High-resolution X-ray tomographic workflow to investigate the stress distribution in vitreous enamel steels.

Vitreous enamel steels (VES) are a class of metal-ceramic composite materials realised with a low carbon steel basement coated by an enamel layer. During the firing phase to adhere the enamel to the metal, several gas bubbles remain entrapped inside the enamel volume modifying its internal structure. In this work high-resolution X-ray computed tomography (micro-CT) was used to investigate these composite materials. The micro-CT reconstructions enabled a detailed investigation of VES minimising the metal artefacts. The tomograms were used to develop finite element models (FEM) of VES by means of a representative volume element (RVE) to evaluate the thermal residual stresses caused by the manufacturing process, as well as the effect of the 3D bubbles distribution on the internal stress patterns after the thermic gradient. The promising results from this study have the potential to inform further research on such composite materials by optimising manufacturing processes for targeted applications.

Vitreous enamel steels are a particular class of composite materials composed by a low carbon steel basement coated by a vitreous enamel layer. Throughout the firing process applied to fix the enamel on the steel substrate, several gas bubbles remain entrapped inside the internal volume of the enamel modifying its internal microstructure. The presence of these bubbles substantially modifies the internal mechanical state of the structure developing residual stresses both among the bubbles and between the enamel-metal surface. However, to date no methods are still available to properly investigate the 3D bubbles morphology, distribution and stress patterns inside these materials. For this reason, in the present study we developed for the first time a high-resolution X-ray computed tomography (micro-CT) protocol able to investigate the vitreous enamel steels full field structure and numerically study their mechanics when the thermal gradient is applied. The micro-CT scans reconstructions allowed the visualisation of the enamel coating structure minimising metal artefacts. Moreover, the scans were postprocessed developing unpreceded 3D reconstructions with which the distribution, the volume and the mean diameter of the bubbles were analysed and defined. Subsequently, full field finite element computational models able to evaluate the thermal residual stresses produced inside the enamel volume were developed. They permitted to investigate the effect of the bubbles distribution on the internal residual stress patterns due to the thermal gradient generated throughout the cooling phase. The promising results from this study have the potential to inform further research on such composite materials by optimising manufacturing processes for targeted applications.

Cement opacity and color as influencing factors on the final shade of metal-free ceramic restorations.

OBJECTIVE: To investigate the influence of opacity and color of luting cements on the final shade of metal-free restorations. MATERIALS AND METHODS: Five resin cement colors in combination with four different thicknesses of CAD/CAM ceramic materials were tested, and a composite substrate was used as dentin color reference (n = 3). Specimen color was measured with a spectrophotometer equipped with an integrating sphere before and after cementation (CIELAB). Cement and ceramic color and opacity (TP) were assessed by measuring the tested ceramic thickness as a 1-mm thick disk for each of the cement shades. The differences in color were evaluated (ΔE00 ). Data were statistically analyzed by a Two-Way ANOVA followed by the Tukey Test for post-hoc comparison (P < .05) and multiple comparison Pearson's test (P < .05); the acceptability and perceptibility threshold were evaluated. RESULTS: Statistically significant influence was found for factors ceramic thickness and cement shade (P < .001). Perceptible and unacceptable color changes were induced on the final restoration by resin cements (ranging from ΔE00  = 0.69 ± 0.54 to ΔE00  = 5.53 ± 0.46), the correlation between factors became strong (r2 > 0.6) in case of mismatch between color and translucency of cement and ceramic. Only the clear shade in combination with the thickest ceramic, resulted in an imperceptible color change (ΔE00  = 0.69 ± 0.54). CONCLUSIONS: The final shade of ceramic restorations can be influenced by resin cements; the magnitude of influence is related to the cement optical properties. CLINICAL SIGNIFICANCE: In order to influence the final shade of a ceramic restoration, a cement more opaque than the restorative material should be used. Conversely, in the case of a fitting shade of the restoration, a cement more translucent than the restoration should be used to avoid undesired changes.

Flexural resistance of CAD-CAM blocks. Part 3: Polymer-based restorative materials for permanent restorations.

