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The adhesion of initial colonizers such asStreptococcus mutansto 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 ofS. mutansto 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 livingS. mutansUA 159 orS. sanguinisSK 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 relevantS. mutansUA 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 betweenS. mutansand 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 betweenS. mutansUA 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.

Recent advances in atomic force microscopy (AFM) have allowed the characterisation of dental-associated biomaterials and biological surfaces with high-resolution and minimal sample preparation. In this context, the topography of dental enamel – the hardest mineralised tissue in the body – has been explored with AFM-based approaches at the micro-scale. With age, teeth are known to suffer changes that can impact their structural stability and function; however, changes in enamel structure because of ageing have not yet been explored with nanoscale resolution. Therefore, the aim of this exploratory work was to optimise an approach to characterise the ultrastructure of dental enamel and determine potential differences in topography, hydroxyapatite (HA) crystal size, and surface roughness at the nanoscale associated to ageing. For this, a total of six teeth were collected from human donors from which enamel specimens were prepared. By employing AC mode imaging, HA crystals were characterised in both transversal and longitudinal orientation with high-resolution in environmental conditions. Sound superficial enamel displayed the presence of a pellicle-like coating on its surface, that was not observable on cleaned specimens. Acidetching exposed crystals that were imaged and morphologically characterised in highresolution at the nanoscale in both the external and internal regions of enamel in older and younger specimens. Our results demonstrated important individual variations in HA crystal width and roughness parameters across the analysed specimens; however, an increase in surface roughness and decrease in HA width was observed for the pooled older external enamel group compared to younger specimens. Overall, high-resolution AFM was an effective approach for the qualitative and quantitative characterisation of human dental enamel ultrastructure at the nanometre range. Future work should focus on exploring the ageing of dental enamel with increased sample sizes to compensate for individual differences as well as other potential confounding factors such as behavioural habits and mechanical forces.

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 to 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, twelve healthy teeth from 50 year-old patients were collected and prepared to obtain crown and root dentin discs. Following demineralization, MGO-derived AGEs were quantified with a competitive ELISA assay. In addition, atomic force microscopy (AFM) nano-indentation was utilized to measure changes in elastic modulus in both peritubular (PT) and intertubular (IT) collagen fibrils. Finally, principal component analysis (PCA) was carried out to determine aging profiles for both crown and root dentin. Results showed increased presence of MGO AGEs in the organic matrix of dentin in the >50 year-old compared to 50 year-old group associated to ultrastructural changes in the organic matrix determined by AFM analysis. Furthermore, PCA loading plots suggested different ‘aging profiles’ in both crown and root dentin, which could potentially 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 nanomechanical behavior of collagen that may impact diagnostic and restorative procedures in the elderly.