Biology subjects

Aguayo, S.

Publications and source records attributed to Aguayo, S..

10 recordsLinked to original sources

bioRxiv2026

Conditioning dentinal collagen by glycation improves long-term bond strength

ObjectivesThis study examined methylglyoxal (MGO) as a collagen crosslinker to reinforce demineralized dentin, enhance its enzymatic resistance, and improve long-term durability of the resin-dentin bond. MethodsDemineralized dentinal collagen films were treated with 0.5, 1, or 3 M MGO and subsequently exposed to collagenase to assess their resistance to enzymatic degradation. MGO-induced crosslink formation was monitored using Attenuated Total Reflectance-Fourier Transform Infrared spectroscopy (ATR-FTIR) spectroscopy by tracking the carbohydrate-associated band at 1180 cm-{superscript 1}. The apparent elastic modulus of the treated specimens was measured using a three-point bending test. For bond strength evaluation, resin-dentin beams were prepared and tested using microtensile bond strength (TBS) to assess the influence of MGO pretreatment on interfacial adhesion. ResultsThe ATR-FTIR spectra demonstrated increased intensity in the carbohydrate double bands (1000-1180 cm-{superscript 1}) in glycated samples compared to the control. Glycation with 3 M MGO exhibited the highest resistance to enzymatic degradation, persisting for up to 60 hours with a 78-fold increase in resistance factor compared to the control group (p < 0.05). Furthermore, glycation with 3 M MGO resulted in a 4-fold increase in elastic modulus compared with the control group. Notably, the functionalized dentin retained its improved mechanical properties even after collagenase exposure, whereas the control group experienced a significant 68.2% reduction in elastic modulus (p = 0.002). While MGO pretreatment did not influence resin infiltration or initial TBS ({approx}30-35 MPa), it maintained its original bond strength after one month of collagenase challenge. In contrast, the control group exhibited a significant reduction, decreasing to 17 {+/-} 5.5 MPa compared with its initial value (p < 0.01). ConclusionMGO demonstrated efficacy in enhancing the mechanical properties and enzymatic stability of collagen as well as improving the resistance of the bonded interface to enzymatic degradation. SignificanceMGO pretreatment maintains the long-term stability of the resin-dentin interface by protecting dentinal collagen from enzymatic degradation, without compromising initial bonding performance. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/701305v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@54ae32org.highwire.dtl.DTLVardef@1787fdforg.highwire.dtl.DTLVardef@130b40org.highwire.dtl.DTLVardef@47c5f9_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Unraveling novel insights into dual-species cariogenic biofilm formation: a comparative analysis on natural vs artificial bioengineered dentin models

Dental caries is the most prevalent biofilm-associated disease affecting billions of people worldwide, including elderly individuals. Conventional biofilm study methods rely on human or animal-derived samples, posing challenges regarding accessibility, cost, and ethical considerations. While in-vitro systems offer a promising alternative, they often fail to replicate the structural characteristics of dentin, which play a crucial role in bacterial adhesion. To bridge this gap, a bioengineered dentin-like construct has recently been developed as a reproducible and accessible model for studying biofilm formation associated with dental aging. Therefore, this study aimed to assess dual-species Streptococcus mutans and Candida albicans biofilm formation on bioengineered dentin substrates and compare it to biofilm formation on natural human aged dentin. For this, S. mutans UA159 and C. albicans (ATCC 90028) were co-cultured on bioengineered and natural dentin slabs, and polymicrobial biofilm formation and EPS production were characterized via high-resolution confocal laser scanning microscopy. Following biofilm formation, image processing was conducted using COMSTAT software to determine biofilm growth parameters. Additionally, fluorescence intensity was quantified via microplate readings, and cell viability was assessed using a Live/Dead viability kit. Overall, results showed similar biofilm formation patterns between the bioengineered and aged dentin, with no significant differences found in biofilm physical properties or viability. These findings suggest that this bioengineered dentin system provides a reliable platform for studying biofilm formation in the context of dental aging, making it a valuable tool for investigating microbial adhesion and cariogenic biofilm development under controlled conditions, potentially facilitating future research in biofilm-related oral diseases. ImportanceDental caries is one of the most common chronic diseases worldwide, which is driven by complex microbial biofilms formed on the tooths surface. However, existing models for studying these biofilms in the laboratory often rely on human or animal tissues, which are difficult to obtain and standardize, and present ethical challenges. In this study, we validate a bioengineered dentin-like model that accurately mimics the microarchitecture of aged human dentin, a key site for root caries in the elderly. By comparing biofilms formed by the clinically significant Streptococcus mutans and Candida albicans on both artificial and natural substrates, we show that the engineered model supports biofilm development under comparable parameters and enables detection of changes in microbial virulence. Overall, this platform provides a reproducible and scalable alternative for studying oral biofilms with potential applications in understanding disease pathogenesis, novel treatment testing, and integration into next-generation organ-on-a-chip systems.