PURPOSE: Concurrently with the growing interest in CAD-CAM systems, several new materials of different chemical nature have become available. As an alternative to ceramics, numerous polymer-based materials have recently been proposed for permanent prosthetic restorations. Aim of this study was to test the CAD-CAM polymer-based materials available on the market, comparing mean flexural strength, Weibull characteristic strength and Weibull modulus. METHODS: Seven types of polymer-based blocks were tested: Lava Ultimate, 3M; Brilliant Crios, Coltene; Cerasmart, GC; Block HC, Shofu; Katana Avencia, Noritake; Grandio Blocs, Voco; Tetric CAD, and Ivoclar-Vivadent. Specimens were cut out from blocks, finished, polished, and tested in a three-point bending test apparatus until failure (n=30). Flexural strength, Weibull characteristic strength, and Weibull modulus were calculated. Flexural strength data were statistically analyzed. ANOVA on Ranks was applied, followed by the Dunn's test for post hoc comparisons (P= 0.05). RESULTS: Flexural strength values (MPa) were measured (mean±standard deviation). Different letters in parentheses label statistically significant differences: Grandio Blocs 266±24(a), Brilliant Crios 259±21(ab); Tetric CAD 254±15(ab); Katana Avencia 241±29(bc); Cerasmart 221±24(cd); Lava Ultimate 196±23(de); Block HC 139±10(e). All the tested materials had flexural strengths greater than 100 MPa, thereby satisfying the requirements of ISO standards for polymer-based materials. For all the tested materials the Weibull characteristic strength was greater than 100 MPa. Weibull modulus ranged between 21.20 (Tetric CAD) and 9.09 (Katana Avencia). CLINICAL SIGNIFICANCE: Even though all the CAD-CAM polymer-based materials marketed in blocks tested in the present study satisfy the requirements of ISO standard for polymer-based materials, their flexural resistance differs significantly. The data presented in the study may be helpful to clinicians for selecting the most appropriate materials for each clinical case.

Evaluation of Antibacterial and Cytotoxicity Properties of Silver Nanowires and Their Composites with Carbon Nanotubes for Biomedical Applications.

In this work, we prepared silver nanowires (AgNWs) via the polyol method in the presence or absence of single wall carbon nanotubes (CNTs) and tested their physicochemical, antibacterial and cytotoxic properties. Results showed that the introduction of CNTs lead to the formation of AgNWs at lower temperature, but the final product characteristics of AgNWs and AgNWs-CNT were not significantly different. AgNWs exhibited antibacterial properties against all the studied bacterial species via the formation of oxygen reactive species (ROS) and membrane damage. Furthermore, AgNWs exhibited a dose-dependent and time-dependent toxicity at concentrations ≥ 10 µg/mL. Fibroblasts appeared to be more resistant than human colorectal adenocarcinoma (Caco-2) and osteoblasts to the toxicity of AgNWs. The cytotoxicity of AgNWs was found to be related to the formation of ROS, but not to membrane damage. Overall, these results suggest that AgNWs are potential antibacterial agents against E. coli, S. aureus, MRSA and S. saprophyticus, but their dosage needs to be adjusted according to the route of administration.

Carbon nanotubes play an important role in the spatial arrangement of calcium deposits in hydrogels for bone regeneration.

Age related bone diseases such as osteoporosis are considered among the main causes of reduced bone mechanical stability and bone fractures. In order to restore both biological and mechanical function of diseased/fractured bones, novel bioactive scaffolds that mimic the bone structure are constantly under development in tissue engineering applications. Among the possible candidates, chitosan-based thermosensitive hydrogel scaffolds represent ideal systems due to their biocompatibility, biodegradability, enhanced antibacterial properties, promotion of osteoblast formation and ease of injection, which makes them suitable for less invasive surgical procedures. As a main drawback, these chitosan systems present poor mechanical performance that could not support load-bearing applications. In order to produce more mechanically-competent biomaterials, the combined addition of hydroxyapatite and carbon nanotubes (CNTs) is proposed in this study. Specifically, the aim of this work is to develop thermosensitive chitosan hydrogels containing stabilised single-walled and multi-walled CNTs, where their effect on the mechanical/physiochemical properties, calcium deposition patterns and ability to provide a platform for the controlled release of protein drugs was investigated. It was found that the addition of CNTs had a significant effect on the sol-gel transition time and significantly increased the resistance to compression for the hydrogels. Moreover, in vitro calcification studies revealed that CNTs played a major role in the spatial arrangements of newly formed calcium deposits in the composite materials studied, suggesting that they may have a role in the way the repair of fragile and/or fractured bones occurs in vivo.

