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Salwa Ibrahim,1,* Adam P Strange,2,* Sebastian Aguayo,2,3 Albatool Shinawi,1 Nabilah Harith,1 Nurjehan Mohamed-Ibrahim,1 Samera Siddiqui,2 Susan Parekh,1 Laurent Bozec4 1Department of Paediatric Dentistry, UCL Eastman Dental Institute, University College London, London, UK; 2Department of Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, University College London, London, UK; 3School of Dentistry, Faculty of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile; 4Faculty of Dentistry, University of Toronto, Toronto, Canada*These authors contributed equally to this workCorrespondence: Laurent BozecFaculty of Dentistry, University of Toronto, 124 Edward Street, Toronto ON M5G 1G6, CanadaEmail L.bozec@utoronto.caIntroduction: Dentinogenesis imperfecta type 1 (OIDI) is considered a relatively rare genetic disorder (1:5000 to 1:45,000) associated with osteogenesis imperfecta. OIDI impacts the formation of collagen fibrils in dentin, leading to morphological and structural changes that affect the strength and appearance of teeth. However, there is still a lack of understanding regarding the nanoscale characterization of the disease, in terms of collagen ultrastructure and mechanical properties. Therefore, this research presents a qualitative and quantitative report into the phenotype and characterization of OIDI in dentin, by using a combination of imaging, nanomechanical approaches.Methods: For this study, 8 primary molars from OIDI patients and 8 primary control molars were collected, embedded in acrylic resin and cut into longitudinal sections. Sections were then demineralized in 37% phosphoric acid using a protocol developed in-house. Initial experiments demonstrated the effectiveness of the demineralization protocol, as the ATR-FTIR spectral fingerprints showed an increase in the amide bands together with a decrease in phosphate content. Structural and mechanical analyses were performed directly on both the mineralized and demineralized samples using a combination of scanning electron microscopy, atomic force microscopy, and Wallace indentation.Results: Mesoscale imaging showed alterations in dentinal tubule morphology in OIDI patients, with a reduced number of tubules and a decreased tubule diameter compared to healthy controls. Nanoscale collagen ultrastructure presented a similar D-banding periodicity between OIDI and controls. Reduced collagen fibrils diameter was also recorded for the OIDI group. The hardness of the (mineralized) control dentin was found to be significantly higher (p

Tarek Ahmed,1 Anthony Nash,2 Kristina EN Clark,3 Marion Ghibaudo,4 Nora H de Leeuw,2 Anne Potter,4 Richard Stratton,3 Helen L Birch,5 Ramona Enea Casse,4 Laurent Bozec1 1Division of Biomaterials and Tissue Engineering, Eastman Dental Institute, University College London, 2Department of Chemistry, University College London, 3Centre for Rheumatology and Connective Tissue Diseases, Division of Medicine, University College London, London, UK; 4L’Oréal Research and Innovation, Aulnay-sous-Bois, France; 5Division of Surgery and Interventional Science, UCL Institute of Orthopaedics and Musculoskeletal Science, University College London, London, UK Abstract: The extracellular matrix of the dermis is a complex, dynamic system with the various dermal components undergoing individual physiologic changes as we age. Age-related changes in the physical properties of collagen were investigated in particular by measuring the effect of aging, most likely due to the accumulation of advanced glycation end product (AGE) cross-links, on the nanomechanical properties of the collagen fibril using atomic force microscope nano-indentation. An age-related decrease in the Young’s modulus of the transverse fibril was observed (from 8.11 to 4.19 GPa in young to old volunteers, respectively, P

Adam P Strange,1 Sebastian Aguayo,1 Tarek Ahmed,1 Nicola Mordan,1 Richard Stratton,2 Stephen R Porter,3 Susan Parekh,4 Laurent Bozec1 1Department of Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, 2Centre for Rheumatology and Connective Tissue Diseases, Royal Free Hospital, UCL Medical School, 3UCL Eastman Dental Institute, 4Department of Pediatrics, UCL Eastman Dental Institute, London, UK Abstract: Scleroderma (or systemic sclerosis, SSc) is a disease caused by excess crosslinking of collagen. The skin stiffens and becomes painful, while internally, organ function can be compromised by the less elastic collagen. Diagnosis of SSc is often only possible in advanced cases by which treatment time is limited. A more detailed analysis of SSc may provide better future treatment options and information of disease progression. Recently, the histological stain picrosirius red showing collagen register has been combined with atomic force microscopy (AFM) to study SSc. Skin from healthy individuals and SSc patients was biopsied, stained and studied using AFM. By investigating the crosslinking of collagen at a smaller hierarchical stage, the effects of SSc were more pronounced. Changes in morphology and Young’s elastic modulus were observed and quantified; giving rise to a novel technique, we have termed “quantitative nanohistology”. An increase in nanoscale stiffness in the collagen for SSc compared with healthy individuals was seen by a significant increase in the Young’s modulus profile for the collagen. These markers of stiffer collagen in SSc are similar to the symptoms experienced by patients, giving additional hope that in the future, nanohistology using AFM can be readily applied as a clinical tool, providing detailed information of the state of collagen. Keywords: rheumatology, adjunct diagnosis, picrosirius red, collagen, nanohistology

