Purpose: Recently, our group found exosome-like extracellular vesicles (EVs) in Apis mellifera honey displaying strong antibacterial effects; however, the underlying mechanism is still not understood. Thus, the aim of this investigation was to characterize the molecular and nanomechanical properties of A. mellifera honey-derived EVs in order to elucidate the mechanisms behind their antibacterial effect, as well as to determine differential antibiofilm properties against relevant oral streptococci. Methods: A. mellifera honey-derived EVs (HEc-EVs) isolated via ultracentrifugation were characterized with Western Blot and ELISA to determine the presence of specific exosomal markers and antibacterial cargo, and atomic force microscopy (AFM) was utilized to explore their ultrastructural and nanomechanical properties via non-destructive immobilization onto poly-L-lysine substrates. Furthermore, the effect of HEc-EVs on growth and biofilm inhibition of S. mutans was explored with microplate assays and compared to S. sanguinis. AFM was utilized to describe ultrastructural and nanomechanical alterations such as cell wall elasticity changes following HEc-EV exposure. Results: Molecular characterization of HEc-EVs identified for the first time important conserved exosome markers such as CD63 and syntenin, and the antibacterial molecules MRJP1, defensin-1 and jellein-3 were found as intravesicular cargo. Nanomechanical characterization revealed that honey-derived EVs were mostly <150nm, with elastic modulus values in the low MPa range, comparable to EVs from other biological sources. Furthermore, incubating oral streptococci with EVs confirmed their antibacterial and antibiofilm capacities, displaying an increased effect on S. mutans compared to S. sanguinis. AFM nanocharacterization showed topographical and nanomechanical alterations consistent with membrane damage on S. mutans. Conclusion: Honey is a promising new source of highly active EVs with exosomal origin, containing a number of antibacterial peptides as cargo molecules. Furthermore, the differential effect of HEC-EVs on S. mutans and S. sanguinis may serve as a novel biofilmmodulating strategy in dental caries.

Methylglyoxal (MGO) is an important molecule derived from glucose metabolism with the capacity of attaching to collagen and generating advanced glycation end products (AGEs), which accumulate in tissues over time and are associated with aging and diseases. However, the accumulation of MGO-derived AGEs in dentin and their effect on the nanomechanical properties of dentinal collagen remain unknown. Thus, the aim of the present study was to quantify MGO-based AGEs in the organic matrix of human dentin as a function of age and associate these changes with alterations in the nanomechanical and ultrastructural properties of dentinal collagen. For this, 12 healthy teeth from <26-y-old and >50-y-old patients were collected and prepared to obtain crown and root dentin discs. Following demineralization, MGO-derived AGEs were quantified with a competitive ELISA. In addition, atomic force microscopy nanoindentation was utilized to measure changes in elastic modulus in peritubular and intertubular collagen fibrils. Finally, principal component analysis was carried out to determine aging profiles for crown and root dentin. Results showed an increased presence of MGO AGEs in the organic matrix of dentin in the >50-y-old specimens as compared with the <26-y-old specimens in crown and root. Furthermore, an increase in peritubular and intertubular collagen elasticity was observed in the >50-y-old group associated with ultrastructural changes in the organic matrix as determined by atomic force microscopy analysis. Furthermore, principal component analysis loading plots suggested different “aging profiles” in crown and root dentin, which could have important therapeutic implications in restorative and adhesive dentistry approaches. Overall, these results demonstrate that the organic matrix of human dentin undergoes aging-related changes due to MGO-derived AGEs with important changes in the nanomechanical behavior of collagen that may affect diagnostic and restorative procedures in older people.

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Currently, there is a variety of laboratory tools and strategies that have been developed to investigate in-vivo processes using in-vitro models. Amongst these, microfabrication represents a disruptive technology that is currently enabling next-generation biomedical research through the development of complex laboratory approaches (e.g., microfluidics), engineering of micrometer scale sensors and actuators (micropillars for traction force microscopy), and the creation of environments mimicking cell, tissue, and organ-specific contexts. Although microfabrication has been around for some time, its application in dental and oral research is still incipient. Nevertheless, in recent years multiple lines of research have emerged that use microfabrication-based approaches for the study of oral diseases and conditions with micro- and nano-scale sensitivities. Furthermore, many investigations are aiming to develop clinically relevant microfabrication-based applications for diagnostics, screening, and oral biomaterial manufacturing. Therefore, the objective of this review is to summarize the current application of microfabrication techniques in oral sciences, both in research and clinics, and to discuss possible future applications of these technologies for in-vitro studies and practical patient care. Initially, this review provides an overview of the most employed microfabrication methods utilized in biomedicine and dentistry. Subsequently, the use of micro- and nano-fabrication approaches in relevant fields of dental research such as endodontic and periodontal regeneration, biomaterials research, dental implantology, oral pathology, and biofilms was discussed. Finally, the current and future uses of microfabrication technology for clinical dentistry and how these approaches may soon be widely available in clinics for the diagnosis, prevention, and treatment of relevant pathologies are presented.

The adhesion of initial colonizers such as Streptococcus mutans to collagen is critical for dentinal and root caries progression. One of the most described pathological and aging-associated changes in collagen, including dentinal collagen, is the generation of advanced glycation end-products (AGEs) such as methylglyoxal (MGO)-derived AGEs. Despite previous reports suggesting that AGEs alter bacterial adhesion to collagen, the biophysics driving oral streptococcal attachment to MGO-modified collagen remains largely understudied. Thus, the aim of this work was to unravel the dynamics of the initial adhesion of S. mutans to type-I collagen in the presence and absence of MGO-derived AGEs, by employing bacterial cell force-spectroscopy with atomic force microscopy (AFM). Type-I collagen gels were treated with 10mM MGO to induce AGE formation, which was characterized with microscopy and ELISA. Subsequently, AFM cantilevers were functionalized with living S. mutans UA 159 or S. sanguinis SK 36 cells and probed against collagen surfaces to obtain force-curves displaying bacterial attachment in real-time, from which the adhesion force, number of events, Poisson analysis, and contour and rupture lengths for each individual detachment event were computed. Furthermore, in-silico docking studies between the relevant S. mutans UA 159 collagen-binding protein SpaP and collagen were computed, in the presence and absence of MGO. Overall, results showed that MGO modification increased both the number and adhesion force of single-unbinding events between S. mutans and collagen, without altering the contour or rupture lengths. Both experimental and in-silico simulations suggest that this effect is due to increased specific and non-specific forces and interactions between S. mutans UA 159 and MGO-modified collagen substrates. In summary, these results suggest that collagen alterations due to glycation and AGE formation may play a role in early bacterial adherence to oral tissues, associated with conditions such as aging or chronic hyperglycemia, amongst others.