Validation of clinically related aging models based on enamel wear.

PURPOSE: Physiological and erosive wear reported in clinical studies were reviewed, and in vitro aging models were developed to simulate and compare the effect of aging on human teeth with the review data obtained from clinical studies. METHODS: A review of clinical studies and randomized clinical trials that quantify enamel wear was performed in the PubMed database. The first in vitro analysis evaluated the effect of mechanical chewing simulation only. Enamel specimens were aged in the chewing simulator (up to 1.2 million cycles) with two occlusal loads (30 and 50 N). In the second in vitro analysis, specimens were aged in two aging models. The first model (MT) simulated mechanical and thermal oral challenges: MT1- 240,000 chewing and 10,000 thermal cycles, MT2- 480,000 chewing and 20,000 thermal cycles, MT3- 1.2 million chewing and 50,000 thermal cycles. The second model (MTA) simulated mechanical, thermal, and acidic oral challenges as follows: MTA1- 240,000 chewing, 10,000 thermal and 3-h acidic cycles; MTA2: 480,000 chewing, 20,000 thermal and 6-h acidic cycles, MTA3- 1.2 million chewing, 50,000 thermal and 15-h acidic cycles. RESULTS: The review included 13 clinical studies evaluating tooth wear (eight physiological and five erosive). The results estimated the annual average physiological wear as 38.4 µm (9.37-51). In comparison, the MT1 showed wear of 60 (24) µm. Also, the average annual erosive wear in the literature was 179.5 µm (70-265) compared to MTA1-induced wear of 209 (14) µm. CONCLUSION: There was wide variation in tooth wear reported in clinical studies, suggesting a critical need for more accurate studies, possibly based on scanning technologies. Despite this, the data reported using the novel aging models are within a range to be considered consistent with and to simulate tooth wear measured in vivo.

Effect of artificial aging on optical properties and crystalline structure of high-translucency zirconia.

PURPOSE: To investigate the effect of different in vitro aging protocols on the optical properties and crystalline structure of high-translucency (HT) zirconia. MATERIALS AND METHODS: Thirty-six specimens of HT and extra-high translucency (XT) zirconia were divided into three groups: control (CO)-no treatment; hydrothermal aging (HA)-autoclave aging for 12.5 h at 134°C, 2 bar; clinically related aging (CRA)-aging in the chewing simulator for 1.2 million cycles, followed by 50,000 thermocycles (5-55°C) and immersion in HCl (pH 1.2) for 15 h. Optical properties, crystalline structure, and surface roughness were analyzed and compared using analysis of variance (5% significance level). RESULTS: There was no statistically significant effect of aging on translucency (p = 0.10), but CRA promoted the development of a high contrast ratio (p = 0.03). Aging did not cause significant color changes for HT (p = 0.65) or XT (p = 0.36). The proportion of monoclinic crystals increased to 40% for HT-zirconia after HA and 5% after CRA. No monoclinic crystals were detected for XT groups. There was no effect of aging on surface roughness (p = 0.77). CONCLUSIONS: Although hydrothermal aging has been widely used to verify zirconia crystalline stability, it did not generate an effect similar to clinically related aging on the optical properties and crystalline structure of zirconia. HA affected the crystalline structure of HT-zirconia, and CRA compromised the optical properties of XT zirconia.

Development of a facile method to compute collagen network pathological anisotropy using AFM imaging.

Type I collagen, a fundamental extracellular matrix (ECM) component, is pivotal in maintaining tissue integrity and strength. It is also the most prevalent fibrous biopolymer within the ECM, ubiquitous in mammalian organisms. This structural protein provides essential mechanical stability and resilience to various tissues, including tendons, ligaments, skin, bone, and dentin. Collagen has been structurally investigated for several decades, and variation to its ultrastructure by histology has been associated with several pathological conditions. The current study addresses a critical challenge in the field of collagen research by providing a novel method for studying collagen fibril morphology at the nanoscale. It offers a computational approach to quantifying collagen properties, enabling a deeper understanding of how collagen type I can be affected by pathological conditions. The application of Fast Fourier Transform (FFT) coupled with Atomic Force Microscope (AFM) imaging distinguishes not only healthy and diseased skin but also holds potential for automated diagnosis of connective tissue disorders (CTDs), contributing to both clinical diagnostics and fundamental research in this area. Here we studied the changes in the structural parameters of collagen fibrils in Ehlers Danlos Syndrome (EDS). We have used skin extracted from genetically mutant mice that exhibit EDS phenotype as our model system (Col1a1Jrt/+ mice). The collagen fibrils were analyzed by AFM based descriptive-structural parameters, coupled with a 2D Fast Fourier Transform(2D-FFT) approach that automated the analysis of AFM images. In addition, each sample was characterized based on its FFT and power spectral density. Our qualitative data showed morphological differences in collagen fibril clarity (clearness of the collagen fibril edge with their neighbouring fibri), D-banding, orientation, and linearity. We have also demonstrated that FFT could be a new tool for distinguishing healthy from tissues with CTDs by measuring the disorganization of fibrils in the matrix. We have also employed FFT to reveal the orientations of the collagen fibrils, providing clinically relevant phenotypic information on their organization and anisotropy. The result of this study can be used to develop a new automated tool for better diagnosis of CTDs.