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 10 mM MGO to induce AGE formation, which was characterized with microscopy and enzyme-linked immunosorbent assay. Subsequently, AFM cantilevers were functionalized with living S. mutans UA 159 or Streptococcus 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 computer simulation 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 nonspecific forces and interactions between S. mutans UA 159 and MGO-modified collagen substrates. In summary, these results suggest that collagen alterations due to aging and glycation may play a role in early bacterial adherence to oral tissues, associated with conditions such as aging or chronic hyperglycemia, among others. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Biofilm-mediated oral diseases such as dental caries and periodontal disease remain highly prevalent in populations worldwide. Biofilm formation initiates with the attachment of primary colonizers onto surfaces, and in the context of caries, the adhesion of oral streptococci to dentinal collagen is crucial for biofilm progression. It is known that dentinal collagen suffers from glucose-associated crosslinking as a function of aging or disease; however, the effect of collagen crosslinking on the early adhesion and subsequent biofilm formation of relevant oral streptococci remains unknown. Therefore, the aim of this work was to determine the impact of collagen glycation on the initial adhesion of primary colonizers such as Streptococcus mutans UA159 and Streptococcus sanguinis SK 36, as well as its effect on the early stages of streptococcal biofilm formation in vitro. Type I collagen matrices were crosslinked with either glucose or methylglyoxal. Atomic force microscopy nanocharacterization revealed morphologic and mechanical changes within the collagen matrix as a function of crosslinking, such as a significantly increased elastic modulus in crosslinked fibrils. Increased nanoadhesion forces were observed for S. mutans on crosslinked collagen surfaces as compared with the control, and retraction curves obtained for both streptococcal strains demonstrated nanoscale unbinding behavior consistent with bacterial adhesin-substrate coupling. Overall, glucose-crosslinked substrates specifically promoted the initial adhesion, biofilm formation, and insoluble extracellular polysaccharide production of S. mutans, while methylglyoxal treatment reduced biofilm formation for both strains. Changes in the adhesion behavior and biofilm formation of oral streptococci as a function of collagen glycation could help explain the biofilm dysbiosis seen in older people and patients with diabetes. Further studies are necessary to determine the influence of collagen crosslinking on the balance between acidogenic and nonacidogenic streptococci to aid in the development of novel preventive and therapeutic treatment against dental caries in these patients. [ABSTRACT FROM AUTHOR]

Denture-associated stomatitis is a common candidal infection that may give rise to painful oral symptoms, as well as be a reservoir for infection at other sites of the body. As poly (methyl methacrylate) (PMMA) remains the main material employed in the fabrication of dentures, the aim of this research was to evaluate the adhesion of Candida albicans cells onto PMMA surfaces by employing an atomic force microscopy (AFM) single-cell force spectroscopy (SCFS) technique. For experiments, tipless AFM cantilevers were functionalized with PMMA microspheres and probed against C. albicans cells immobilized onto biopolymer-coated substrates. Both a laboratory strain and a clinical isolate of C. albicans were used for SCFS experiments. Scanning electron microscopy (SEM) and AFM imaging of C. albicans confirmed the polymorphic behavior of both strains, which was dependent on growth culture conditions. AFM force-spectroscopy results showed that the adhesion of C. albicans to PMMA is morphology dependent, as hyphal tubes had increased adhesion compared with yeast cells (P < 0.05). C. albicans budding mother cells were found to be nonadherent, which contrasts with the increased adhesion observed in the tube region. Comparison between strains demonstrated increased adhesion forces for a clinical isolate compared with the lab strain. The clinical isolate also had increased survival in blood and reduced sensitivity to complement opsonization, providing additional evidence of strain-dependent differences in Candida-host interactions that may affect virulence. In conclusion, PMMA-modified AFM probes have shown to be a reliable technique to characterize the adhesion of C. albicans to acrylic surfaces. [ABSTRACT FROM AUTHOR]

