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Osteocytes play a role as mechanosensory cells by sensing flow-induced mechanical stimuli applied on their cell processes. High-resolution imaging of osteocyte processes and the canalicular wall are necessary for the analysis of this mechanosensing mechanism. Focused ion beam-scanning electron microscopy (FIB-SEM) enabled the visualization of the structure at the nanometer scale with thousands of serial-section SEM images. We applied machine learning for the automatic semantic segmentation of osteocyte processes and canalicular wall and performed a morphometric analysis using three-dimensionally reconstructed images.Six-week-old-mice femur were used. Osteocyte processes and canaliculi were observed at a resolution of 2 nm/voxel in a 4 × 4 μm region with 2000 serial-section SEM images. Machine learning was used for automatic semantic segmentation of the osteocyte processes and canaliculi from serial-section SEM images. The results of semantic segmentation were evaluated using the dice similarity coefficient (DSC). The segmented data were reconstructed to create three-dimensional images and a morphological analysis was performed.The DSC was 83%. Using the segmented data, a three-dimensional image of approximately 3.5 μm in length was reconstructed. The morphometric analysis revealed that the median osteocyte process diameter was 73.8 ± 18.0 nm, and the median pericellular fluid space around the osteocyte process was 40.0 ± 17.5 nm.We used machine learning for the semantic segmentation of osteocyte processes and canalicular wall for the first time, and performed a morphological analysis using three-dimensionally reconstructed images.

Nanocrystal-based processing has attracted significant interest for the fabrication of highly functional materials with controlled crystallinity and fine porous structures. In this study, we focused on the template-free synthesis of nanocrystal-based materials with size-tailored pores using layered nickel hydroxide intercalated with acrylate anions. Polymerization of the acrylates encouraged interconnection of the nanocrystals and the formation of homogeneous gel networks. Cryogels after freeze-drying had pores with an average diameter from 4.8 nm (mesoscale) to 68.9 nm (macroscale). It was found that the surface characteristics of starting nanocrystals determined the phase separation tendency of interconnected species from the reaction media and resultant porous structures. We believe that the present study can enable the design of template-free nanocrystal-based porous materials., The present work was partially supported by JSPS KAKENHI (JP20K15368), the MEXT Leading Initiative for Excellent Young Researchers, the Foundation for the Promotion of Ion Engineering, and the Izumi Science and Technology Foundation (2019-J-112).

Osteocytes form a three-dimensional (3D) cellular network within the mineralized bone matrix. The cellular network has important roles in mechanosensation and mechanotransduction related to bone homeostasis. We visualized the embedded osteocyte network in chick calvariae and observed the flow-induced Ca2+ signaling in osteocytes using 3D time-lapse imaging. In response to the flow, intracellular Ca2+ ([Ca2+]i) significantly increased in developmentally mature osteocytes in comparison with young osteocytes in the bone matrix. To investigate the differences in response between young and developmentally mature osteocytes in detail, we evaluated the expression of osteocyte-related genes using the osteocyte-like cell line MLO-Y4, which was 3D-cultured within type I collagen gels. We found that the c-Fos, Cx43, Panx3, Col1a1, and OCN mRNA levels significantly increased on day 15 in comparison with day 7. These findings indicate that developmentally mature osteocytes are more responsive to mechanical stress than young osteocytes and have important functions in bone formation and remodeling.

The collagen network acts as a scaffold for calcification and its three-dimensional structure influences bone strength. It is therefore important to observe the collagen network in detail and three-dimensionally. In this study, we observed the collagen network of chick embryonic calvariae in membranous bone three-dimensionally using orthogonally arranged FIB-SEM. A 25 × 25 μm area of chick embryonic calvaria was observed at a high resolution (25 nm per pixel). The inside of the bone (i.e. the primary calcified tissue), the bone cells (i.e. the osteoblasts and the osteocytes), the organelles, and the collagen fibrils were observed in detail. These structures were observed three-dimensionally using the Amira software program. In addition, the collagen fibrils of the bone were automatically extracted using the XTracing extension software program, and three-dimensional morphometry was performed. Almost all of the collagen fibrils ran along the longitudinal axis of the trabecular bone. We found that the regularity of the collagen fibril orientation was less remarkable in the osteoblast layer, which contained numerous osteoblasts. The collagen fibril orientation started to show regularity toward the central bone layer, which contained few bone cells.