Quantitative nanohistology of aging dermal collagen.

The skin is the largest organ in the body and is essential for protecting us from environmental stressors such as UV radiation, pollution, and pathogens. As we age, our skin undergoes complex changes that can affect its function, appearance, and health. These changes result from intrinsic (chronological) and extrinsic (environmental) factors that can cause damage to the skin's cells and extracellular matrix. As higher-resolution microscopical techniques, such as Atomic Force Microscopy (AFM), are being deployed to support histology, it is possible to explore the biophysical properties of the dermal scaffold's constituents, such as the collagen network. In this study, we demonstrate the use of our AFM-based quantitative nanohistology, performed directly on unfixed cryosections of 30 donors (female, Caucasian), to differentiate between dermal collagen from different age groups and anatomical sites. The initial 420 (10 × 10 µm2) Atomic Force Microscopy images were segmented into 42,000 (1 × 1 µm2) images before being classified according to four pre-defined empirical collagen structural biomarkers to quantify the structural heterogeneity of the dermal collagen. These markers include interfibrillar gap formation, undefined collagen structure, and registered or unregistered dense collagen fibrillar network with evident D-banding. The structural analysis was also complemented by extensive nanoindentation (∼1,000 curves) performed on individual fibrils from each section, yielding 30,000 indentation curves for this study. Principal Component Analysis was used to reduce the complexity of high-dimensional datasets. The % prevalence of the empirical collagen structural biomarkers between the papillary and reticular dermis for each section proves determinant in differentiating between the donors as a function of their age or the anatomical site (cheek or breast). A case of abnormal biological aging validated our markers and nanohistology approach. This case also highlighted the difference between chronological and biological aging regarding dermal collagen phenotyping. However, quantifying the impact of chronic and pathological conditions on the structure and function of collagen at the sub-micron level remains challenging and lengthy. By employing tools such as the Atomic Force Microscope as presented here, it is possible to start evaluating the complexity of the dermal matrix at the nanoscale and start identifying relevant collagen morphology which could be used toward histopathology standards.

Enhanced detoxification of Cr6+ by Shewanella oneidensis via adsorption on spherical and flower-like manganese ferrite nanostructures.

Maximizing the safe removal of hexavalent chromium (Cr6+) from waste streams is an increasing demand due to the environmental, economic and health benefits. The integrated adsorption and bio-reduction method can be applied for the elimination of the highly toxic Cr6+ and its detoxification. This work describes a synthetic method for achieving the best chemical composition of spherical and flower-like manganese ferrite (MnxFe3-xO4) nanostructures (NS) for Cr6+ adsorption. We selected NS with the highest adsorption performance to study its efficiency in the extracellular reduction of Cr6+ into a trivalent state (Cr3+) by Shewanella oneidensis (S. oneidensis) MR-1. MnxFe3-xO4 NS were prepared by a polyol solvothermal synthesis process. They were characterised by powder X-ray diffraction (XRD), transmission electron microscopy (TEM), X-ray photoelectron spectrometry (XPS), dynamic light scattering (DLS) and Fourier transform-infrared (FTIR) spectroscopy. The elemental composition of MnxFe3-xO4 was evaluated by inductively coupled plasma atomic emission spectroscopy. Our results reveal that the oxidation state of the manganese precursor significantly affects the Cr6+ adsorption efficiency of MnxFe3-xO4 NS. The best adsorption capacity for Cr6+ is 16.8 ± 1.6 mg Cr6+/g by the spherical Mn0.22+Fe2.83+O4 nanoparticles at pH 7, which is 1.4 times higher than that of Mn0.8Fe2.2O4 nanoflowers. This was attributed to the relative excess of divalent manganese in Mn0.22+Fe2.83+O4 based on our XPS analysis. The lethal concentration of Cr6+ for S. oneidensis MR-1 was 60 mg L-1 (determined by flow cytometry). The addition of Mn0.22+Fe2.83+O4 nanoparticles to S. oneidensis MR-1 enhanced the bio-reduction of Cr6+ 2.66 times compared to the presence of the bacteria alone. This work provides a cost-effective method for the removal of Cr6+ with a minimum amount of sludge production.