P2RX7 purinoceptor: a therapeutic target for ameliorating the symptoms of duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is the most common inherited muscle disease, leading to severe disability and death in young men. Death is caused by the progressive degeneration of striated muscles aggravated by sterile inflammation. The pleiotropic effects of the mutant gene also include cognitive and behavioral impairments and low bone density. Current interventions in DMD are palliative only as no treatment improves the long-term outcome. Therefore, approaches with a translational potential should be investigated, and key abnormalities downstream from the absence of the DMD product, dystrophin, appear to be strong therapeutic targets. We and others have demonstrated that DMD mutations alter ATP signaling and have identified P2RX7 purinoceptor up-regulation as being responsible for the death of muscles in the mdx mouse model of DMD and human DMD lymphoblasts. Moreover, the ATP-P2RX7 axis, being a crucial activator of innate immune responses, can contribute to DMD pathology by stimulating chronic inflammation. We investigated whether ablation of P2RX7 attenuates the DMD model mouse phenotype to assess receptor suitability as a therapeutic target. METHODS AND FINDINGS: Using a combination of molecular, histological, and biochemical methods and behavioral analyses in vivo we demonstrate, to our knowledge for the first time, that genetic ablation of P2RX7 in the DMD model mouse produces a widespread functional attenuation of both muscle and non-muscle symptoms. In dystrophic muscles at 4 wk there was an evident recovery in key functional and molecular parameters such as improved muscle structure (minimum Feret diameter, p < 0.001), increased muscle strength in vitro (p < 0.001) and in vivo (p = 0.012), and pro-fibrotic molecular signatures. Serum creatine kinase (CK) levels were lower (p = 0.025), and reduced cognitive impairment (p = 0.006) and bone structure alterations (p < 0.001) were also apparent. Reduction of inflammation and fibrosis persisted at 20 mo in leg (p = 0.038), diaphragm (p = 0.042), and heart muscles (p < 0.001). We show that the amelioration of symptoms was proportional to the extent of receptor depletion and that improvements were observed following administration of two P2RX7 antagonists (CK, p = 0.030 and p = 0.050) without any detectable side effects. However, approaches successful in animal models still need to be proved effective in clinical practice. CONCLUSIONS: These results are, to our knowledge, the first to establish that a single treatment can improve muscle function both short and long term and also correct cognitive impairment and bone loss in DMD model mice. The wide-ranging improvements reflect the convergence of P2RX7 ablation on multiple disease mechanisms affecting skeletal and cardiac muscles, inflammatory cells, brain, and bone. Given the impact of P2RX7 blockade in the DMD mouse model, this receptor is an attractive target for translational research: existing drugs with established safety records could potentially be repurposed for treatment of this lethal disease.

Three-dimensional local measurements of bone strain and displacement: comparison of three digital volume correlation approaches.

Different digital volume correlation (DVC) approaches are currently available or under development for bone tissue micromechanics. The aim of this study was to compare accuracy and precision errors of three DVC approaches for a particular three-dimensional (3D) zero-strain condition. Trabecular and cortical bone specimens were repeatedly scanned with a micro-computed tomography (CT). The errors affecting computed displacements and strains were extracted for a known virtual translation, as well as for repeated scans. Three DVC strategies were tested: two local approaches, based on fast-Fourier-transform (DaVis-FFT) or direct-correlation (DaVis-DC), and a global approach based on elastic registration and a finite element (FE) solver (ShIRT-FE). Different computation subvolume sizes were tested. Much larger errors were found for the repeated scans than for the virtual translation test. For each algorithm, errors decreased asymptotically for larger subvolume sizes in the range explored. Considering this particular set of images, ShIRT-FE showed an overall better accuracy and precision (a few hundreds microstrain for a subvolume of 50 voxels). When the largest subvolume (50-52 voxels) was applied to cortical bone, the accuracy error obtained for repeated scans with ShIRT-FE was approximately half of that for the best local approach (DaVis-DC). The difference was lower (250 microstrain) in the case of trabecular bone. In terms of precision, the errors shown by DaVis-DC were closer to the ones computed by ShIRT-FE (differences of 131 microstrain and 157 microstrain for cortical and trabecular bone, respectively). The multipass computation available for DaVis software improved the accuracy and precision only for the DaVis-FFT in the virtual translation, particularly for trabecular bone. The better accuracy and precision of ShIRT-FE, followed by DaVis-DC, were obtained with a higher computational cost when compared to DaVis-FFT. The results underline the importance of performing a quantitative comparison of DVC methods on the same set of samples by using also repeated scans, other than virtual translation tests only. ShIRT-FE provides the most accurate and precise results for this set of images. However, both DaVis approaches show reasonable results for large nodal spacing, particularly for trabecular bone. Finally, this study highlights the importance of using sufficiently large subvolumes, in order to achieve better accuracy and precision.

