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Acta Biomaterialia Sep 2023The development of treatment strategies for skeletal diseases relies on the understanding of bone mechanical properties in relation to its structure at different length...
The development of treatment strategies for skeletal diseases relies on the understanding of bone mechanical properties in relation to its structure at different length scales. At the microscale, indention techniques can be used to evaluate the elastic, plastic, and fracture behaviour of bone tissue. Here, we combined in situ high-resolution SRµCT indentation testing and digital volume correlation to elucidate the anisotropic crack propagation, deformation, and fracture of ovine cortical bone under Berkovich and spherical tips. Independently of the indenter type we observed significant dependence of the crack development due to the anisotropy ahead of the tip, with lower strains and smaller crack systems developing in samples indented in the transverse material direction, where the fibrillar bone ultrastructure is largely aligned perpendicular to the indentation direction. Such alignment allows to accommodate the strain energy, inhibiting crack propagation. Higher tensile hoop strains generally correlated with regions that display significant cracking radial to the indenter, indicating a predominant Mode I fracture. This was confirmed by the three-dimensional analysis of crack opening displacements and stress intensity factors along the crack front obtained for the first time from full displacement fields in bone tissue. The X-ray beam significantly influenced the relaxation behaviour independent of the tip. Raman analyses did not show significant changes in specimen composition after irradiation compared to non-irradiated tissue, suggesting an embrittlement process that may be linked to damage of the non-fibrillar organic matrix. This study highlights the importance of three-dimensional investigation of bone deformation and fracture behaviour to explore the mechanisms of bone failure in relation to structural changes due to ageing or disease. STATEMENT OF SIGNIFICANCE: Characterising the three-dimensional deformation and fracture behaviour of bone remains essential to decipher the interplay between structure, function, and composition with the aim to improve fracture prevention strategies. The experimental methodology presented here, combining high-resolution imaging, indentation testing and digital volume correlation, allows us to quantify the local deformation, crack propagation, and fracture modes of cortical bone tissue. Our results highlight the anisotropic behaviour of osteonal bone and the complex crack propagation patterns and fracture modes initiating by the intricate stress states beneath the indenter tip. This is of wide interest not only for the understanding of bone fracture but also to understand other architectured (bio)structures providing an effective way to quantify their toughening mechanisms in relation to their main mechanical function.
Topics: Sheep; Animals; Synchrotrons; Anisotropy; Bone and Bones; Cortical Bone; Fractures, Bone; Stress, Mechanical
PubMed: 37127075
DOI: 10.1016/j.actbio.2023.04.038 -
Biology Apr 2023Cortical bone microstructure assessment in biological and forensic anthropology can assist with the estimation of age-at-death and animal-human differentiation, for...
Cortical bone microstructure assessment in biological and forensic anthropology can assist with the estimation of age-at-death and animal-human differentiation, for example. Osteonal structures within cortical bone are the key feature under analysis, with osteon frequency and metric parameters providing crucial information for the assessment. Currently, the histomorphological assessment consists of a time-consuming manual process for which specific training is required. Our work investigates the feasibility of automatic analysis of human bone microstructure images through the application of deep learning. In this paper, we use a U-Net architecture to address the semantic segmentation of such images into three classes: intact osteons, fragmentary osteons, and background. Data augmentation was used to avoid overfitting. We evaluated our fully automatic approach using a sample of 99 microphotographs. The contours of intact and fragmentary osteons were traced manually to provide ground truth. The Dice coefficients were 0.73 for intact osteons, 0.38 for fragmented osteons, and 0.81 for background, giving an average of 0.64. The Dice coefficient of the binary classification osteon-background was 0.82. Although further refinement of the initial model and tests with larger datasets are needed, this study provides, to the best of our knowledge, the first proof of concept for the use of computer vision and deep learning for differentiating both intact and fragmentary osteons in human cortical bone. This approach has the potential to widen and facilitate the use of histomorphological assessment in the biological and forensic anthropology communities.
PubMed: 37106819
DOI: 10.3390/biology12040619 -
Frontiers in Bioengineering and... 2023Osteons are composed of concentric lamellar structure, the concentric lamellae are composed of periodic thin and thick sub-lamellae, and every 5 sub-lamellae is a cycle,...