Correction: Enhanced detoxification of Cr6+ by Shewanella oneidensis via adsorption on spherical and flower-like manganese ferrite nanostructures.

[This corrects the article DOI: 10.1039/D2NA00691J.].

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 neighbouring 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 the accumulation of AGEs. STATEMENT OF SIGNIFICANCE: In humans, Advanced Glycation End-products (AGEs) are naturally produced as a result of aging process. There is an evident lack of knowledge in the basic science literature explaining the biomechanical impact of AGE-mediated crosslinks on the functional and structural properties of collagen at both the nanoscale (single fibrils) and mesoscale (bundles of fibrils). This research, demonstrates how it is possible to harness this natural phenomenon in vitro to enhance the properties of engineered collagen fibrils and scaffolds. This study identifies unique variations in the properties of collagen at nanoscale and mesoscale following accumulation of AGEs. In their approach, they investigate the unique properties conferred to collagen, namely enhanced water sorption, differential elastic modulus, and finally sigmoidal proteolytic degradation behavior.

Violin Varnishes: Microstructure and Nanomechanical Analysis.

The aim of the current work is twofold: to demonstrate the application of in situ non-invasive imaging by portable atomic force microscopy (AFM) on the surfaces of a violin and to integrate compositional and mechanical analysis at the nano scale level on model samples of varnished wood. These samples were prepared according to traditional recipes by an Italian lute-maker family well practised in the art. Samples of oil and spirit-based varnishes on maple wood, naturally and accelerated light aged, were studied. AFM was used to measure the nanomechanical properties of the model samples and established that the spirit-based varnish was stiffer than the oil-based. Synchrotron radiation micro- Fourier Transform Infra-red analysis of the layer structure revealed that stiffer spirit-based varnish showed less penetration into the wood than the oil-based. Further PeakForce Quantitative Nanomechanical Mapping (QNM) demonstrated a difference in adhesion values between the oil- and spirit-based samples.

Protocol for a Case Control Study to Evaluate Oral Health as a Biomarker of Child Exposure to Adverse Psychosocial Experiences.

BACKGROUND: The early identification of children who have experienced adversity is critical for the timely delivery of interventions to improve coping and reduce negative consequences. Self-report is the usual practice for identifying children with exposure to adversity. However, physiological characteristics that signal the presence of disease or other exposures may provide a more objective identification strategy. This protocol describes a case-control study that assesses whether exposure to adversity is more common in children with tooth enamel anomalies compared to children without such anomalies. METHODS: For 150 mother-child pairs from a pediatric dental clinic in Toronto, Canada, maternal interviews will assess the child's adverse and resilience-building experiences. Per child, one (exfoliated or extracted) tooth will be assessed for suspected enamel anomalies. If anomalies are present, the child is a case, and if absent, the child is a control. Tooth assessment modalities will include usual practice for dental exams (visual assessment) and modalities with greater sensitivity to identify anomalies. CONCLUSION: If structural changes in children's teeth are associated with exposure to adversity, routine dental exams could provide an opportunity to screen children for experiences of adversity. Affected children could be referred for follow-up.

Reprogramming bone progenitor identity and potency through control of collagen density and oxygen tension.