bioengineering↗

bioRxiv2025

Royal Jelly Derived Extracellular Vesicles Modulate Microglial Nanomechanics and Inflammatory Responses

BACKGROUND: Microglia, the brain's resident immune cells, undergo profound mechanical and functional changes upon activation contributing to neuroinflammation, a pathological signature of many neurological diseases. Thus, new anti-inflammatory treatment options are needed that tackle these mechanobiological alterations in microglia, which remain strongly understudied. In this context, extracellular vesicles (EVs) are crucial mediators of intercellular and interkingdom communication, yet their influence on the mechanobiological properties of recipient cells remains largely unknown. Honeybee-derived Royal Jelly EVs (RJEVs) have demonstrated remarkable anti-inflammatory properties, but their impact on microglial cellular nanomechanics and uptake mechanisms remains unclear. RESULTS: In this study, we used a multi-disciplinary approach to analyze the resulting biological and nanomechanical changes following the activation of human microglia and the potential effect of RJEV treatment on these mechanobiological parameters. We observed that LPS treatment was associated with decreased cellular Young's modulus, increased membrane fluidity, and enhanced motility of microglia, indicating a more migratory and pro-inflammatory phenotype. Additionally, LPS exposure altered cellular EV uptake mechanisms by shifting preference from an equilibrium of four mechanisms to the predominance of macropinocytosis and clathrin-dependent endocytosis. Remarkably, RJEV treatment counteracted these mechanobiological changes by, in turn, increasing microglial stiffness, reducing motility, and decreasing secretion of pro-inflammatory cytokines. CONCLUSION: This is the first study to demonstrate that microglial activation state dictates EV uptake mechanisms and to establish a direct link between inflammation, cellular and membrane mechanics, and EV-mediated modulation. Our findings highlight RJEVs as promising candidates for regulating neuroinflammation by targeting microglial mechanobiology as well as opening new strategies for EV-based therapeutics.

neuroscience↗

bioRxiv2024

Biofilm formation on glycated collagen modulates Streptococcus mutans bacterial extracellular vesicle production and cargo

Streptococcus mutans is the major microbial etiological agent of dental caries and can adhere to surfaces such as type-I collagen, present in dentin and periodontal tissues. Recent studies have characterized planktonic S. mutans bacterial extracellular vesicles (bEVs) and demonstrated environmental-induced changes due to sugar presence or pH alterations. However, to date there are no studies exploring if surface-derived changes - such as tissue glycation - can modulate bEV production in the context of oral biofilm formation in the elderly. Therefore, the aim of this work was to determine the role of biofilm formation and collagen glycation on the morphology and composition of S. mutans bEVs. For this, bEVs from S. mutans biofilms on native and glycated collagen surfaces were isolated, characterized, and compared to bEVs from planktonic cells. Nanoparticle tracking analysis and microscopy confirmed bEV production and showed that bEVs from biofilms are smaller in size and less abundant than those from planktonic cells. Furthermore, proteome analysis revealed that S. mutans biofilm formation on native and glycated collagen led to the enrichment of several key virulence proteins such as Eno, LuxS, Tpx, and ScrB. Also, a shift towards proteins involved in metabolic processes was found in bEVs following biofilm formation on collagen surfaces, whereas glucan metabolism proteins were overexpressed in vesicles from the planktonic state. These results demonstrate that biofilm formation, as well as the glycation of collagen associated with aging and hyperglycemia, can modulate bEV characteristics and cargo and could play a central role in S. mutans virulence and the development of diseases such as dental caries and periodontal disease.