Unveiling the potential of diffusion model-based framework with transformer for hyperspectral image classification.

Hyperspectral imaging has gained popularity for analysing remotely sensed images in various fields such as agriculture and medical. However, existing models face challenges in dealing with the complex relationships and characteristics of spectral-spatial data due to the multi-band nature and data redundancy of hyperspectral data. To address this limitation, we propose a novel approach called DiffSpectralNet, which combines diffusion and transformer techniques. The diffusion method is able extract diverse and meaningful spectral-spatial features, leading to improvement in HSI classification. Our approach involves training an unsupervised learning framework based on the diffusion model to extract high-level and low-level spectral-spatial features, followed by the extraction of intermediate hierarchical features from different timestamps for classification using a pre-trained denoising U-Net. Finally, we employ a supervised transformer-based classifier to perform the HSI classification. We conduct comprehensive experiments on three publicly available datasets to validate our approach. The results demonstrate that our framework significantly outperforms existing approaches, achieving state-of-the-art performance. The stability and reliability of our approach are demonstrated across various classes in all datasets.

Full-field strain distribution in hierarchical electrospun nanofibrous poly-L(lactic) acid/collagen scaffolds for tendon and ligament regeneration: A multiscale study.

Regeneration of injured tendons and ligaments (T/L) is a worldwide need. In this study electrospun hierarchical scaffolds made of a poly-L (lactic) acid/collagen blend were developed reproducing all the multiscale levels of aggregation of these tissues. Scanning electron microscopy, microCT and tensile mechanical tests were carried out, including a multiscale digital volume correlation analysis to measure the full-field strain distribution of electrospun structures. The principal strains (εp1 and εp3) described the pattern of strains caused by the nanofibers rearrangement, while the deviatoric strains (εD) revealed the related internal sliding of nanofibers and bundles. The results of this study confirmed the biomimicry of such electrospun hierarchical scaffolds, paving the way to further tissue engineering and clinical applications.

Biofabrication of nanocomposite-based scaffolds containing human bone extracellular matrix for the differentiation of skeletal stem and progenitor cells.

Autograft or metal implants are routinely used in skeletal repair. However, they fail to provide long-term clinical resolution, necessitating a functional biomimetic tissue engineering alternative. The use of native human bone tissue for synthesizing a biomimetic material ink for three-dimensional (3D) bioprinting of skeletal tissue is an attractive strategy for tissue regeneration. Thus, human bone extracellular matrix (bone-ECM) offers an exciting potential for the development of an appropriate microenvironment for human bone marrow stromal cells (HBMSCs) to proliferate and differentiate along the osteogenic lineage. In this study, we engineered a novel material ink (LAB) by blending human bone-ECM (B) with nanoclay (L, Laponite®) and alginate (A) polymers using extrusion-based deposition. The inclusion of the nanofiller and polymeric material increased the rheology, printability, and drug retention properties and, critically, the preservation of HBMSCs viability upon printing. The composite of human bone-ECM-based 3D constructs containing vascular endothelial growth factor (VEGF) enhanced vascularization after implantation in an ex vivo chick chorioallantoic membrane (CAM) model. The inclusion of bone morphogenetic protein-2 (BMP-2) with the HBMSCs further enhanced vascularization and mineralization after only seven days. This study demonstrates the synergistic combination of nanoclay with biomimetic materials (alginate and bone-ECM) to support the formation of osteogenic tissue both in vitro and ex vivo and offers a promising novel 3D bioprinting approach to personalized skeletal tissue repair. Supplementary Information: The online version contains supplementary material available at 10.1007/s42242-023-00265-z.