Osteons are composed of concentric lamellar structure, the concentric lamellae are composed of periodic thin and thick sub-lamellae, and every 5 sub-lamellae is a cycle, the periodic helix angle of mineralized collagen fibers in two adjacent sub-lamellae is 30°. Four biomimetic models with different fiber helix angles were established and fabricated according to the micro-nano structure of osteon. The effects of the fiber periodic helical structure on impact characteristic and energy dissipation of multi-layer biomimetic composite were investigated. The calculation results indicated that the stress distribution, contact characteristics and fiber failur during impact, and energy dissipation of the composite are affected by the fiber helix angle. The stress concentration of composite materials under external impact can be effectively improved by adjusting the fiber helix angle when the material composition and material performance parameters are same. Compared with the sample30, the maximum stress of sample60 and sample90 increases by 38.1% and 69.8%, respectively. And the fiber failure analysis results shown that the model with a fiber helix angle of 30° has a better resist impact damage. The drop-weight test results shown that the impact damage area of the specimen with 30° helix angle is smallest among the four types of biomimetic specimens. The periodic helical structure of mineralized collagen fibers in osteon can effectively improve the impact resistance of cortical bone. The research results can provide useful guidance for the design and manufacture of high-performance, impact-resistant biomimetic composite materials.
PubMed: 37091332
DOI: 10.3389/fbioe.2023.999137 -
Hua Xi Kou Qiang Yi Xue Za Zhi = Huaxi... Apr 2023This study aimed to investigate the effect of new biomimetic micro/nano surfaces on the osteoclastic differentiation of RAW264.7 macrophages by simulating natural...
OBJECTIVES
This study aimed to investigate the effect of new biomimetic micro/nano surfaces on the osteoclastic differentiation of RAW264.7 macrophages by simulating natural osteons for the design of concentric circular structures and modifying graphene oxide (GO).
METHODS
The groups were divided into smooth titanium surface group (SS), concentric microgrooved titanium surface group (CMS), and microgroove modified with GO group (GO-CMS). The physicochemical properties of the material surfaces were studied using scanning electron microscopy (SEM), contact-angle measurement, atomic force microscopy, X-ray photoelectron spectroscopy analysis, and Raman spectroscopy. The effect of the modified material surface on the cell biological behavior of RAW264.7 was investigated by cell-activity assay, SEM, and laser confocal microscopy. The effect on the osteoclastic differentiation of macrophages was investiga-ted by tartrate-resistant acid phosphatase (TRAP) immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR) experiments.
RESULTS
Macrophages were arranged in concentric circles along the microgrooves, and after modification with GO, the oxygen-containing groups on the surface of the material increased and hydrophilicity increased. Osteoclasts in the GO-CMS group were small in size and number and had the lowest TRAP expression. Although it promoted the proliferation of macrophages in the GO-CMS group, the expression of osteoclastic differentiation-related genes was lower than that in the SS group, and the difference was statistically significant (<0.05).
CONCLUSIONS
Concentric circular microgrooves restricted the fusion of osteoclasts and the formation of sealing zones. Osteomimetic concentric microgrooves modified with GO inhibited the osteoclastic differentiation of RAW 264.7 macrophages.
Topics: Graphite; Titanium; Haversian System; Macrophages; Cell Differentiation; Oxides; Surface Properties
PubMed: 37056182
DOI: 10.7518/hxkq.2023.2022354 -
Journal of Synchrotron Radiation May 2023Palaeohistological analysis has numerous applications in understanding the palaeobiology of extinct dinosaurs. Recent developments of synchrotron-radiation-based X-ray...
Palaeohistological analysis has numerous applications in understanding the palaeobiology of extinct dinosaurs. Recent developments of synchrotron-radiation-based X-ray micro-tomography (SXMT) have allowed the non-destructive assessment of palaeohistological features in fossil skeletons. Yet, the application of the technique has been limited to specimens on the millimetre to micrometre scale because its high-resolution capacity has been obtained at the expense of a small field of view and low X-ray energy. Here, SXMT analyses of dinosaur bones with widths measuring ∼3 cm under a voxel size of ∼4 µm at beamline BL28B2 at SPring-8 (Hyogo, Japan) are reported, and the advantages of virtual-palaeohistological analyses with large field of view and high X-ray energy are explored. The analyses provide virtual thin-sections visualizing palaeohistological features comparable with those obtained by traditional palaeohistology. Namely, vascular canals, secondary osteons and lines of arrested growth are visible in the tomography images, while osteocyte lacunae are unobservable due to their micrometre-scale diameter. Virtual palaeohistology at BL28B2 is advantageous in being non-destructive, allowing multiple sampling within and across skeletal elements to exhaustively test the skeletal maturity of an animal. Continued SXMT experiments at SPring-8 should facilitate the development of SXMT experimental procedures and aid in understanding the paleobiology of extinct dinosaurs.
Topics: Animals; Fossils; Dinosaurs; Synchrotrons; X-Rays; X-Ray Microtomography
PubMed: 37026390
DOI: 10.1107/S1600577523001790 -
Biology Mar 2023In forensic anthropology, the differential diagnosis between peri- and postmortem bone fractures is mainly based on macroscopic criteria. In contrast, studies focusing...