microbiology↗

bioRxiv2023

Microfabrication-based engineering of biomimetic dentin-like constructs to simulate dental aging

1.Human dentin is a highly organized dental tissue displaying a complex microarchitecture consisting of micrometer-sized tubules encased in a mineralized type-I collagen matrix. As such, it serves as an important substrate for the adhesion of microbial colonizers and oral biofilm formation in the context of dental caries disease, including root caries in the elderly. Despite this issue, there remains a current lack of effective biomimetic in-vitro dentin models that facilitate the study of oral microbial adhesion by considering the surface architecture at the micro- and nanoscales. Therefore, the aim of this study was to develop a novel in-vitro microfabricated biomimetic dentin surface that simulates the complex surface microarchitecture of exposed dentin. For this, a combination of soft lithography microfabrication and biomaterial science approaches were employed to construct a micropitted PDMS substrate functionalized with mineralized type-I collagen. These dentin analogues were subsequently glycated with methylglyoxal (MGO) to simulate dentin matrix aging in-vitro and analyzed utilizing an interdisciplinary array of techniques including atomic force microscopy (AFM), elemental analysis, and electron microscopy. AFM force-mapping demonstrated that the nanomechanical properties of the biomimetic constructs were within the expected biological parameters, and that mineralization was mostly predominated by hydroxyapatite deposition. Finally, dual-species biofilms of Streptococcus mutans and Candida albicans were grown and characterized on the biofunctionalized PDMS microchips, demonstrating biofilm specific morphologic characteristics and confirming the suitability of this model for the study of early biofilm formation under controlled conditions. Overall, we expect that this novel biomimetic dentin model could serve as an in-vitro platform to study oral biofilm formation or dentin-biomaterial bonding in the laboratory without the need for animal or human tooth samples in the future.

bioengineering↗

bioRxiv2023

Quantitative Nanohistology of aging dermal collagen

While the external signs of skin aging have been well-defined throughout history, much less is known about aging within the ultrastructure of our skin. Our skin, the largest organ in our body, is structured by collagen through fibrils or large sheets. With the increased use of nanometrology tools in histology, it is now possible to explore how the aging process affects collagen at its most fundamental level, the collagen fibril. Here, we show how atomic force microscopy-based quantitative nanohistology can differentiate skin from different age groups and anatomical sites. Following the definition of specific collagen biomarkers at the nanoscale, we used a segmentation approach to quantify the prevalence of 4 structural biomarkers over a dataset of 42,000 images (30 donors) complemented by extensive nanomechanical analyses (30,000 indentation curves) on histological sections. Our results demonstrate that specific age-related collagen fingerprints could be found when comparing the % prevalence of each marker between the papillary and reticular dermis. A case of abnormal biological aging validated our markers and nanohistology approach. This first extensive study focusing on defining signs of dermal aging at the nanoscale proves we are all unique to our dermal collagen ultrastructure.

biophysics↗

bioRxiv2022

Nanoscale dynamics of streptococcal adhesion to AGE-modified collagen

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.

microbiology↗

bioRxiv2022

Extracellular Vesicles derived from Apis mellifera Royal Jelly promote wound healing by modulating inflammation and cellular responses