Characterisation of a metallic foam-cement composite under selected loading conditions.

An open-cell metallic foam was employed as an analogue material for human trabecular bone to interface with polymethyl methacrylate (PMMA) bone cement to produce composite foam-cement interface specimens. The stress-displacement curves of the specimens were obtained experimentally under tension, shear, mixed tension and shear (mixed-mode), and step-wise compression loadings. In addition, under step-wise compression, an image-guided failure assessment (IGFA) was used to monitor the evolution of micro-damage of the interface. Microcomputed tomography (µCT) images were used to build a subject-specific model, which was then used to perform finite element (FE) analysis under tension, shear and compression. For tension-shear loading conditions, the strengths of the interface specimens were found to increase with the increase of the loading angle reaching the maximum under shear loading condition, and the results compare reasonably well with those from bone-cement interface. Under compression, however, the mechanical strength measured from the foam-cement interface is much lower than that from bone-cement interface. Furthermore, load transfer between the foam and the cement appears to be poor under both tension and compression, hence the use of the foam should be discouraged as a bone analogue material for cement fixation studies in joint replacements.

A Comprehensive Mechanical Characterization of Subject-Specific 3D Printed Scaffolds Mimicking Trabecular Bone Architecture Biomechanics.

This study presents a polymeric scaffold designed and manufactured to mimic the structure and mechanical compressive characteristics of trabecular bone. The morphological parameters and mechanical behavior of the scaffold were studied and compared with trabecular bone from bovine iliac crest. Its mechanical properties, such as modulus of elasticity and yield strength, were studied under a three-step monotonic compressive test. Results showed that the elastic modulus of the scaffold was 329 MPa, and the one for trabecular bone reached 336 MPa. A stepwise dynamic compressive test was used to assess the behavior of samples under various loading regimes. With microcomputed tomography (µCT), a three-dimensional reconstruction of the samples was obtained, and their porosity was estimated as 80% for the polymeric scaffold and 88% for trabecular bone. The full-field strain distribution of the samples was measured using in situ µCT mechanics and digital volume correlation (DVC). This provided information on the local microdeformation mechanism of the scaffolds when compared to that of the tissue. The comprehensive results illustrate the potential of the fabricated scaffolds as biomechanical templates for in vitro studies. Furthermore, there is potential for extending this structure and fabrication methodology to incorporate suitable biocompatible materials for both in vitro and in vivo clinical applications.

Open-porous magnesium-based scaffolds withstand in vitro corrosion under cyclic loading: A mechanistic study.

The successful application of magnesium (Mg) alloys as biodegradable bone substitutes for critical-sized defects may be comprised by their high degradation rate resulting in a loss of mechanical integrity. This study investigates the degradation pattern of an open-porous fluoride-coated Mg-based scaffold immersed in circulating Hanks' Balanced Salt Solution (HBSS) with and without in situ cyclic compression (30 N/1 Hz). The changes in morphological and mechanical properties have been studied by combining in situ high-resolution X-ray computed tomography mechanics and digital volume correlation. Although in situ cyclic compression induced acceleration of the corrosion rate, probably due to local disruption of the coating layer where fatigue microcracks were formed, no critical failures in the overall scaffold were observed, indicating that the mechanical integrity of the Mg scaffolds was preserved. Structural changes, due to the accumulation of corrosion debris between the scaffold fibres, resulted in a significant increase (p < 0.05) in the material volume fraction from 0.52 ± 0.07 to 0.47 ± 0.03 after 14 days of corrosion. However, despite an increase in fibre material loss, the accumulated corrosion products appear to have led to an increase in Young's modulus after 14 days as well as lower third principal strain (εp3) accumulation (-91000 ± 6361 µÎµ and -60093 ± 2414 µÎµ after 2 and 14 days, respectively). Therefore, this innovative Mg scaffold design and composition provide a bone replacement, capable of sustaining mechanical loads in situ during the postoperative phase allowing new bone formation to be initially supported as the scaffold resorbs.

Effect of radiation-induced damage of trabecular bone tissue evaluated using indentation and digital volume correlation.

