Effect of plate orientation on apparent thickness of mineral plates by transmission electron microscopy.

PURPOSE: Transmission electron microscopy (TEM) is widely used to study the ultrastructure of bone. The mineral of bone occurs as polycrystalline mineral plates about 3 to 6 nm in thickness. A problem in using TEM to make quantitative analyses of bone is that the orientation of the plates with respect to the plane of the section being imaged is expected to affect their apparent thickness. The purpose of this study was to test if this was true, if the apparent thickness of plates changed substantially as a result of tilt of the section. METHODS: We prepared TEM sections of samples of cortical human bone by ion beam milling, orienting one section parallel to the collagen fibril axes and one perpendicular to them. We obtained TEM bright field and HAADF images of these sections, tilting the sections up to ± 20° at 2° intervals and measuring the apparent thickness of individual mineral platelets at each angle of tilt. RESULTS: Thickness appears to double as section is tilted ± 20°. True thickness of plates is determined by tilting the section along an axis parallel to the plate orientation and determining the minimum apparent thickness. However, as plates are tilted away from minimum-thickness orientation, they become less well-resolved, disappearing when tilted more than 20°. We therefore also measured apparent thickness of only the darkest (most electron scattering) plate images in an untilted section and obtained the same average thickness as that obtained by tilting. CONCLUSION: We conclude that tilting of the section is not necessary to obtain an accurate measurement of the thickness of mineral plates.

Tooth acellular extrinsic fibre cementum incremental lines in humans are formed by parallel branched Sharpey's fibres and not by its mineral phase.

In humans, the growth pattern of the acellular extrinsic fibre cementum (AEFC) has been useful to estimate the age-at-death. However, the structural organization behind such a pattern remains poorly understood. In this study tooth cementum from seven individuals from a Mexican modern skeletal series were analyzed with the aim of unveiling the AEFC collagenous and mineral structure using multimodal imaging approaches. The organization of collagen fibres was first determined using: light microscopy, transmission electron microscopy (TEM), electron tomography, and plasma FIB scanning electron microscopy (PFIB-SEM) tomography. The mineral properties were then investigated using: synchrotron small-angle X-ray scattering (SAXS) for T-parameter (correlation length between mineral particles); synchrotron X-ray diffraction (XRD) for L-parameter (mineral crystalline domain size estimation), alignment parameter (crystals preferred orientation) and lattice parameters a and c; as well as synchrotron X-ray fluorescence for spatial distribution of calcium, phosphorus and zinc. Results show that Sharpey's fibres branched out fibres that cover and uncover other collagen bundles forming aligned arched structures that are joined by these same fibres but in a parallel fashion. The parallel fibres are not set as a continuum on the same plane and when they are superimposed project the AEFC incremental lines due to the collagen birefringence. The orientation of the apatite crystallites is subject to the arrangement of the collagen fibres, and the obtained parameter values along with the elemental distribution maps, revealed this mineral tissue as relatively homogeneous. Therefore, no intrinsic characteristics of the mineral phase could be associated with the alternating AEFC incremental pattern.

Impact of palladium/palladium hydride conversion on electrochemical CO2 reduction via in-situ transmission electron microscopy and diffraction.

Electrochemical conversion of CO2 offers a sustainable route for producing fuels and chemicals. Pd-based catalysts are effective for converting CO2 into formate at low overpotentials and CO/H2 at high overpotentials, while undergoing poorly understood morphology and phase structure transformations under reaction conditions that impact performance. Herein, in-situ liquid-phase transmission electron microscopy and select area diffraction measurements are applied to track the morphology and Pd/PdHx phase interconversion under reaction conditions as a function of electrode potential. These studies identify the degradation mechanisms, including poisoning and physical structure changes, occurring in PdHx/Pd electrodes. Constant potential density functional theory calculations are used to probe the reaction mechanisms occurring on the PdHx structures observed under reaction conditions. Microkinetic modeling reveals that the intercalation of *H into Pd is essential for formate production. However, the change in electrochemical CO2 conversion selectivity away from formate and towards CO/H2 at increasing overpotentials is due to electrode potential dependent changes in the reaction energetics and not a consequence of morphology or phase structure changes.