The biophysical microenvironment of the cell is being increasingly used to control cell signaling and to direct cell function. Herein, engineered 3D tuneable biomimetic scaffolds are used to control the cell microenvironment of Adipose-derived Mesenchymal Stromal Cells (AMSC), which exhibit a collagen density-specific profile for early and late stage bone cell lineage status. Cell potency was enhanced when AMSCs were cultured within low collagen density environments in hypoxic conditions. A transitional culture containing varied collagen densities in hypoxic conditions directed differential cell fate responses. The early skeletal progenitor identity (PDPN+CD146-CD73+CD164+) was rescued in the cells which migrated into low collagen density gels, with cells continuously exposed to the high collagen density gels displaying a transitioned bone-cartilage-stromal phenotype (PDPN+CD146+CD73-CD164-). This study uncovers the significant contributions of the physical and physiological cell environment and highlights a chemically independent methodology for reprogramming and isolating skeletal progenitor cells from an adipose-derived cell population.

Compromised dental cells viability following teeth-whitening exposure.

This study aimed to assess the viability of dental cells following time-dependent carbamide peroxide teeth-whitening treatments using an in-vitro dentin perfusion assay model. 30 teeth were exposed to 5% or 16% CP gel (4 h daily) for 2-weeks. The enamel organic content was measured with thermogravimetry. The time-dependent viability of human dental pulp stem cells (HDPSCs) and gingival fibroblast cells (HGFCs) following either indirect exposure to 3 commercially available concentrations of CP gel using an in-vitro dentin perfusion assay or direct exposure to 5% H2O2 were investigated by evaluating change in cell morphology and by hemocytometry. The 5% and 16% CP produced a significantly lower (p < 0.001) enamel protein content (by weight) when compared to the control. The organic content in enamel varied accordingly to the CP treatment: for the 16% and 5% CP treatment groups, a variation of 4.0% and 5.4%, respectively, was observed with no significant difference. The cell viability of HDPSCs decreased exponentially over time for all groups. Within the limitation of this in-vitro study, we conclude that even low concentrations of H2O2 and CP result in a deleterious change in enamel protein content and compromise the viability of HGFCs and HDPSCs. These effects should be observed in-vivo.

Measuring the elastic modulus of soft culture surfaces and three-dimensional hydrogels using atomic force microscopy.

Growing interest in exploring mechanically mediated biological phenomena has resulted in cell culture substrates and 3D matrices with variable stiffnesses becoming standard tools in biology labs. However, correlating stiffness with biological outcomes and comparing results between research groups is hampered by variability in the methods used to determine Young's (elastic) modulus, E, and by the inaccessibility of relevant mechanical engineering protocols to most biology labs. Here, we describe a protocol for measuring E of soft 2D surfaces and 3D hydrogels using atomic force microscopy (AFM) force spectroscopy. We provide instructions for preparing hydrogels with and without encapsulated live cells, and provide a method for mounting samples within the AFM. We also provide details on how to calibrate the instrument, and give step-by-step instructions for collecting force-displacement curves in both manual and automatic modes (stiffness mapping). We then provide details on how to apply either the Hertz or the Oliver-Pharr model to calculate E, and give additional instructions to aid the user in plotting data distributions and carrying out statistical analyses. We also provide instructions for inferring differential matrix remodeling activity in hydrogels containing encapsulated single cells or organoids. Our protocol is suitable for probing a range of synthetic and naturally derived polymeric hydrogels such as polyethylene glycol, polyacrylamide, hyaluronic acid, collagen, or Matrigel. Although sample preparation timings will vary, a user with introductory training to AFM will be able to use this protocol to characterize the mechanical properties of two to six soft surfaces or 3D hydrogels in a single day.

Controlled Environment Neutron Radiography of Moisture Sorption/Desorption in Nanocellulose-Treated Cotton Painting Canvases.

Nanocellulose-based materials have recently been used to consolidate degraded cotton painting canvases. Canvas-supported paintings consist of materials that are sensitive to moisture and especially susceptible to environmental fluctuations in temperature and relative humidity (RH). These environmental fluctuations occur in uncontrolled environments found in historic houses and palaces and can lead to hydrolytic degradation and mechanical damage to canvases. To simulate this situation in an experimental setting, canvas samples were mounted in a custom-made closed-cell and subjected to programmed cycles of RH at a controlled temperature while exposed to the neutron beam. Results are presented for both untreated samples and those treated with a polar consolidant, cellulose nanofibrils (CNF(aq)) in water, and an apolar consolidant, a composite of persilylated methyl cellulose with surface silylated cellulose nanocrystals (MC+CNC(h)) in heptane. They were then compared with changes in ionic conductivities as measured by dielectric analysis (DEA) with the same cyclic RH program and temperature. Although the samples were exposed to the same experimental conditions, they presented treatment-specific responses. CNF-treated canvas showed higher hygroscopicity than the untreated sample and facilitated moisture diffusion across the sample to areas not exposed to the environment. A sample treated with MC+CNC(h) retarded moisture diffusion during the increase in RH and could, therefore, afford protection to moisture absorption in uncontrolled environments. Thus, the experimental setup and resulting data provide a pilot study demonstrating the potential of neutron radiography in following and comparing real-time moisture diffusion dynamics in untreated and nanocellulose-consolidated cotton canvases and assisting in validating the overall benefit of the treatment.