Introduction The intercellular network of cell-cell communication among osteocytes is mediated by gap junctions. Gap junctional intercellular communication (GJIC) is thought to play an important role in the integration and synchronization of bone remodeling. To further understand the mechanism of bone development it is important to quantify the difference in the GJIC capacity of young and developmentally mature osteocytes. Materials and methods We first established an embryonic chick calvaria growth model to show the growth of the calvaria in embryos at 13 to 21 days of age. We then applied a fluorescence recovery after photobleaching (FRAP) technique to compare the difference in the GJIC capacity of young osteocytes with that of developmentally mature osteocytes. Finally, we quantified the dye (Calcein) diffusion from the FRAP data using a mathematic model of simple diffusion which was also used to identify simple diffusion GJIC pattern cells (fitted model) and accelerated diffusion GJIC pattern cells (non-fitted model). Results The relationship between the longest medial-lateral length of the calvaria (frontal bone) and the embryonic age fit a logarithmic growth model: length = 5.144 × ln(day) − 11.340. The morphometric data during osteocyte differentiation showed that the cellular body becomes more spindle-shaped and that the cell body volume decreased by approximately 22% with an increase in the length of the processes between the cells. However, there were no significant differences in the cellular body surface area or in the distance between the mass centres of the cells. The dye-displacement rate in young osteocytes was significantly higher than that in developmentally mature osteocytes: dye displacement only occurred in 26.88% of the developmentally mature osteocytes, while it occurred in 64.38% of the young osteocytes. Additionally, in all recovered osteocytes, 36% of the developmentally mature osteocytes comprised non-fitted model cells while 53.19% of the young osteocytes were the non-fitted model, which indicates the active transduction of dye molecules. However, there were no statistically significant differences between the young and developmentally mature osteocytes with regard to the diffusion coefficient, permeability coefficient, or permeance of the osteocyte processes, which were 3.93 ± 3.77 (× 10− 8 cm2/s), 5.12 ± 4.56 (× 10− 5 cm2/s) and 2.99 ± 2.47 (× 10− 13 cm2/s) (mean ± SD), respectively. Conclusions These experiments comprehensively quantified the GJIC capacity in the embryonic chick calvaria and indicated that the cell-cell communication capacity of the osteocytes in the embryonic chick calvaria was related to their development.

In the bone, collagen fibrils form a lamellar structure called the "twisted plywood-like model." Because of this unique structure, bone can withstand various mechanical stresses. However, the formation of this structure has not been elucidated because of the difficulty of observing the collagen fibril production of the osteoblasts via currently available methods. This is because the formation occurs in the very limited space between the osteoblast layer and bone matrix. In this study, we used ultra-high-voltage electron microscopy (UHVEM) to observe collagen fibril production three-dimensionally. UHVEM has 3-MV acceleration voltage and enables us to use thicker sections. We observed collagen fibrils that were beneath the cell membrane of osteoblasts elongated to the outside of the cell. We also observed that osteoblasts produced collagen fibrils with polarity. By using AVIZO software, we observed collagen fibrils produced by osteoblasts along the contour of the osteoblasts toward the bone matrix area. Immediately after being released from the cell, the fibrils run randomly and sparsely. But as they recede from the osteoblast, the fibrils began to run parallel to the definite direction and became thick, and we observed a periodical stripe at that area. Furthermore, we also observed membrane structures wrapped around filamentous structures inside the osteoblasts. The filamentous structures had densities similar to the collagen fibrils and a columnar form and diameter. Our results suggested that collagen fibrils run parallel and thickly, which may be related to the lateral movement of the osteoblasts. UHVEM is a powerful tool for observing collagen fibril production.