Osseointegration of functionally graded Ti6Al4V porous implants: Histology of the pore network.

The additive manufacturing of titanium into porous geometries offers a means to generate low-stiffness endosseous implants with a greater surface area available for osseointegration. In this work, selective laser melting was used to produce gyroid-based scaffolds with a uniform pore size of 300 µm or functionally graded pore size from 600 µm to 300 µm. Initial in vitro assessment with Saos-2 cells showed favourable cell proliferation at pore sizes of 300 and 600 µm. Following implantation into rabbit tibiae, early histological observations at four weeks indicated some residual inflammation alongside neovessel infiltration into the scaffold interior and some early apposition of mineralized bone tissue. At twelve weeks, both scaffolds were filled with a mixture of adipocyte-rich marrow, micro-capillaries, and mineralized bone tissue. X-ray microcomputed tomography showed a higher bone volume fraction (BV/TV) and percentage of bone-implant contact (BIC) in the implants with 300 µm pores than in the functionally graded specimens. In functionally graded specimens, localized BV/TV measurement was observed to be higher in the innermost region containing smaller pores (estimated at 300-400 µm) than in larger pores at the implant exterior. The unit cell topology of the porous implant was also observed to guide the direction of bone ingrowth by conducting along the implant struts. These results suggest that in vivo experimentation is necessary alongside parametric optimization of functionally graded porous implants to predict short-term and long-term bone apposition.

Ultrastructure and Nanoporosity of Human Bone Shown with Correlative On-Axis Electron and Spectroscopic Tomographies.

Mineralized collagen fibrils are the building block units of bone at the nanoscale. While it is known that collagen fibrils are mineralized both inside their gap zones (intra-fibrillar mineralization) and on their outer surfaces (extra-fibrillar mineralization), a clear visualization of this architecture in three dimensions (3D), combining structural and compositional information over large volumes, but without compromising the resolution, remains challenging. In this study, we demonstrate the use of on-axis Z-contrast electron tomography (ET) with correlative energy-dispersive X-ray spectroscopy (EDX) tomography to examine rod-shaped samples with diameters up to 700 nm prepared from individual osteonal lamellae in the human femur. Our work mainly focuses on two aspects: (i) low-contrast nanosized circular spaces ("holes") observed in sections of bone oriented perpendicular to the long axis of a long bone, and (ii) extra-fibrillar mineral, especially in terms of morphology and spatial relationship with respect to intra-fibrillar mineral and collagen fibrils. From our analyses, it emerges quite clearly that most "holes" are cross-sectional views of collagen fibrils. While this had been postulated before, our 3D reconstructions and reslicing along meaningful two-dimensional (2D) cross-sections provide a direct visual confirmation. Extra-fibrillar mineral appears to be composed of thin plates that are interconnected and span over several collagen fibrils, confirming that mineralization is cross-fibrillar, at least for the extra-fibrillar phase. EDX tomography shows mineral signatures (Ca and P) within the gap zones, but the signal appears weaker than that associated with the extra-fibrillar mineral, pointing toward the existence of dissimilarities between the two types of mineralization.

Liquid Transmission Electron Microscopy for Probing Collagen Biomineralization.

Collagen biomineralization is fundamental to hard tissue assembly. While studied extensively, collagen mineralization processes are not fully understood, with the majority of theories derived from electron microscopy (EM) under static, dehydrated, or frozen conditions, unlike the liquid phase environment where mineralization occurs. Herein, novel liquid transmission EM (TEM) strategies are presented, in which collagen mineralization was explored in liquid for the first time via TEM. Custom thin-film enclosures were employed to visualize the mineralization of reconstituted collagen fibrils in a calcium phosphate and polyaspartic acid solution to promote intrafibrillar mineralization. TEM highlighted that at early time points precursor mineral particles attached to collagen and progressed to crystalline mineral platelets aligned with fibrils at later time points. This aligns with observations from other techniques and validates the liquid TEM approach. This work provides a new liquid imaging approach for exploring collagen biomineralization, advancing toward understanding disease pathogenesis and remineralization strategies for hard tissues.