ILC1 drive intestinal epithelial and matrix remodelling.

Organoids can shed light on the dynamic interplay between complex tissues and rare cell types within a controlled microenvironment. Here, we develop gut organoid cocultures with type-1 innate lymphoid cells (ILC1) to dissect the impact of their accumulation in inflamed intestines. We demonstrate that murine and human ILC1 secrete transforming growth factor ß1, driving expansion of CD44v6+ epithelial crypts. ILC1 additionally express MMP9 and drive gene signatures indicative of extracellular matrix remodelling. We therefore encapsulated human epithelial-mesenchymal intestinal organoids in MMP-sensitive, synthetic hydrogels designed to form efficient networks at low polymer concentrations. Harnessing this defined system, we demonstrate that ILC1 drive matrix softening and stiffening, which we suggest occurs through balanced matrix degradation and deposition. Our platform enabled us to elucidate previously undescribed interactions between ILC1 and their microenvironment, which suggest that they may exacerbate fibrosis and tumour growth when enriched in inflamed patient tissues.

Microporous Biodegradable Films Promote Therapeutic Angiogenesis.

Peripheral arterial disease and critical limb ischemia are common symptoms of cardiovascular disease. Vascular surgery is used to create a bypass around occluded blood vessels to improve blood flow to ischemic muscle, thus avoiding the need for amputation. Attempts to vascularize tissues by therapeutic angiogenesis using delivery of exogenous angiogenic agents are underwhelming. A material-based approach that provides an endogenous stimulus capable of promoting angiogenesis and increased tissue perfusion would provide a paradigm shift in treatment options available. It is reported here that microporous biodegradable films produced using thermally induced phase separation provide a localized biophysical stimulus of proangiogenic genes in vivo that is associated with increased blood vessel density and restoration of blood flow to ischemic tissue. These findings show, for the first time, that acellular, nonfunctionalized biodegradable biomaterials can provide an innovative, material-based approach for therapeutic angiogenesis to enhance tissue reperfusion in vivo.

A Synergistic Relationship between Polycaprolactone and Natural Polymers Enhances the Physical Properties and Biological Activity of Scaffolds.

Biomaterials for tissue engineering include natural and synthetic polymers, but their clinical application is still limited due to various disadvantages associated with the use of these polymers. This uncertainty of the polymeric approach in tissue engineering launches an opportunity to address a key question: can we eliminate the disadvantages of both natural and synthetic polymers by combining them to form a synergistic relationship? To answer this question, we fabricated scaffolds from elastin, collagen, fibrin, and electrospun polycaprolactone (PCL) with different ratios. The material characterization of these scaffolds investigated degradation, water contact angle, angiogenesis by an ex ovo chorion allantoic membrane (CAM) assay, and mechanical and structural properties. Biological activity and specific differentiation pathways (MSC, adipogenic, osteogenic, myogenic, and chondrogenic) were studied by using human adipose-derived stem cells. Results indicated that all composite polymers degraded at a different rate, thus affecting their mechanical integrity. Cell-based assays demonstrated continual proliferative and viable properties of the cells on all seeded scaffolds with the particular initiation of a differentiation pathway among which the PCL/collagen/fibrin composite was the most angiogenic material with maximum vasculature. We were able to tailor the physical and biological properties of PCL-based composites to form a synergistic relationship for various tissue regeneration applications.

In Vivo Relationship between the Nano-Biomechanical Properties of Streptococcal Polysaccharide Capsules and Virulence Phenotype.