In forensic anthropology, the differential diagnosis between peri- and postmortem bone fractures is mainly based on macroscopic criteria. In contrast, studies focusing on bone histology are very scarce. In a recent publication, we showed that (perimortem) fractures in fresh human bones exhibit a different osteonal microcracking pattern than (postmortem) damage in dry bones. In the current work, we explored whether this osteonal microcracking pattern is distinctive of the vitality of (perimortem) fresh bone fractures. To this end, we compared the number, length and structural distribution of microcracks in vital humeral fractures from forensic autopsy cases with experimentally reproduced, three point-bending fractures in fresh and dry human humeri. Half of the fresh experimental bones were fractured whilst applying axial compression, i.e., attempting to simulate intra vitam conditions more accurately. The results showed a similar osteonal microcracking pattern between vital fractures and experimental fractures of fresh humeri subjected to axial compression. Interestingly, this pattern was significantly different from the one observed in the experimental fractures of fresh humeri without axial compression and dry humeri. This supports our hypothesis that the osteonal microcracking pattern can potentially be used as a marker for vital perimortem trauma, providing a histomorphometric tool for fracture timing.
PubMed: 36979091
DOI: 10.3390/biology12030399 -
Royal Society Open Science Mar 2023Here we examine the tibial microstructure of modern and fossil angulate tortoises to assess the histology and growth from the late Miocene-early Pliocene, Pleistocene...
Here we examine the tibial microstructure of modern and fossil angulate tortoises to assess the histology and growth from the late Miocene-early Pliocene, Pleistocene through to modern forms. The cross-sections of all the tibiae sampled revealed highly vascularized, uninterrupted, fibrolamellar bone tissue during early ontogeny, which suggests that early growth was fast. However, later in ontogeny, growth was slower, as indicated by the deposition of parallel-fibred bone tissue in the outer cortex, and even ceased periodically, as indicated by lines of arrested growth. Comparative analyses of the growth rates of the tortoises from different time periods showed that the tortoises from the late Miocene-early Pliocene Langebaanweg locality and from Diepkloof Rock Shelter had relatively slower growth rates under less optimal growth conditions. Additionally, these prehistoric specimens show extensive remodelling, and several generations of secondary osteons further suggest functional and/or metabolic stresses on the skeleton. Palaeoenvironmental reconstructions suggest that it was mostly cooler and drier with seasonal fluctuations in late Miocene-early Pliocene, and it is likely that responded to these conditions by having a lower rate of growth as compared with their modern counterparts, which thrive in the current prevailing more favourable Mediterranean type of climate.
PubMed: 36908987
DOI: 10.1098/rsos.230064 -
International Journal of Molecular... Mar 2023The repair of orthopedic and maxillofacial defects in modern medicine currently relies heavily on the use of autograft, allograft, void fillers, or other structural...
The repair of orthopedic and maxillofacial defects in modern medicine currently relies heavily on the use of autograft, allograft, void fillers, or other structural material composites. This study examines the in vitro osteo regenerative potential of polycaprolactone (PCL) tissue scaffolding, fabricated via a three-dimensional (3D) additive manufacturing technology, i.e., a pneumatic micro extrusion (PME) process. The objectives of this study were: (i) To examine the innate osteoinductive and osteoconductive potential of 3D-printed PCL tissue scaffolding and (ii) To perform a direct in vitro comparison of 3D-printed PCL scaffolding with allograft Allowash cancellous bone cubes with regards to cell-scaffold interactions and biocompatibility with three primary human bone marrow (hBM) stem cell lines. This study specifically examined cell survival, cell integration, intra-scaffold cell proliferation, and differentiation of progenitor cells to investigate the potential of 3D-printed PCL scaffolds as an alternative to allograft bone material for the repair of orthopedic injuries. We found that mechanically robust PCL bone scaffolds can be fabricated via the PME process and the resulting material did not elicit detectable cytotoxicity. When the widely used osteogenic model SAOS-2 was cultured in PCL extract medium, no detectable effect was observed on cell viability or proliferation with multiple test groups showing viability ranges of 92.2% to 100% relative to a control group with a standard deviation of ±10%. In addition, we found that the honeycomb infill pattern of the 3D-printed PCL scaffold allowed for superior mesenchymal stem-cell integration, proliferation, and biomass increase. When healthy and active primary hBM cell lines, having documented in vitro growth rates with doubling times of 23.9, 24.67, and 30.94 h, were cultured directly into 3D-printed PCL scaffolds, impressive biomass increase values were observed. It was found that the PCL scaffolding material allowed for biomass increase values of 17.17%, 17.14%, and 18.18%, compared to values of 4.29% for allograph material cultured under identical parameters. It was also found that the honeycomb scaffold infill pattern was superior to the cubic and rectangular matrix structures, and provided a superior microenvironment for osteogenic and hematopoietic progenitor cell activity and auto-differentiation of primary hBM stem cells. Histological and immunohistochemical studies performed in this work confirmed the regenerative potential of PCL matrices in the orthopedic setting by displaying the integration, self-organization, and auto-differentiation of hBM progenitor cells within the matrix. Differentiation products including mineralization, self-organizing "proto-osteon" structures, and in vitro erythropoiesis were observed in conjunction with the documented expression of expected bone marrow differentiative markers including CD-99 (>70%), CD-71 (>60%), and CD-61 (>5%). All of the studies were conducted without the addition of any exogenous chemical or hormonal stimulation and exclusively utilized the abiotic and inert material polycaprolactone; setting this work apart from the vast majority of contemporary investigations into synthetic bone scaffold fabrication In summary, this study demonstrates the unique clinical potential of 3D-printed PCL scaffolds for stem cell expansion and incorporation into advanced microstructures created via PME manufacturing to generate a physiologically inert temporary bony defect graft with significant autograft features for enhanced end-stage healing.