Nanoscale implant anchorage aided by cement line deposition into titanium dioxide nanotubes.

The success of titanium dental implants relies on osseointegration, the load-bearing connection between bone tissue and the device that, in contact osteogenesis, comprises the deposition of bony cement line matrix onto the implant surface. Titanium dioxide nanotubes (NTs) are considered a promising surface for improved osseointegration, yet the mechanisms of cement line integration with such features remains elusive. Herein, we illustrate cement line deposition into NTs on the surface of titanium implants with two underlaying microstructures: a machined surface or a blasted/acid etched surface placed in the tibiae of Wistar rats. After retrieval, scanning electron microscopy of tissue reflected from the implant surface indicated minimal incursion of the cement line matrix into the NTs. To investigate this further, focused ion beam was utilized to prepare cross-sectional samples that could be characterized using scanning transmission electron microscopy. The cement line matrix covered NTs regardless of underlying microstructure, which was further confirmed by elemental analysis. In some instances, cement line infiltration into the NTs was noted, which reveals a mechanism of nanoscale anchorage. This study is the first to demonstrate cement line deposition into titanium NTs, suggesting nano-anchorage as a mechanism for the success of the NT modified surfaces in vivo.

Bone structure and composition in a hyperglycemic, obese, and leptin receptor-deficient rat: Microscale characterization of femur and calvarium.

Metabolic abnormalities, such as diabetes mellitus and obesity, can impact bone quantity and/or bone quality. In this work, we characterize bone material properties, in terms of structure and composition, in a novel rat model with congenic leptin receptor (LepR) deficiency, severe obesity, and hyperglycemia (type 2 diabetes-like condition). Femurs and calvaria (parietal region) from 20-week-old male rats are examined to probe bones formed both by endochondral and intramembranous ossification. Compared to the healthy controls, the LepR-deficient animals display significant alterations in femur microarchitecture and in calvarium morphology when analyzed by micro-computed X-ray tomography (micro-CT). In particular, shorter femurs with reduced bone volume, combined with thinner parietal bones and shorter sagittal suture, point towards a delay in the skeletal development of the LepR-deficient rodents. On the other hand, LepR-deficient animals and healthy controls display analogous bone matrix composition, which is assessed in terms of tissue mineral density by micro-CT, degree of mineralization by quantitative backscattered electron imaging, and various metrics extrapolated from Raman hyperspectral images. Some specific microstructural features, i.e., mineralized cartilage islands in the femurs and hyper-mineralized areas in the parietal bones, also show comparable distribution and characteristics in both groups. Overall, the altered bone microarchitecture in the LepR-deficient animals indicates compromised bone quality, despite the normal bone matrix composition. The delayed development is also consistent with observations in humans with congenic Lep/LepR deficiency, making this animal model a suitable candidate for translational research.

Functionalization of 3D Printed Scaffolds Using Polydopamine and Silver Nanoparticles for Bone-Interfacing Applications.

The prevention of bacterial colonization and the stimulation of osseointegration are two major requirements for bone-interfacing materials to reduce the incidence of complications and promote the restoration of the patient's health. The present investigation developed an effective, two-step functionalization of 3D printed scaffolds intended for bone-interfacing applications using a simple polydopamine (PDA) dip-coating method followed by the formation of silver nanoparticles (AgNPs) after a second coating step in silver nitrate. 3D printed polymeric substrates coated with a ∼20 nm PDA layer and 70 nm diameter AgNPs proved effective in hindering Staphylococcus aureus biofilm formation, with a 3000-8000-fold reduction in the number of bacterial colonies formed. The implementation of porous geometries significantly accelerated osteoblast-like cell growth. Microscopy characterization further elucidated homogeneity, features, and penetration of the coating inside the scaffold. A proof-of-concept coating on titanium substrates attests to the transferability of the method to other materials, broadening the range of applications both in and outside the medical sector. The antibacterial efficiency of the coating is likely to lead to a decrease in the number of bacterial infections developed after surgery in the presence of these coatings on prosthetics, thus translating to a reduction in revision surgeries and improved health outcomes.

