advent of revolutionary omics technologies. Accordingly, a consensus has been reached that dental caries is a community-scale metabolic disorder, and its etiology is beyond a single causative organism. This conclusion was based on a variety of microbiome studies following the flow of information along the central dogma of biology from genomic data to the end products of metabolism. These studies were facilitated by the unprecedented growth of the next- generation sequencing tools and omics techniques, such as metagenomics and metatranscriptomics, to estimate the community composition of oral microbiome and its functional potential. Furthermore, the rapidly evolving proteomics and metabolomics platforms, including nuclear magnetic resonance spectroscopy and/or mass spectrometry coupled with chromatography, have enabled precise quantification of the translational outcomes. Although the majority supports 'conserved functional changes' as indicators of dysbiosis, it remains unclear how caries dynamics impact the microbiota functions and vice versa, over the course of disease onset and progression. What compounds the situation is the host-microbiota crosstalk. Genome-wide association studies have been undertaken to elucidate the interaction of host genetic variation with the microbiome. However, these studies are challenged by the complex interaction of host genetics and environmental factors. All these complementary approaches need to be orchestrated to capture the key players in this multifactorial disease. Herein, we critically review the milestones in caries research focusing on the state-of-art singular and integrative omics studies, supplemented with a bibliographic network analysis to address the oral microbiome, the host factors, and their interactions. Additionally, we highlight gaps in the dental literature and shed light on critical future research questions and study designs that could unravel the complexities of dental caries, the most globally widespread disease.

Bioinspired caries preventive strategy via customizable pellicles of saliva-derived protein/peptide constructs.

Dental caries has been the most widespread chronic disease globally associated with significant health and financial burdens. Caries typically starts in the enamel, which is a unique tissue that cannot be healed or regrown; nonetheless, new preventive approaches have limitations and no effective care has developed yet. Since enamel is a non-renewable tissue, we believe that the intimate overlaying layer, the acquired enamel pellicle (AEP), plays a crucial lifetime protective role and could be employed to control bacterial adhesion and dental plaque succession. Based on our identified AEP whole proteome/peptidome, we investigated the bioinhibitory capacities of the native abundant proteins/peptides adsorbed in pellicle-mimicking conditions. Further, we designed novel hybrid constructs comprising antifouling and antimicrobial functional domains derived from statherin and histatin families, respectively, to attain synergistic preventive effects. Three novel constructs demonstrated significant multifaceted bio-inhibition compared to either the whole saliva and/or its native proteins/peptides via reducing biomass fouling and inducing biofilm dispersion beside triggering bacterial cell death. These data are valuable to bioengineer precision-guided enamel pellicles as an efficient and versatile prevention remedy. In conclusion, integrating complementary acting functional domains of salivary proteins/peptides is a novel translational approach to design multifunctional customizable enamel pellicles for caries prevention.

Keratinocyte-Specific Peptide-Based Surfaces for Hemidesmosome Upregulation and Prevention of Bacterial Colonization.

Percutaneous devices like orthopedic prosthetic implants for amputees, catheters, and dental implants suffer from high infection rates. A critical aspect mediating peri-implant infection of dental implants is the lack of a structural barrier between the soft tissue and the implant surface which could impede bacteria access and colonization of exposed implant surfaces. Parafunctional soft tissue regeneration around dental implants is marked by a lack of hemidesmosome formation and thereby weakened mechanical attachment. In response to this healthcare burden, a simultaneously hemidesmosome-inducing, antimicrobial, multifunctional implant surface was engineered. A designer antimicrobial peptide, GL13K, and a laminin-derived peptide, LamLG3, were coimmobilized with two different surface fractional areas. The coimmobilized peptide surfaces showed antibiofilm activity against Streptococcus gordonii while enhancing proliferation, hemidesmosome formation, and mechanical attachment of orally derived keratinocytes. Notably, the coatings demonstrated specific activation of keratinocytes: the coatings showed no effects on gingival fibroblasts which are known to impede the quality of soft tissue attachment to dental implants. These coatings demonstrated stability and retained activity against mechanical and thermochemical challenges, suggesting their intraoral durability. Overall, these multifunctional surfaces may be able to reduce peri-implantitis rates and enhance the success rates of all percutaneous devices via strong antimicrobial activity and enhanced soft tissue attachment to implants.