This paper describes the application of atomic force microscopy (AFM) for the imaging of collagen denaturation as observed in parchment. Parchment is prepared from processed animal skin and collagen is the main component. Large collections in national archives, libraries and religious institutions contain numerous documents written on parchment. Their preservation presents an unsolved problem for conservators. The main challenge is to assess the state of collagen and to detect what conservators refer to as the pre-gelatinised state, which can cause surface cracking resulting in a loss of text and can increase the vulnerability of parchment to aqueous cleaning agents. Atomic force microscopy (AFM) was first used within the Improved Damage Assessment of Parchment (IDAP) project, enabling the characterisation of the collagen structure within parchment at the nanoscale. Damage categories were also established based on the extent of the ordered collagen structure that was observed in the AFM images. This paper describes the work following the IDAP project, where morphological changes in the fibres due to both artificial and natural ageing were observed and linked to observations made by AFM. It also explores the merits and drawbacks of different approaches used for sample preparation and the possibility of using a portable AFM for imaging directly on the surface of documents. A case study on a manuscript from the 18th century is presented. [ABSTRACT FROM AUTHOR]

Adhesion of bacteria to dental implant surfaces is the critical initial step in the process of biofilm colonization; however, the specific nanoadhesive interactions occurring during the first contact between bacterial cells and biomaterial substrates remain poorly understood. In this report, we utilize single-cell force spectroscopy to characterize the dynamics of the initial interaction between living Staphylococcus aureus cells and machined titanium surfaces at the nanoscale. Values for maximum adhesion force were found to increase from 0-s (–0.27 ± 0.30 nN) to 60-s (–9.15 ± 0.78 nN) surface delays, with similar results observed for total adhesion work (7.39 ± 2.38 and 988.06 ± 117.08 aJ, respectively). Single unbinding events observed at higher surface delays were modeled according to the wormlike chain model, obtaining molecular contour-length predictions of 314.06 ± 9.27 nm. Average single-bond rupture forces of −0.95 ± 0.04 nN were observed at increased contact times. Short- and long-range force components of bacterial adhesion were obtained by Poisson analysis of single unbinding event peaks, yielding values of −0.75 ± 0.04 and −0.58 ± 0.15 nN, respectively. Addition of 2-mg/mL chlorhexidine to the buffer solution resulted in the inhibition of specific adhesive events but an increased overall adhesion force and work. These results suggest that initial attachment of S. aureus to smooth titanium is mostly mediated by short-range attractive forces observed at higher surface delays. [ABSTRACT FROM AUTHOR]

Mineralised tissues, such as bone, consist of two material phases: collagen protein fibrils that form the structural models upon which the mineral, calcium hydroxyapatite, is subsequently deposited. Collagen and mineral are removed in a three-dimensional manner by osteoclasts during bone turnover in skeletal growth or repair, and matrix proteins are replaced by the synthetic activity of osteoblasts and then calcify. The resolution of atomic force microscopy and use of unmodified, fully calcified samples has enabled the imaging of the overall bone and dentine structure, including collagen and mineral phases. Mineral crystals, in the diameter size range of 225nm up to 1.4mm, were found in unmodified bone and dentine respectively. D-banded collagen is observed in dentine after acid treatment and in bone after osteoclast-mediated matrix resorption; axial periodicity values of approximately 67 and 69nm are observed, respectively. These experimental approaches have enabled the structure of mineralised tissues to be examined in native samples and will facilitate the study of bone structure in important clinical disorders of the skeleton, such as osteoporosis. [ABSTRACT FROM AUTHOR]

Near-field photothermal Fourier transform infra-red microspectroscopy, which utilizes atomic force microscopy (AFM)-type temperature sensors, is being developed with the aim of achieving a spatial resolution higher than the diffraction limit. Here we report on a new implementation of the technique. Sensitivity of the technique is assessed by recording infra-red spectra from small quantities of analytes and thin films. A photothermomechanical approach, which utilizes conventional AFM probes as temperature sensors, is also discussed based on preliminary results. Early indication suggests that the photothermal approach is more sensitive than the thermomechanical one. [ABSTRACT FROM AUTHOR]