Evaluation of Vitamin D isolated or Associated with Teriparatide in Peri-Implant Bone Repair in Tibia of Orchiectomized Rats.

(1) Background: The objective of this study was to evaluate the morphometry of peri-implant bone tissue in orchiectomized rats, treated with vitamin D isolated or associated with teriparatide. (2) Methods: 24 rats were divided into 4 groups: ORQ-orchiectomy, without drug treatment, ORQ+D-orchiectomy, treated with vitamin D, ORQTERI-orchiectomy, treated with teriparatide and ORQTERI+D-orchiectomy, treated with teriparatide + vitamin D. Each animal received an implant in the tibial metaphysis. Euthanasia occurred 60 days after implant surgery. Computed microtomography (micro-CT) was performed to evaluate the parameters of volume and percentage of bone volume (BV, BV/TV), trabecular thickness (Tb.Th), number and separation of trabeculae (Tb.N, Tb.Sp) and percentage of total porosity (Po-tot). Data were subjected to 1-way ANOVA and Tukey post-test, with a significance level of 5%. (3) Results: For the parameters BV, BV/TV, Tb.Th, the ORQTERI+D group showed the highest values in relation to the other groups and for Po-tot, the lowest values were for ORQTERI+D. For Tb.Sp and Tb.N, there was no statistically significant difference when comparing intragroup results (p > 0.05). (4) Conclusions: It was possible to conclude that treatment with vitamin D associated with teriparatide increases bone volume and improves bone quality.

Unraveling the Supramolecular Structure and Nanoscale Dislocations of Bacterial Cellulose Ribbons Using Correlative Super-Resolution Light and Electron Microscopy.

Cellulose is a structural linear polysaccharide that is naturally produced by plants and bacteria, making it the most abundant biopolymer on Earth. The hierarchical structure of cellulose from the nano- to microscale is intimately linked to its biosynthesis and the ability to process this sustainable resource for materials applications. Despite this, the morphology of bacterial cellulose microfibrils and their assembly into higher order structures, as well as the structural origins of the alternating crystalline and disordered supramolecular structure of cellulose, have remained elusive. In this work, we employed high-resolution transmission electron and atomic force microscopies to study the morphology of bacterial cellulose ribbons at different levels of its structural hierarchy and provide direct visualization of nanometer-wide microfibrils. The non-persistent twisting of cellulose ribbons was characterized in detail, and we found that twists are associated with nanostructural defects at the bundle and microfibril levels. To investigate the structural origins of the persistent disordered regions that are present along cellulose ribbons, we employed a correlative super-resolution light and electron microscopy workflow and observed that the disordered regions that can be seen in super-resolution fluorescence microscopy largely correlated with the ribbon twisting observed in electron microscopy. Unraveling the hierarchical assembly of bacterial cellulose and the ultrastructural basis of its disordered regions provides insights into its biosynthesis and susceptibility to hydrolysis. These findings are important to understand the cell-directed assembly of cellulose, develop new cellulose-based nanomaterials, and develop more efficient biomass conversion strategies.

Multimodal and Multiscale Characterization of the Bone-Bacteria Interface in a Case of Medication-Related Osteonecrosis of the Jaw.

