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

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

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

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

3D Characterization of Human Nano-osseointegration by On-Axis Electron Tomography without the Missing Wedge

Three-dimensional (3D) visualization of bone-implant interfaces via electron tomography (ET) has contributed to the novel perspective of nano-osseointegration and offers evidential support for nanoscaled biomaterial surface modification. Conventional single-axis ET provides a relatively large field of view of the human bone to titanium implant interface showing bone structure arrangement near the interface. However, the “missing wedge” associated with conventional single-axis ET leads to artifacts and elongation in the reconstruction, limiting the resolution and fidelity of reconstructions, as well as the ability to extract quantitative information from nanostructured interfaces. On-axis ET, performed by 180° rotation of a needle-shaped sample, is a promising method to solve this problem. In this work, we present the first application of on-axis ET for investigation of human bone and laser-modified titanium implant interfaces without the missing wedge. This work demonstrates a near artifact-free 3D visualization of the nanotopographies of the implant surface oxide layer and bone growth into these features. Complementary electron energy-loss spectroscopy (EELS) mapping was used to illustrate the gradual intermixing of carbon and calcium (characteristic elements of bone) with the nanoscaled oxide layer of the implant surface. Ultimately, this approach serves as direct evidence of nano-osseointegration and as a potential platform to evaluate differently structured implant surfaces

3D Characterization of Human Nano-osseointegration by On-Axis Electron Tomography without the Missing Wedge

Three-dimensional (3D) visualization of bone-implant interfaces via electron tomography (ET) has contributed to the novel perspective of nano-osseointegration and offers evidential support for nanoscaled biomaterial surface modification. Conventional single-axis ET provides a relatively large field of view of the human bone to titanium implant interface showing bone structure arrangement near the interface. However, the “missing wedge” associated with conventional single-axis ET leads to artifacts and elongation in the reconstruction, limiting the resolution and fidelity of reconstructions, as well as the ability to extract quantitative information from nanostructured interfaces. On-axis ET, performed by 180° rotation of a needle-shaped sample, is a promising method to solve this problem. In this work, we present the first application of on-axis ET for investigation of human bone and laser-modified titanium implant interfaces without the missing wedge. This work demonstrates a near artifact-free 3D visualization of the nanotopographies of the implant surface oxide layer and bone growth into these features. Complementary electron energy-loss spectroscopy (EELS) mapping was used to illustrate the gradual intermixing of carbon and calcium (characteristic elements of bone) with the nanoscaled oxide layer of the implant surface. Ultimately, this approach serves as direct evidence of nano-osseointegration and as a potential platform to evaluate differently structured implant surfaces

High-Resolution Visualization of the Osteocyte Lacuno-Canalicular Network Juxtaposed to the Surface of Nanotextured Titanium Implants in Human

Osseointegration is controlled by a number of cellular mechanisms. Although factors governing bone formation are well-understood, the maintenance of bone at the bone-implant interface is less clear. Of some interest is the role of osteocytes, which via mechanotransduction are believed to be responsible for mechanical loading-based remodelling events in bone. Using a resin cast etching technique, we investigated the osteocyte lacuno-canalicular network adjacent to nanostructured titanium human dental implants after four years in clinical function. Correlative electron microscopy showed nanoscale osteocyte processes extending directly onto the implant surface. Calcium signal mapping via electron energy loss spectroscopy (EELS) showed apatite ingrowth into the nanotextured surface, while the apatite platelet <i>c</i>-axis was oriented approximately parallel to the collagen fibril direction. Furthermore, Z-contrast electron tomography demonstrated that natural bone-osteocyte and engineered bone-implant interfaces are similar in ultrastructural morphology. The present ultrastructural observation of multiple connections between osteocyte canaliculi and the nanotextured surface oxide suggests that osteocytes contribute toward the maintenance of osseointegration in long-term clinical function

Histology.

Undecalcified toluidine blue-stained sections of the (A) machined and (B) laser-modified implants. Both types of implants are vertically and unicortically positioned in the tibia. The two upper threads are located in the original cortical bone (Ct.B). Endosteal bone downgrowth (white arrows) is observed, extending downwards to the level of the third/fourth thread, whereas the remaining threads are mainly located in the bone marrow (Ma) compartment. Occasionally, periosteal bone formation (black arrows) is observed, reaching up to the implant flange yet remaining below the level of the cover screw. Bone interfacing the (C, E) machined and (D, F) laser-modified implants, at the level of the upper cortical thread (C-D) and the level of the endosteal threads (E-F). For the machined implants, a separation is frequently detected (white asterisk) between bone and implant. The laser-modified surface revealed bone in direct contact. For all laser-modified implants, fracture lines (white arrowheads) were observed in the bone within the threads at short distances (typically 30–50 μm) from the implant surface and running parallel to the implant surface in the thread valley.</p

Multiple stepwise linear regression model controlled for the confounding factors.

<p>The data are pooled for the machined and laser-modified implants (<i>n</i> = 20).</p

Histology.

<p>Undecalcified toluidine blue-stained sections of the (A, C, E, G) machined and (B, D, F, H) laser-modified implants. At the bone-implant interface, morphological features of bone formation and remodelling can be clearly observed in different threads in the cortical bone and the endosteal and bone marrow compartments. (A-B) Areas of ongoing remodelling. Osteoclasts (black arrows), osteoblasts (white arrows) and osteocytes (some of which are indicated by white arrowheads) are located in areas undergoing active remodelling at the interface. Blood vessels are indicated by black arrowheads. (C-D) For both implant types, mature bone occupies the endosteal threads, consisting of densely packed osteons (black arrows), with central blood vessels, surrounded by concentric bone lamellae and mature osteocytes. (E-F) Generally, the lower three threads of both implant types are occupied by bone marrow (Ma). Islands of newly formed, immature bone (black arrows) characterised by intense toluidine blue staining and large rounded osteocytes, which indicate an early stage of bone formation, are sometimes detected in the lower threads. This type of bone appears to be formed <i>de novo</i> and not as an extension from the endosteum. (G-H) Some of the bone marrow threads (Ma) show condensations of haematopoietic (black arrows) as well as relatively large and lightly stained mesenchymal-like cells (white arrows) adjacent to the implant surfaces.</p