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

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.

Hydrophobic and antimicrobial dentin: A peptide-based 2-tier protective system for dental resin composite restorations.

Dental caries, i.e., tooth decay mediated by bacterial activity, is the most widespread chronic disease worldwide. Carious lesions are commonly treated using dental resin composite restorations. However, resin composite restorations are prone to recurrent caries, i.e., reinfection of the surrounding dental hard tissues. Recurrent caries is mainly a consequence of waterborne and/or biofilm-mediated degradation of the tooth-restoration interface through hydrolytic, acidic and/or enzymatic challenges. Here we use amphipathic antimicrobial peptides to directly coat dentin to provide resin composite restorations with a 2-tier protective system, simultaneously exploiting the physicochemical and biological properties of these peptides. Our peptide coatings modulate dentin's hydrophobicity, impermeabilize it, and are active against multispecies biofilms derived from caries-active individuals. Therefore, the coatings hinder water penetration along the otherwise vulnerable dentin/restoration interface, even after in vitro aging, and increase its resistance against degradation by water, acids, and saliva. Moreover, they do not weaken the resin composite restorations mechanically. The peptide-coated highly-hydrophobic dentin is expected to notably improve the service life of resin composite restorations and to enable the development of entirely hydrophobic restorative systems. The peptide coatings were also antimicrobial and thus, they provide a second tier of protection preventing re-infection of tissues in contact with restorations. STATEMENT OF SIGNIFICANCE: We present a technology using designer peptides to treat the most prevalent chronic disease worldwide; dental caries. Specifically, we used antimicrobial amphipathic peptides to coat dentin with the goal of increasing the service life of the restorative materials used to treat dental caries, which is nowadays 5 years on average. Water and waterborne agents (enzymes, acids) degrade restorative materials and enable re-infection at the dentin/restoration interface. Our peptide coatings will hinder degradation of the restoration as they produced highly hydrophobic and antimicrobial dentin/material interfaces. We anticipate a high technological and economic impact of our technology as it can notably reduce the lifelong dental bill of patients worldwide. Our findings can enable the development of restorations with all-hydrophobic and so, more protective components.

Present and future of tissue engineering scaffolds for dentin-pulp complex regeneration.

More than two thirds of the global population suffers from tooth decay, which results in cavities with various levels of lesion severity. Clinical interventions to treat tooth decay range from simple coronal fillings to invasive root canal treatment. Pulp capping is the only available clinical option to maintain the pulp vitality in deep lesions, but irreversible pulp inflammation and reinfection are frequent outcomes for this treatment. When affected pulp involvement is beyond repair, the dentist has to perform endodontic therapy leaving the tooth non-vital and brittle. On-going research strategies have failed to overcome the limitations of existing pulp capping materials so that healthy and progressive regeneration of the injured tissues is attained. Preserving pulp vitality is crucial for tooth homeostasis and durability, and thus, there is a critical need for clinical interventions that enable regeneration of the dentin-pulp complex to rescue millions of teeth annually. The identification and development of appropriate biomaterials for dentin-pulp scaffolds are necessary to optimize clinical approaches to regenerate these hybrid dental tissues. Likewise, a deep understanding of the interactions between the micro-environment, growth factors, and progenitor cells will provide design basis for the most fitting scaffolds for this purpose. In this review, we first introduce the long-lasting clinical dental problem of rescuing diseased tooth vitality, the limitations of current clinical therapies and interventions to restore the damaged tissues, and the need for new strategies to fully revitalize the tooth. Then, we comprehensively report on the characteristics of the main materials of naturally-derived and synthetically-engineered polymers, ceramics, and composite scaffolds as well as their use in dentin-pulp complex regeneration strategies. Finally, we present a series of innovative smart polymeric biomaterials with potential to overcome dentin-pulp complex regeneration challenges.