Medication-related osteonecrosis of the jaw (MRONJ) is a known side effect of bisphosphonates (BPs). Although bacterial infection is usually present, the etiology of MRONJ remains unknown. Here we apply a multimodal and multiscale (micro-to-nano) characterization approach to investigate the interface between necrotic bone and bacteria in MRONJ. A non-necrotic bone sample was used as control. Both necrotic and non-necrotic bone samples were collected from the jaw of a female individual affected by MRONJ after using BPs for 23 years. For the first time, resin cast etching was used to expose bacteria at the necrotic site. The bone-bacteria interface was also resolved at the nanoscale by scanning transmission electron microscopy (STEM). Nanosized particulates, likely corresponding to degraded bone mineral, were often noted in close proximity to or enclosed by the bacteria. STEM also revealed that the bone-bacteria interface is composed of a hypermineralized front fading into a highly disordered region, with decreasing content of calcium and phosphorus, as assessed by electron energy loss spectroscopy (EELS). This, combined with the variation in calcium, phosphorus, and carbon across the necrotic bone-bacteria interface evaluated by scanning electron microscopy (SEM)-energy dispersive X-ray spectroscopy (EDX) and the lower mineral-to-matrix ratio measured by micro-Raman spectroscopy in necrotic bone, indicates the absence of a mineralization front in MRONJ. It appears that the bone-bacteria interface originates not only from uncontrolled mineralization but also from the direct action of bacteria degrading the bone matrix. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Fabrication of succinate-alginate xerogel films for in vitro coupling of osteogenesis and neovascularization.

The osseointegration of metallic implants is reliant on a cascade of molecular interactions and the delivery of macromolecules to the implant environment that occurs before substantial bone formation. Early blood vessel formation is a requisite first step in the healing timeline for osteoid formation, where vascular development can be accelerated as a result of controlled hypoxic conditioning. In this study, alginate-derived xerogel films containing varied concentrations of disodium succinate salt which has been shown to induce pseudohypoxia (short-term hypoxic effects while maintaining an oxygenated environment) were developed. Xerogels were characterized for their morphology, succinate release over time and cellular response with osteoblast-mimicking Saos-2 and human umbilical vein endothelial cells (HUVEC). Scanning electron microscopy revealed a multiscale topography that may favour osseointegration and alamarBlue assays indicated no cytotoxic effects during in vitro proliferation of Saos-2 cells. pH measurements of eluted succinate reach 95 % of peak value after 7 h of immersion for all gels containing 10 mM of succinate or less, and 60 % within the first 40 min. In vitro exposure of HUVECs to succinate-conditioned media increased the net concentration of total proteins measured by bicinchoninic acid (BCA) assay and maintains stable vascular endothelial growth factor (VEGF) and extracellular platelet-derived growth factor (PDGF) for vessel formation through comparison of enzyme-linked immunosorbent assays (ELISAs) of the culture media and cell lysate. Tube formation assays also showed a sustained increase in tube diameter across the first 48 h of HUVEC culture when succinate concentrations of 1 and 10 µM in the xerogel. Overall, the succinate-alginate films serve as a prospective organic coating for bone-interfacing implant materials which may induce temporary pseudohypoxic conditions favourable for early angiogenesis and bone regeneration in vivo at succinate concentrations of 1 or 10 µM.

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice.

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-Å-10 Å. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

Atom probe tomography for biomaterials and biomineralization.

