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.

Three-Dimensional Insights into Interfacial Segregation at the Atomic Scale in a Nanocrystalline Glass-Ceramic.

The distribution of dopant atoms plays a key role in the effectiveness of doping, thereby requiring delicate characterizations. In this study, we found that energy-dispersive X-ray spectroscopy (EDX) and electron energy loss spectroscopy (EELS) techniques in scanning transmission electron microscopy (STEM) were not adequate to reveal the distribution of yttrium and the chemical composition of the ZrO2/SiO2 heterophase interface in an yttrium-doped ZrO2-SiO2 nanocrystalline glass-ceramic. Atom probe tomography (APT) is rarely utilized to characterize ceramics due to some inherent difficulties. However, we successfully revealed the three-dimensional distribution of ZrO2 nanocrystallites and SiO2 matrix at the atomic scale with APT under optimized and well-controlled conditions. We also found that the ZrO2 nanocrystallites had a special core-shell structure, with a thin Zr/Si interfacial layer as a shell and a ZrO2 solid solution as a core. Yttrium dopants showed interfacial segregation at both ZrO2 grain boundaries and the ZrO2/SiO2 heterophase interfaces.

Ti-5Al-5Mo-5V-3Cr bone implants with dual-scale topography: a promising alternative to Ti-6Al-4V.

Modifications to the compositional, topographical and morphological aspects of bone implants can lead to improved osseointegration, thus increasing the success of bone implant procedures. This study investigates the creation of dual-scale topography on Ti-5Al-5Mo-5V-3Cr (Ti5553), an alloy not presently used in the biomedical field, and compares it to Ti-6Al-4V (Ti64), the most used Ti alloy for bone implants. Dual-scale surface topography was obtained by combining selective laser melting (SLM) and electrochemical anodization, which resulted in micro- and nanoscale surface features, respectively. Ti5553 and Ti64 samples were manufactured by SLM and showed comparable surface topography. Subsequent electrochemical anodization succeeded in forming titania nanotubes (TNTs) on both alloys, with larger nanotubes obtained with Ti5553 at all investigated anodization voltages. At an anodization voltage of 40 V, a minimum time of 20 min was necessary to have nanotube formation on the surface of either alloy, while only nanopores were evident for shorter times. Seeded Saos-2 cells showed ideal interactions with surface-modified structures, with filopodia extending to both surface microparticles characteristic of SLM and to the interior of TNTs. Attractiveness of Ti5553 lies in its lower elastic modulus (E = 72 GPa) compared to Ti64, which should mitigate stress-shielding phenomena in vivo. This, combined with the analogous results obtained in terms of dual-scale surface topography and cell-substrate interaction, could indicate Ti5553 as a promising alternative to the widely-employed Ti64 for bone implant device manufacturing.

Bone hierarchical organization through the lens of materials science: Present opportunities and future challenges.

Multiscale characterization is essential to better understand the hierarchical architecture of bone and an array of analytical methods contributes to exploring the various structural and compositional aspects. Incorporating X-ray tomography, X-ray scattering, vibrational spectroscopy, and atom probe tomography alongside electron microscopy provides a comprehensive approach, offering insights into the diverse levels of organization within bone. X-ray scattering techniques reveal information about collagen-mineral spatial relationships, while X-ray tomography captures 3D structural details, especially at the microscale. Electron microscopy, such as scanning and transmission electron microscopy, extends resolution to the nanoscale, showcasing intricate features such as collagen fibril organization. Additionally, atom probe tomography achieves sub-nanoscale resolution and high chemical sensitivity, enabling detailed examination of bone composition. Despite various technical challenges, a correlative approach allows for a comprehensive understanding of bone material properties. Real-time investigations through in situ and in operando approaches shed light on the dynamic processes in bone. Recently developed techniques such as liquid, in situ transmission electron microscopy provide insights into calcium phosphate formation and collagen mineralization. Mechanical models developed in the effort to link structure, composition, and function currently remain oversimplified but can be improved. In conclusion, correlative analytical platforms provide a holistic perspective of bone extracellular matrix and are essential for unraveling the intricate interplay between structure and composition within bone.

Mesoscale characterization of osseointegration around an additively manufactured genistein-coated implant.

Given the hierarchical nature of bone and bone interfaces, osseointegration, namely the formation of a direct bone-implant contact, is best evaluated using a multiscale approach. However, a trade-off exists between field of view and spatial resolution, making it challenging to image large volumes with high resolution. In this study, we combine established electron microscopy techniques to probe bone-implant interfaces at the microscale and nanoscale with plasma focused ion beam-scanning electron microscopy (PFIB-SEM) tomography to evaluate osseointegration at the mesoscale. This characterization workflow is demonstrated for bone response to an additively manufactured Ti-6Al-4V implant which combines engineered porosity to facilitate bone ingrowth and surface functionalization via genistein, a phytoestrogen, to counteract bone loss in osteoporosis. SEM demonstrated new bone formation at the implant site, including in the internal implant pores. At the nanoscale, scanning transmission electron microscopy and energy-dispersive X-ray spectroscopy confirmed the gradual nature of the bone-implant interface. By leveraging mesoscale analysis with PFIB-SEM tomography that captures large volumes of bone-implant interface with nearly nanoscale resolution, the presence of mineral ellipsoids varying in size and orientation was revealed. In addition, a well-developed lacuno-canalicular network and mineralization fronts directed both towards the implant and away from it were highlighted.

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.

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.

Bone without borders - Monetite-based calcium phosphate guides bone formation beyond the skeletal envelope.

Calcium phosphates (CaP) represent an important class of osteoconductive and osteoinductive biomaterials. As proof-of-concept, we show how a multi-component CaP formulation (monetite, beta-tricalcium phosphate, and calcium pyrophosphate) guides osteogenesis beyond the physiological envelope. In a sheep model, hollow dome-shaped constructs were placed directly over the occipital bone. At 12 months, large amounts of bone (∼75%) occupy the hollow space with strong evidence of ongoing remodelling. Features of both compact bone (osteonal/osteon-like arrangements) and spongy bone (trabeculae separated by marrow cavities) reveal insights into function/need-driven microstructural adaptation. Pores within the CaP also contain both woven bone and vascularised lamellar bone. Osteoclasts actively contribute to CaP degradation/removal. Of the constituent phases, only calcium pyrophosphate persists within osseous (cutting cones) and non-osseous (macrophages) sites. From a translational perspective, this multi-component CaP opens up exciting new avenues for osteotomy-free and minimally-invasive repair of large bone defects and augmentation of the dental alveolar ridge.

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.

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.

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.

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.

From tissue retrieval to electron tomography: nanoscale characterization of the interface between bone and bioactive glass.

The success of biomaterials for bone regeneration relies on many factors, among which osseointegration plays a key role. Biogran (BG) is a bioactive glass commonly employed as a bone graft in dental procedures. Despite its use in clinical practice, the capability of BG to promote osseointegration has never been resolved at the nanoscale. In this paper, we present the workflow for characterizing the interface between newly formed bone and BG in a preclinical rat model. Areas of bone-BG contact were first identified by backscattered electron imaging in a scanning electron microscope. A focused ion beam in situ lift-out protocol was employed to prepare ultrathin samples for transmission electron microscopy analysis. The bone-BG gradual interface, i.e. the biointerphase, was visualized at the nanoscale with unprecedented resolution thanks to scanning transmission electron microscopy. Finally, we present a method to view the bone-BG interface in three dimensions using electron tomography.