Osteocytes form a three-dimensional (3D) cellular network within the mineralized bone matrix. The cellular network has important roles in mechanosensation and mechanotransduction related to bone homeostasis. We visualized the embedded osteocyte network in chick calvariae and observed the flow-induced Ca

To ensure safety, lithium ion batteries (LIBs) are used with control circuits that restrict their operation voltages within an appropriate range. However, because of the capacity variation among LIB cells and the potential failure of battery control circuits, the behavior of LIBs at voltages higher or lower than the safe range should be fully clarified. Previous studies have focused primarily on over-charge and less on over-discharge (ODC), but the risk of ODC seems to be growing due to the wide spread and long-term use of LIBs. ODC has been reported to generate gas, but the gas species detected have varied. It has also been reported that ODC causes Cu to dissolve from the anode current collector, and that deep ODC would destroy SEI films. We examined the structural change of the SEI film by ODC as well as the gas species generated to elucidate how ODC generates gas. Furthermore, we performed isotopic labeling of the electrolyte to find the origin of the gas. We prepared LiMn2O4/carbon-based LIBs with aluminum-laminate packaging by using LiPF6 and an electrolyte consisting of EC and DEC. A fresh cell and a cycled cell with a capacity that had degraded to 40% were discharged to 0.5 V. The amount of ODC-generated gas was estimated from the cell-volume change. The gas volume was 2.1 cc in the fresh cell and 33.3 cc in the degraded cell, indicating that degraded cells suffer from a higher amount of gas evolution. The generated gas was analyzed by GC-TCD (carrier gas: Ar) and QMS. The generated gas was dominantly H2 (>75%), including a small amount of CxHy and COz molecules, which was in contrast to previous studies. Moreover, QMS detected a trace of CuCO, which might be produced by a reaction between the dissolved Cu atoms and the electrolyte. The difference in the major gas species may be due to the sample materials but could also be due to the gas analysis conditions such as the carrier gas species and mass scan ranges. The thickness of the SEI films in the anode was evaluated by AES with Ar+ sputtering. The SEI thickness, which we define as the depth at which the carbon proportion reaches 50%, was about 40 nm before ODC and 25 nm after ODC. Additionally, XPS measurements on the anodes revealed that ODC reduced the Li-related SEI components. These results indicate that ODC causes SEI to decompose. We also confirmed with cross-sectional SEM-EDX that ODC roughened the Cu anode current collector, thus causing Cu atoms to diffuse into the carbon active materials. To investigate the origin of the hydrogen involved in the generated gas, we prepared two types of cells with deuterated EC (series A) and standard hydrogenated EC (series B). We cycled them at 45oC until their capacity faded to 50%. Since EC is known to decompose more easily than DEC, the series-A samples were expected to have SEI films containing D atoms after the cycling, which we confirmed by cross-sectional TOF-SIMS. We also prepared a third type of cells (series C) by replacing the electrolyte of the cycled series-B cells with that containing deuterated EC. The three types of cells were discharged to 0.5 V, and the evolved gas was analyzed by QMS. In the series-A samples, a small amount of HD and D2 molecules were detected, but the majority consisted of H2 molecules. In the series-B samples, only H2 molecules were detected as expected. The molecules generated in the series-C samples were primarily H2. These results indicate that the origin of the generated H2 is not the SEI components derived from the electrolyte (not from EC, at least), and that the H2-gas evolution is not a result of direct decomposition of the electrolyte. The origin of the H2 is presumably hydrogen that is derived from the cathode and incorporated into the SEI on the anode. In summary, we found that ODC destroys SEI films on the anode and generates H2 gas. The H2 does not seem to be derived from the electrolyte (EC); rather, H-impurities in the cathode active material are likely to be the H2 source. The volume of the ODC-induced gas increases with cycles. This tendency is attributable to a thicker SEI with H atoms and a higher anode potential upon ODC of the degraded cells. Suppressing the residual H-concentration in the cathode and SEI growth seems to be an effective way to eliminate the gas generation upon ODC.