Biominerals and biomaterials are part of our daily lives, from our skeleton and teeth to coral reefs and carbon-capturing single-cell organisms in the oceans, to engineered ceramics comprising our toothpaste and bone replacements. Many biominerals are hierarchically structured with remarkable material properties that arise from their unique combination of organic and inorganic components. Such structural hierarchy is often formed through a process of biomineralization. However, many fundamental questions remain regarding mineralization events in bones or teeth, and near biomaterials, partly due to the challenges in characterizing three-dimensional (3D) structure and chemical composition simultaneously at the nanometer scale. Atom probe tomography (APT) is a 3D characterization technique that combines both sub-nanometer spatial resolution and compositional sensitivity down to tens of parts per million. While APT is well-established in application to conventional engineering materials, recent years have seen its expansion into biomineralization research. Here, we focus our review on APT applications to biominerals, biomaterials and biointerfaces, providing a high-level summary of findings, as well as a primer on theory and best practices specific to the biomineralization community. We show that APT is a promising characterization tool, where its unique ability to quantify 3D chemical composition is not only complementary to other microscopy techniques but could become an integral part of biomaterial research. With the emerging trends of correlative and cryogenic workflow, notwithstanding the challenges outlined herein, APT has the potential to improve understanding of a broader range of biomaterials, while deriving innovative perspectives on clinical applications and strategies for biomaterial design. STATEMENT OF SIGNIFICANCE: Atom probe tomography (APT) is a three-dimensional characterization technique that can provide quantitative elemental and isotopic analysis with sub-nanometer resolution and compositional sensitivity down to tens of parts per million. These capabilities make it uniquely positioned for the analysis of biomineralized materials, both natural and synthetic. Here, we review the various applications of APT to the field of biomineralization, including applications in biominerals, biomaterials, biointerfaces and other biological materials, such as cells or proteins. A brief but comprehensive summary of the relevant technical concepts, limitations, and future perspectives to enable growth in this field are also included. Although APT is relatively new to the field of biomineralization, it has shown the potential to transform our basic understanding of biomineralization mechanisms and better inform biomaterials design.

PTH 1-34-functionalized bioactive glass improves peri-implant bone repair in orchiectomized rats: Microscale and ultrastructural evaluation.

The objective of this work was to investigate the use of Biogran® functionalized with parathyroid hormone (PTH) 1-34 by sonochemistry for the local delivery of this anabolic agent to the implant site. The effects of Biogran® and topical administration of PTH 1-34 on peri-implant bone regeneration were evaluated from the microscale to ultrastructural levels in healthy (SHAM) and orchiectomized (ORQ). While some animals only received a titanium implant in their tibial metaphyses (CLOT group), in others the peri-implant defect was first filled with Biogran® either without or with PTH 1-34 functionalization (BG and BGPTH groups, respectively) prior to implant installation. Osseointegration was characterized from a biomechanical perspective by measuring the removal torque with the counter-torque technique. Micro-CT was used to evaluate the percentage of bone volume, trabecular thickness, number and separation, and bone-implant contact (BIC). Dynamics of new bone formation were assessed by measuring fluorochrome area, daily mineral apposition rate, and neoformed bone area using confocal laser microscopy. RT-PCR was performed to evaluate ALP and osteocalcin expression. The interface between newly formed bone and Biogran® was examined using scanning electron microscopy (SEM) and scanning transmission electron microscopy (STEM) at the micro-and nanoscale, respectively, while elemental analyses were completed in SEM with energy-dispersive X-ray spectroscopy (EDS). STEM imaging demonstrated the intimate attachment of bone to Biogran® (nanoscale level). Overall, the results suggest that the effectiveness of the topical administration of PTH 1-34 at the implant site seems enhanced in osteoporotic bone, promoting peri-implant bone regeneration to comparable levels in healthy conditions.

Bone mineral organization at the mesoscale: A review of mineral ellipsoids in bone and at bone interfaces.