In common with many bacterial pathogens, Streptococcus pneumoniae has a polysaccharide capsule which facilitates immune evasion and determines virulence. Recent data have shown that the closely related Streptococcus mitis also expresses polysaccharide capsules including those with an identical chemical structure to S. pneumoniae capsular serotypes. We utilized 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 differences in virulence potential. When comparing S. mitis and S. pneumoniae strains with identical capsule serotypes, S. mitis strains were susceptible to neutrophil killing, and electron microscopy and AFM demonstrated significant morphological differences. Force-volume mapping using AFM showed distinct force-curve profiles for the center and edge areas of encapsulated streptococcal strains. This "edge effect" was not observed in unencapsulated bacteria 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 similar biomechanical characteristics. This infers a potential relationship between capsule biochemistry and nanomechanics, independent of bacterial strain. Overall, this study demonstrates that it is possible to investigate reproducibly the mechanistic, structural, and mechanical properties of both the capsule and the body of individual living bacterial cells and relate the data to virulence phenotypes. We have demonstrated that using nanomechanics to investigate individual bacterial cells we can now begin to identify the surface properties bacterial pathogens require to avoid host-mediated immunity.

The mechanobiology of tendon fibroblasts under static and uniaxial cyclic load in a 3D tissue engineered model mimicking native extracellular matrix.

Tendon mechanobiology plays a vital role in tendon repair and regeneration; however, this mechanism is currently poorly understood. We tested the role of different mechanical loads on extracellular matrix (ECM) remodelling gene expression and the morphology of tendon fibroblasts in collagen hydrogels, designed to mimic native tissue. Hydrogels were subjected to precise static or uniaxial loading patterns of known magnitudes and sampled to analyse gene expression of known mechano-responsive ECM-associated genes (Collagen I, Collagen III, Tenomodulin, and TGF-ß). Tendon fibroblast cytomechanics was studied under load by using a tension culture force monitor, with immunofluorescence and immunohistological staining used to examine cell morphology. Tendon fibroblasts subjected to cyclic load showed that endogenous matrix tension was maintained, with significant concomitant upregulation of ECM remodelling genes, Collagen I, Collagen III, Tenomodulin, and TGF-ß when compared with static load and control samples. These data indicate that tendon fibroblasts acutely adapt to the mechanical forces placed upon them, transmitting forces across the ECM without losing mechanical dynamism. This model demonstrates cell-material (ECM) interaction and remodelling in preclinical a platform, which can be used as a screening tool to understand tendon regeneration.

Phenotypic Properties of Collagen in Dentinogenesis Imperfecta Associated with Osteogenesis Imperfecta.

INTRODUCTION: 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<0.05) than that of the OIDI (mineralized) dentine. Both the exposed peri- and intratubular dentinal collagen presented bimodal elastic behaviors (Young's moduli). The control samples presented a stiffening of the intratubular collagen when compared to the peritubular collagen. In case of the OIDI, this stiffening in the collagen between peri- and intratubular dentinal collagen was not observed and the exposed collagen presented overall a lower elasticity than the control samples. CONCLUSION: This study presents a systematic approach to the characterization of collagen structure and properties in OIDI as diagnosed in dentin. Structural markers for OIDI at the mesoscale and nanoscale were found and correlated with an observed lack of increased elastic moduli of the collagen fibrils in the intratubular OIDI dentin. These findings offer an explanation of how structural changes in the dentin could be responsible for the failure of some adhesive restorative materials as observed in patients affected by OIDI.

Engineering of a Functional Tendon Using Collagen As a Natural Polymer.

Reconstruction of a tendon rupture is surgically challenging as each end of the tendon retracts, leaving a substantial gap and direct repair is often not feasible. A tendon graft is required to bridge this defect and restore function. Presently, these gaps are filled with auto-, allo-, or synthetic grafts, but they all have clinical limitations. To address this issue, we developed tissue-engineered grafts by a rapid process using compressed type I collagen, which is the most dominant protein in the tendon. However, biomechanical properties were found to be unsuitable to withstand complete load-bearing in vivo. Hence, a modified suture technique was previously developed to reduce the load on the engineered collagen graft to aid integration in vivo. Using this technique, we tested engineered collagen grafts in vivo on a lapine model in three groups up to 12 weeks without immobilization. Gross observation at 3 and 12 weeks showed the bridge integrated without adhesions with a significant increase in the mechanical, structural and histological properties as compared to 1 week. Insertion of a tissue-engineered collagen graft using a novel load-bearing suture technique which partially loads in vivo showed integration, greater mechanical strength and no adhesion formation in the time period tested. This collagen graft has inherent advantages as compared to the present-day tendon grafts.