Exposure to X-ray radiation for an extended amount of time can cause damage to the bone tissue and therefore affect its mechanical properties. Specifically, high-resolution X-ray Computed Tomography (XCT), in both synchrotron and lab-based systems, has been employed extensively for evaluating bone micro-to-nano architecture. However, to date, it is still unclear how long exposures to X-ray radiation affect the mechanical properties of trabecular bone, particularly in relation to lab-XCT systems. Indentation has been widely used to identify local mechanical properties such as hardness and elastic modulus of bone and other biological tissues. The purpose of this study is therefore, to use indentation and XCT-based investigative tools such as digital volume correlation (DVC) to assess the microdamage induced by long exposure of trabecular bone tissue to X-ray radiation and how this affects its local mechanical properties. Trabecular bone specimens were indented before and after X-ray exposures of 33 and 66 h, where variation of elastic modulus was evaluated at every stage. The resulting elastic modulus was decreased, and micro-cracks appeared in the specimens after the first long X-ray exposure and crack formation increased after the second exposure. High strain concentration around the damaged tissue exceeding 1% was also observed from DVC analysis. The outcomes of this study show the importance of designing appropriate XCT-based experiments in lab systems to avoid degradation of the bone tissue mechanical properties due to radiation and these results will help to inform future studies that require long X-ray exposure for in situ experiments or generation of reliable subject-specific computational models.

Digital volume correlation for the characterization of musculoskeletal tissues: Current challenges and future developments.

Biological tissues are complex hierarchical materials, difficult to characterise due to the challenges associated to the separation of scale and heterogeneity of the mechanical properties at different dimensional levels. The Digital Volume Correlation approach is the only image-based experimental approach that can accurately measure internal strain field within biological tissues under complex loading scenarios. In this minireview examples of DVC applications to study the deformation of musculoskeletal tissues at different dimensional scales are reported, highlighting the potential and challenges of this relatively new technique. The manuscript aims at reporting the wide breath of DVC applications in the past 2 decades and discuss future perspective for this unique technique, including fast analysis, applications on soft tissues, high precision approaches, and clinical applications.

Nonlinear micro finite element models based on digital volume correlation measurements predict early microdamage in newly formed bone.

Bone regeneration in critical-sized defects is a clinical challenge, with biomaterials under constant development aiming at enhancing the natural bone healing process. The delivery of bone morphogenetic proteins (BMPs) in appropriate carriers represents a promising strategy for bone defect treatment but optimisation of the spatial-temporal release is still needed for the regeneration of bone with biological, structural, and mechanical properties comparable to the native tissue. Nonlinear micro finite element (µFE) models can address some of these challenges by providing a tool able to predict the biomechanical strength and microdamage onset in newly formed bone when subjected to physiological or supraphysiological loads. Yet, these models need to be validated against experimental data. In this study, experimental local displacements in newly formed bone induced by osteoinductive biomaterials subjected to in situ X-ray computed tomography compression in the apparent elastic regime and measured using digital volume correlation (DVC) were used to validate µFE models. Displacement predictions from homogeneous linear µFE models were highly correlated to DVC-measured local displacements, while tissue heterogeneity capturing mineralisation differences showed negligible effects. Nonlinear µFE models improved the correlation and showed that tissue microdamage occurs at low apparent strains. Microdamage seemed to occur next to large cavities or in biomaterial-induced thin trabeculae, independent of the mineralisation. While localisation of plastic strain accumulation was similar, the amount of damage accumulated in these locations was slightly higher when including material heterogeneity. These results demonstrate the ability of the nonlinear µFE model to capture local microdamage in newly formed bone tissue and can be exploited to improve the current understanding of healing bone and mechanical competence. This will ultimately aid the development of BMPs delivery systems for bone defect treatment able to regenerate bone with optimal biological, mechanical, and structural properties.

Investigating the Fibrillar Ultrastructure and Mechanics in Keloid Scars Using In Situ Synchrotron X-ray Nanomechanical Imaging.