Much debate still revolves around bone architecture, especially at the nano- and microscale. Bone is a remarkable material where high strength and toughness coexist thanks to an optimized composition of mineral and protein and their hierarchical organization across several distinct length scales. At the nanoscale, mineralized collagen fibrils act as building block units. Despite their key role in biological and mechanical functions, the mechanisms of collagen mineralization and the precise arrangement of the organic and inorganic constituents in the fibrils remains not fully elucidated. Advances in three-dimensional (3D) characterization of mineralized bone tissue by focused ion beam-scanning electron microscopy (FIB-SEM) revealed mineral-rich regions geometrically approximated as prolate ellipsoids, much larger than single collagen fibrils. These structures have yet to become prominently recognized, studied, or adopted into biomechanical models of bone. However, they closely resemble the circular to elliptical features previously identified by scanning transmission electron microscopy (STEM) in two-dimensions (2D). Herein, we review the presence of mineral ellipsoids in bone as observed with electron-based imaging techniques in both 2D and 3D with particular focus on different species, anatomical locations, and in proximity to natural and synthetic biomaterial interfaces. This review reveals that mineral ellipsoids are a ubiquitous structure in all the bones and bone-implant interfaces analyzed. This largely overlooked hierarchical level is expected to bring different perspectives to our understanding of bone mineralization and mechanical properties, in turn shedding light on structure-function relationships in bone. STATEMENT OF SIGNIFICANCE: In bone, the hierarchical organization of organic (mainly collagen type I) and inorganic (calcium-phosphate mineral) components across several length scales contributes to a unique combination of strength and toughness. However, aspects related to the collagen-mineral organization and to mineralization mechanisms remain unclear. Here, we review the presence of mineral prolate ellipsoids across a variety of species, anatomical locations, and interfaces, both natural and with synthetic biomaterials. These mineral ellipsoids represent a largely unstudied feature in the organization of bone at the mesoscale, i.e., at a level connecting nano- and microscale. Thorough understanding of their origin, development, and structure can provide valuable insights into bone architecture and mineralization, assisting the treatment of bone diseases and the design of bio-inspired materials.

Ionic liquid treatment for efficient sample preparation of hydrated bone for scanning electron microscopy.

This study presents a new protocol for preparing bone samples for scanning electron microscopy (SEM) using a room temperature ionic liquid (RTIL) treatment method. RTIL-based solutions can be adopted as an alternative to lengthy and laborious traditional means of preparation for SEM due to their unique low-vapour pressure and conductive properties. Applied to biological samples, RTILs can be used quickly and efficiently to observe hydrated, unfixed structures in typical SEM systems. This first-time feasibility study of the optimization of this protocol for bone was explored through various SEM modalities using two distinct ionic liquids, 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMI][BF4]) and 1-butyl-3-methyl imidazolium tetrafluoroborate ([BMI][BF4]), at varying concentrations of 5, 10, and 25 % v/v in aqueous solution through an addition-based method. Based on qualitative observations in the SEM, a 60-second solution addition treatment of 10 % v/v [BMI][BF4] performed the best in imaging hydrated, unfixed bone samples, resulting in minimal charge buildup and no solution pooling on the surface. The treatment was applied effectively to a variety of bone samples, notably flat and polished, as well as highly topographical bone fracture surfaces of both healthy and osteoporotic human bone samples. In comparison to conventionally dehydrated bone, the RTIL treatment better preserved the natural bone structure, resulting in minimal microcracking in observed structures.

Resonance vibration interventions in the femur: Experimental-numerical modelling approaches.

MOTIVE: External vibration excitation might be key to many novel non-surgical interventions for pathologies in the musculoskeletal system and in other parts of the human organism. Lack of understanding about vibration patterns, their controllability, and reproducibility are three limitations of ongoing research. This study establishes a bovine vibration model and animal model replacements for future research. METHODS: We used biological samples (n=5) and one polyurethane sample of the bovine femur. Mechanical resonance was measured experimentally and analysed numerically by finite element method. MAIN RESULTS: The experiments obtained 5 distinct mode shapes for the biological sample set, with standard deviation < 7.5%. Finite element analysis of the biological samples can replicate experimental mode shape deflection. The use of polyurethane changes resonance character but results are also good approximations of the biological samples. CONCLUSIONS: A model of the bovine femur with consistent resonance behaviour is presented with alternatives (polyurethane and finite element analysis) that can serve in reducing the number of necessary biological samples. Future work will be to adapt results to human anatomy. Of clinical interest will be to influence bone pathologies such as post-surgical non-union, or bone functionality as part of haematopoiesis and endocrine secretion.