Topics: Humans; Bone Marrow Cells; Caproates; Osteogenesis; Polyesters; Printing, Three-Dimensional; Tissue Engineering; Tissue Scaffolds; Mesenchymal Stem Cells
PubMed: 36902373
DOI: 10.3390/ijms24054940 -
Acta Biomaterialia May 2023Bone fragility is a profound complication of type 1 diabetes mellitus (T1DM), increasing patient morbidity. Within the mineralized bone matrix, osteocytes build a...
Bone fragility is a profound complication of type 1 diabetes mellitus (T1DM), increasing patient morbidity. Within the mineralized bone matrix, osteocytes build a mechanosensitive network that orchestrates bone remodeling; thus, osteocyte viability is crucial for maintaining bone homeostasis. In human cortical bone specimens from individuals with T1DM, we found signs of accelerated osteocyte apoptosis and local mineralization of osteocyte lacunae (micropetrosis) compared with samples from age-matched controls. Such morphological changes were seen in the relatively young osteonal bone matrix on the periosteal side, and micropetrosis coincided with microdamage accumulation, implying that T1DM drives local skeletal aging and thereby impairs the biomechanical competence of the bone tissue. The consequent dysfunction of the osteocyte network hampers bone remodeling and decreases bone repair mechanisms, potentially contributing to the enhanced fracture risk seen in individuals with T1DM. STATEMENT OF SIGNIFICANCE: Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease that causes hyperglycemia. Increased bone fragility is one of the complications associated with T1DM. Our latest study on T1DM-affected human cortical bone identified the viability of osteocytes, the primary bone cells, as a potentially critical factor in T1DM-bone disease. We linked T1DM with increased osteocyte apoptosis and local accumulation of mineralized lacunar spaces and microdamage. Such structural changes in bone tissue suggest that T1DM speeds up the adverse effects of aging, leading to the premature death of osteocytes and potentially contributing to diabetes-related bone fragility.
Topics: Humans; Osteocytes; Diabetes Mellitus, Type 1; Aging; Bone and Bones; Apoptosis
PubMed: 36878337
DOI: 10.1016/j.actbio.2023.02.037 -
IUCrJ Mar 2023Bone is a complex, biological tissue made up primarily of collagen fibrils and biomineral nanoparticles. The importance of hierarchical organization in bone was realized...
Bone is a complex, biological tissue made up primarily of collagen fibrils and biomineral nanoparticles. The importance of hierarchical organization in bone was realized early on, but the actual interplay between structural features and the properties on the nanostructural and crystallographic level is still a matter of intense discussion. Bone is the only mineralized tissue that can be remodeled and, at the start of the formation of new bone during this process, a structure called a cement line is formed on which regular bone grows. Here, the orientational relationship of nanostructural and crystallographic constituents as well as the structural properties of both nanostructural and crystallographic constituents around cement lines and the Haversian system in human lamellar bone are investigated. A combination of small- and wide-angle X-ray scattering tensor tomography is employed together with diffraction tomography and synchrotron computed tomography to generate a multi-modal image of the sample. This work shows that the mineral properties vary as a function of the distance to the Haversian canal and, importantly, shows that the cement line has differing mineral properties from the surrounding lamellar bone, in particular with respect to crystallite size and degree of orientation. Cement lines make up a significant portion of the bone matrix despite their small size, hence the reported findings on an altered mineral structure, together with the spatial modulation around the Haversian canal, have implications for the formation and mechanics of bone.
Topics: Humans; Haversian System; Collagen; Bone and Bones; Bone Matrix; Minerals
PubMed: 36786504
DOI: 10.1107/S2052252523000866