Apis mellifera Royal Jelly (RJ) is a well-known remedy in traditional medicine around the world and its versatile effects range from antibacterial to anti-inflammatory properties and pro-regenerative properties. Several active compounds have been identified, however, the mechanisms of action still remain widely unknown. As a glandular product, RJ has been shown to contain a substantial number of extracellular vesicles (EVs) and in this study, we aimed to investigate the extent of involvement of RJEVs in wound healing associated effects. Molecular analysis of RJEVs verified the presence of important conserved exosomal markers such as CD63 and syntenin, as well as cargo molecules MRJP1, defensin-1 and jellein-3. RJEV internalization analysis demonstrated the involvement of membrane fusion as well as macropinocytosis or clathrin-dependent endocytosis into mammalian cells. Furthermore, RJEVs have demonstrated to modulate MSCs differentiation and secretome, as well as decrease LPS-induced inflammation in RAW 264.7 macrophages by blocking the MAPK pathway. In vivo studies confirmed anti-bacterial effects of RJEVs, and demonstrated an acceleration of wound healing in a splinted mouse model. Summarizing, this study suggests that RJEVs of potentially exosomal origin play a crucial role in the known effects of RJ by modulating the inflammatory phase and cellular response in wound healing.

bioengineering↗

bioRxiv2022

Modulation of the Biophysical and Biochemical Properties of Collagen by Glycation for Tissue Engineering Applications

The structural and functional properties of collagen are modulated by the presence of intramolecular and intermolecular crosslinks. Advanced Glycation End-products (AGEs) can produce intermolecular crosslinks by bonding the free amino groups of neighboring proteins. In this research, the following hypothesis is explored: The accumulation of AGEs in collagen decreases its proteolytic degradation rates while increasing its stiffness. Fluorescence Lifetime Imaging (FLIM) and Fourier-transform infrared spectroscopy (FTIR) detect biochemical changes in collagen scaffolds during the glycation process. The accumulation of AGEs increases exponentially in the collagen scaffolds as a function of Methylglyoxal (MGO) concentration by performing autofluorescence measurement and competitive ELISA. Glycated scaffolds absorb water at a much higher rate confirming the direct affinity between AGEs and interstitial water within collagen fibrils. In addition, the topology of collagen fibrils as observed by Atomic Force Microscopy (AFM) is a lot more defined following glycation. The elastic modulus of collagen fibrils decreases as a function of glycation, whereas the elastic modulus of collagen scaffolds increases. Finally, the enzymatic degradation of collagen by bacterial collagenase shows a sigmoidal pattern with a much slower degradation rate in the glycated scaffolds. This study identifies unique variations in the properties of collagen following accumulation of AGEs.

bioengineering↗

bioRxiv2019

Investigating the biochemical properties of Streptococcal polysaccharide capsules using atomic force microscopy

In common with many bacterial pathogens, Streptococcus pneumoniae has a polysaccharide capsule, which facilitates immune evasion and is a key virulence determinant. However, recent data has shown that the closely related Streptococcus mitis can also express polysaccharide capsules including those with an identical chemical structure to S. pneumoniae capsular serotypes. We have used atomic force microscopy (AFM) techniques to investigate the biophysical properties of S. mitis and S. pneumoniae strains expressing the same capsular serotypes that might relate to their differences in virulence potential. When comparing S. mitis and S. pneumoniae strains with identical capsule serotypes S. mitis strains were more susceptible to neutrophil killing and imaging using electron microscopy and AFM demonstrated significant morphological differences. Force-volume mapping using AFM showed distinct force-curve profiles for the centre and edge areas of encapsulated S. pneumoniae and S. mitis strains. This \"edge effect\" was not observed in the unencapsulated streptococcal strains and in an unencapsulated Staphylococcus aureus strain, and therefore was a direct representation of the mechanical properties of the bacterial capsule. When two strains of S. mitis and S. pneumoniae expressed an identical capsular serotype, they presented also similar biomechanical characteristics. This would infer a potential relationship between capsule biochemistry and nanomechanics, independent of the bacterial strains. Overall, AFM was an effective tool to explore the biophysical properties of bacterial capsules of living bacteria by reproducibly quantifying the elastic and adhesive properties of bacterial cell surfaces. Using AFM to investigate capsule differences over a wider range of strains and capsular serotypes of streptococci and correlate the data with phenotypic differences will elucidate how the biophysical properties of the capsule can influence its biological role during infection.

microbiology↗