Fibrotic scarring is prevalent in a range of collagenous tissue disorders. Understanding the role of matrix biophysics in contributing to fibrotic progression is important to develop therapies, as well as to elucidate biological mechanisms. Here, we demonstrate how microfocus small-angle X-ray scattering (SAXS), with in situ mechanics and correlative imaging, can provide quantitative and position-resolved information on the fibrotic matrix nanostructure and its mechanical properties. We use as an example the case of keloid scarring in skin. SAXS mapping reveals heterogeneous gradients in collagen fibrillar concentration, fibril pre-strain (variations in D-period) and a new interfibrillar component likely linked to proteoglycans, indicating evidence of a complex 3D structure at the nanoscale. Furthermore, we demonstrate a proof-of-principle for a diffraction-contrast correlative imaging technique, incorporating, for the first time, DIC and SAXS, and providing an initial estimate for measuring spatially resolved fibrillar-level strain and reorientation in such heterogeneous tissues. By application of the method, we quantify (at the microscale) fibrillar reorientations, increases in fibrillar D-period variance, and increases in mean D-period under macroscopic tissue strains of ~20%. Our results open the opportunity of using synchrotron X-ray nanomechanical imaging as a quantitative tool to probe structure-function relations in keloid and other fibrotic disorders in situ.

Comparison of bone formation mediated by bone morphogenetic protein delivered by nanoclay gels with clinical techniques (autograft and InductOs®) in an ovine bone model.

Development of a growth factor delivery vehicle providing appropriate temporal-spatial release together with an appropriate preclinical large animal model to evaluate bone formation is critical in the development of delivery strategies for bone tissue regeneration. Smectite nanoclays such as LAPONITE™ possess unique thixotropic and protein retention properties offering promise for use in growth factor delivery in bone repair and regeneration. This study has examined bone formation mediated by a clinically approved growth factor delivery system (InductOs®) in combination with Laponite gel in an aged female ovine femoral condyle defect preclinical model (10 weeks). Two different designs, one containing a low volume of Laponite gel (LLG) in combination with the InductOs® absorbable collagen sponge (ACS), the other in which Laponite gel formed the implant (HLG), were compared against InductOs® alone and an autograft positive control. Thus, five groups: (i) empty defect, (ii) autograft, (iii) BMP2 + ACS, (iv) BMP2 + ACS + LLG and (v) BMP2 + HLG + ACS were examined in 9 mm × 12 mm defects performed bilaterally in the medial femoral condyles of 24 aged (>5 years) sheep. Bone formation within the defect was assessed using micro-computed tomography (micro-CT), digital volume correlation (DVC) for biomechanical characterisation as well as histology. The autograft and InductOs® mediated enhanced bone formation (p < 0001) compared to blank controls, while no significant differences were observed between the Laponite/Collagen/BMP delivery vehicles. However, the current study illustrated the excellent biocompatibility of Laponite and its ability to deliver localised active BMP-2, with the opportunity for improved efficacy with further optimisation. Interestingly, DVC-computed strain distributions indicated that the regenerated bone structure is mechanically adapted to bear external loads from the early remodelling stages of the bone reparation cascade. The current studies of selected nanoclay delivery platforms for BMP, assessed in a clinically relevant large animal model auger well for the development of bone fracture therapeutics for an ageing population.

Collagen pre-strain discontinuity at the bone-Cartilage interface.

The bone-cartilage unit (BCU) is a universal feature in diarthrodial joints, which is mechanically-graded and subjected to shear and compressive strains. Changes in the BCU have been linked to osteoarthritis (OA) progression. Here we report existence of a physiological internal strain gradient (pre-strain) across the BCU at the ultrastructural scale of the extracellular matrix (ECM) constituents, specifically the collagen fibril. We use X-ray scattering that probes changes in the axial periodicity of fibril-level D-stagger of tropocollagen molecules in the matrix fibrils, as a measure of microscopic pre-strain. We find that mineralized collagen nanofibrils in the calcified plate are in tensile pre-strain relative to the underlying trabecular bone. This behaviour contrasts with the previously accepted notion that fibrillar pre-strain (or D-stagger) in collagenous tissues always reduces with mineralization, via reduced hydration and associated swelling pressure. Within the calcified part of the BCU, a finer-scale gradient in pre-strain (0.6% increase over ~50µm) is observed. The increased fibrillar pre-strain is linked to prior research reporting large tissue-level residual strains under compression. The findings may have biomechanical adaptative significance: higher in-built molecular level resilience/damage resistance to physiological compression, and disruption of the molecular-level pre-strains during remodelling of the bone-cartilage interface may be potential factors in osteoarthritis-based degeneration.